WO2018120842A1 - 一种双功能分子及其应用 - Google Patents

一种双功能分子及其应用 Download PDF

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WO2018120842A1
WO2018120842A1 PCT/CN2017/096592 CN2017096592W WO2018120842A1 WO 2018120842 A1 WO2018120842 A1 WO 2018120842A1 CN 2017096592 W CN2017096592 W CN 2017096592W WO 2018120842 A1 WO2018120842 A1 WO 2018120842A1
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amino acid
acid sequence
bsab
set forth
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PCT/CN2017/096592
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French (fr)
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陈帅
朱化星
廖远平
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上海欣百诺生物科技有限公司
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Priority claimed from CN201611258667.7A external-priority patent/CN108264566B/zh
Priority claimed from CN201611260818.2A external-priority patent/CN108264562B/zh
Priority claimed from CN201611256643.8A external-priority patent/CN108264557B/zh
Priority claimed from CN201611260781.3A external-priority patent/CN108264560B/zh
Application filed by 上海欣百诺生物科技有限公司 filed Critical 上海欣百诺生物科技有限公司
Priority to EP17886498.9A priority Critical patent/EP3575319A4/en
Priority to US16/474,554 priority patent/US20230242876A1/en
Publication of WO2018120842A1 publication Critical patent/WO2018120842A1/zh

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a bifunctional molecule and an application thereof.
  • T lymphocytes are derived from the thymus (Thymus), so they are called T cells.
  • Mature T cells exist in the thymus-dependent region of peripheral immune organs, occupying a central position in adaptive cellular immune responses, and also play an important auxiliary role in thymus-dependent antigen-induced humoral immune responses.
  • T cells can be divided into Cytotoxic T lymphocyte (CTL), Helper T cell (Th) and Regulatory T cell (Treg).
  • CTL expresses CD8, which is the main effector cell of adaptive cellular immunity.
  • CD8-positive T cells Complete activation and efficient amplification of CD8-positive T cells is the basis for their effective killing of target cells, depending on the role of the dual signaling pathway: MHC I/endogenous antigen on the surface of antigen-presenting cells (APC)
  • APC antigen-presenting cells
  • the peptide complex specifically recognizes the TCR/CD3 complex expressed by T cells, causing CD3 to interact with the cytoplasmic segment of the co-receptor CD8, activating the protein tyrosine kinase linked to the tail of the cytoplasmic segment, and immunizing the CD3 cytoplasmic region Tyrosine phosphorylation in the immune tyrosine-based activation motif (ITAM) initiates a signaling cascade that activates transcription factors that allow T cell activation, which is T cell activation.
  • TAM immune tyrosine-based activation motif
  • the first signal at the same time, co-stimulatory molecules on the surface of T cells (such as CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM- 3, TIGIT, BTLA, etc. can be associated with co-stimulatory molecular ligands on the surface of APC cells (eg CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1, PD-L2, HVEM, etc.
  • co-stimulatory molecules on the surface of T cells such as CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM- 3, TIGIT, BTLA, etc.
  • co-stimulatory molecular ligands on the surface of APC cells eg CD80, CD86, 4-1BBL, B7RP
  • the second signal (co-stimulatory signal): wherein CD28, 4-1BB, ICOS, OX40, GITR, CD40L and CD27 belong to a positive costimulatory molecule, and the corresponding ligands (CD80, CD86, 4-1BBL, B7RP-1, OX40L) , the second signal generated by the interaction of GITRL, CD40, CD70, etc.
  • T cells can lead to complete activation of T cells; and CTLA-4, PD-1, LAG-3, TIM-3, TIGIT and BTLA
  • the second signal (negative costimulatory signal) produced by interaction with the corresponding ligand (CD80, CD86, PD-L1, PD-L2, Galectin-9, HVEM, etc.) is mainly down-regulated and terminated. Activation of T cells.
  • an activated monoclonal full-length antibody against positive co-stimulatory molecules such as CD28, anti-4-1BB and anti-ICOS has been designed and constructed (US Patent 20100168400A1; US Patent 20100183621A1; US Patent 009193789B2) or a blocking type monoclonal full-length antibody against negative co-stimulatory molecules such as PD-1, anti-CTLA-4 and anti-LAG-3 (World Patent 2013173223A1; US Patent 007452535B2; US Patent 2015116539A1)
  • the combination of CD3 full-length antibodies provides a complete double for T cells.
  • the combination of the two monoclonal full-length antibodies still has some shortcomings in specific applications, such as significantly increasing the workload and production cost of recombinant antibody expression and purification, and must be optimized for in vitro activation and expansion of T cells.
  • the relative proportions of the two full length antibodies when two full-length antibodies are used in combination, in order to promote receptor activation, it is necessary to add a higher concentration of the antibody solution or coat the antibody onto the culture plate or the microsphere to enhance its activation effect on the receptor.
  • a bifunctional molecule comprising a first functional domain capable of binding to and activating a T cell surface CD3 molecule and a second functional domain capable of binding and activating a T cell surface CD28 molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating T cell surface CD3 molecules and CD28 molecules, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD3 antibody and the second functional domain is an anti-CD28 antibody.
  • the antibody is a small molecule antibody.
  • the antibody is selected from the group consisting of a Fab antibody, an Fv antibody or a single chain antibody (scFv).
  • the first functional domain and the second functional domain are connected by a connection segment.
  • the linked fragment may have an amino acid number of ⁇ 2.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO: 17.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD, and the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is shown in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second domain.
  • the first functional domain is an anti-CD3 single chain antibody
  • the second functional domain is an anti-CD28 single chain antibody
  • the single chain antibody comprises a heavy chain variable region and a light chain variable region.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of a single-chain antibody against CD3 is set forth in SEQ ID NO.
  • amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is also set forth in SEQ ID NO.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is set forth in SEQ ID NO.
  • a bifunctional molecule comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and activating a T cell positive costimulatory molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating T cell surface CD3 molecules and T cell co-stimulation Molecules, thereby producing a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD3 antibody and the second functional domain is an antibody against a T cell positive costimulatory molecule.
  • the antibody is a small molecule antibody.
  • the antibody is selected from the group consisting of a Fab antibody, an Fv antibody or a single chain antibody (scFv).
  • the first functional domain and the second functional domain are connected by a connection segment.
  • the linked fragment may have an amino acid number of ⁇ 2.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second domain.
  • the first functional domain is a single chain antibody against CD3, and the second domain is a single chain antibody against a T cell positive costimulatory molecule, the single chain antibody comprising a heavy chain variable region and a light chain Variable zone.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the single-chain antibody of the anti-T cell positive costimulatory molecule may be a single chain antibody against 4-1BB, a single chain antibody against ICOS, a single chain antibody against OX40, a single chain antibody against GITR, anti-CD40L Any of a single-chain antibody or a single-chain antibody against CD27.
  • amino acid sequence of the heavy chain variable region of the anti-4-1BB single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-4-1BB single-chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-4-1BB single-chain antibody is shown in SEQ ID NO.
  • the amino acid sequence of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD27 single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is also exemplified as SEQ ID NO. 43, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 55, SEQ ID NO. Or as shown in any of SEQ ID NO.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is any one of SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 61 or SEQ ID NO. Shown.
  • a bifunctional molecule comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and activating a T cell positive costimulatory molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating a T cell surface CD3 molecule and a T cell positive costimulatory molecule, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD3 antibody and the second functional domain is a ligand extracellular domain domain of a T cell positive costimulatory molecule.
  • the antibody is a small molecule antibody.
  • the antibody is selected from the group consisting of a Fab antibody, an Fv antibody or a single chain antibody (scFv).
  • the first functional domain and the second functional domain are connected by a connection segment.
  • the linked fragment may have an amino acid number of ⁇ 2.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second domain.
  • the first functional domain is an anti-CD3 single chain antibody comprising a heavy chain variable region and a light chain variable region.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the ligand extracellular domain of the T cell positive costimulatory molecule is selected from the group consisting of a 4-1BBL extracellular domain domain, a B7RP-1 extracellular domain domain, an OX40L extracellular domain domain, and a GITRL extracellular domain. Any of the domains or the CD70 extracellular domain.
  • amino acid sequence of the 4-1BBL extracellular domain domain is set forth in SEQ ID NO.
  • amino acid sequence of the B7RP-1 extracellular domain domain is set forth in SEQ ID NO.
  • amino acid sequence of the OX40L extracellular domain domain is set forth in SEQ ID NO.
  • amino acid sequence of the GITRL extracellular domain domain is set forth in SEQ ID NO.
  • amino acid sequence of the CD70 extracellular domain domain is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is also exemplified by SEQ ID NO. 149, SEQ ID NO. 153, SEQ ID NO. 157, SEQ ID NO. 161 or SEQ ID NO. Either shown.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is as set forth in any one of SEQ ID NO. 151, SEQ ID NO. 155, SEQ ID NO. 159, SEQ ID NO. 163 or SEQ ID NO.
  • a bifunctional molecule comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and blocking a T cell negative costimulatory molecule .
  • the bifunctional molecule is capable of binding and blocking T cell negative costimulatory molecules while binding to and activating T cell surface CD3 molecules, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD3 antibody and the second functional domain is an antibody against a T cell negative costimulatory molecule.
  • the antibody is a small molecule antibody.
  • the antibody is selected from the group consisting of a Fab antibody, an Fv antibody or a single chain antibody (scFv).
  • the first functional domain and the second functional domain are connected by a connection segment.
  • the linked fragment may have an amino acid number of ⁇ 2.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second domain.
  • the first functional domain is a single chain antibody against CD3, and the second domain is a single chain antibody against an anti-T cell negative costimulatory molecule, the single chain antibody comprising a heavy chain variable region and a light chain Variable zone.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the single-chain antibody of the anti-T cell negative costimulatory molecule may be a single-chain antibody against PD-1, a single-chain antibody against CTLA-4, a single-chain antibody against LAG-3, and anti-TIM-3. Any of a single-chain antibody, a single-chain antibody against TIGIT, or a single-chain antibody against BTLA.
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-TIGIT single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single chain antibody is set forth in SEQ ID NO. 242.
  • the amino acid sequence of the single-chain antibody against PD-1 is set forth in SEQ ID NO.
  • the amino acid sequence of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-LAG-3 single-chain antibody is as SEQ ID NO. 251.
  • the amino acid sequence of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-TIGIT single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is SEQ ID NO. 218, SEQ ID NO. 222, SEQ ID NO. 226, SEQ ID NO. 230, SEQ ID NO. 234 or SEQ. Shown as any of ID NO.238.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is any one of SEQ ID NO. 220, SEQ ID NO. 224, SEQ ID NO. 228, SEQ ID NO. 232, SEQ ID NO. 236 or SEQ ID NO. Shown.
  • a polynucleotide encoding the aforementioned bifunctional molecule.
  • an expression vector comprising the aforementioned polynucleotide is provided.
  • a host cell which is transformed with the aforementioned expression vector.
  • a method for producing the aforementioned bifunctional molecule comprises: constructing an expression vector containing a bifunctional molecule gene sequence, and then transforming an expression vector containing a bifunctional molecule gene sequence into a host cell to induce expression, The bifunctional molecule is isolated from the expression product.
  • the expression vector is pcDNA3.1.
  • the host cell uses Chinese hamster ovary cells (CHO).
  • a ninth aspect of the invention there is provided the use of the aforementioned bifunctional molecule for the preparation of a T cell in vitro amplification agent.
  • a T cell in vitro amplification agent comprising the aforementioned bifunctional molecule.
  • a method for expanding T cells in vitro comprising the step of: acting on said T cells by said bifunctional molecule.
  • the method can be for non-therapeutic purposes.
  • the present invention has the following beneficial effects:
  • the present invention binds and activates a first functional domain of a T cell surface CD3 molecule and a second functional domain capable of binding and activating a T cell surface CD28 molecule to be fused to the same protein peptide chain to form a bifunctional molecule, using eukaryotic cells
  • the expression system is produced, the expression product has a single structure, the purification process is simple, the protein yield is high, the preparation process and the product are stable; and the anti-CD3 monoclonal full-length antibody and the anti-CD28 monoclonal full-length antibody are used in combination, and the two antibodies need to be separately expressed and purified.
  • the preparation process is more complicated, and the workload and production cost are significantly increased.
  • the bifunctional molecule of the present invention is a single protein, and the anti-CD3 full-length antibody is combined with the anti-CD28 full-length antibody, and the T cell is better in vitro, the protein is used less, and the use is simple and can be adopted.
  • the solution form is added directly without the need to optimize the relative proportions of the two full length antibodies.
  • the present invention also binds and activates a first functional domain of a T cell surface CD3 molecule and a second functional domain capable of binding and activating a T cell positive costimulatory molecule to be fused to the same protein peptide chain to form a bifunctional molecule, using true
  • the nuclear cell expression system produces a single structure, the purification process is simple, the protein yield is high, the preparation process and the product are stable; and the anti-CD3 monoclonal full-length antibody and the anti-T cell positive costimulatory molecule monoclonal full-length antibody are used in combination.
  • the two antibodies need to be separately expressed and purified, the preparation process is more complicated, and the workload and production cost are significantly increased.
  • the bifunctional molecule of the present invention is a single protein, and the anti-CD3 full-length antibody is combined with the anti-T cell positive costimulatory molecule full-length antibody, and the T cell activation and amplification effect is better, and the protein dosage is less. It is easy to use and can be directly added as a solution without optimizing the relative proportions of the two full-length antibodies.
  • the present invention also binds and activates a first domain of a T cell surface CD3 molecule and a ligand extracellular domain of a T cell positive costimulatory molecule fused to the same protein peptide chain to form a bifunctional molecule, using eukaryotic cells
  • the expression system is produced, the expression product has a single structure, the purification process is simple, the protein yield is high, the preparation process and the product are stable; and the anti-CD3 monoclonal full-length antibody and the anti-T cell positive costimulatory molecule monoclonal full-length antibody are used in combination, two The antibodies need to be separately expressed and purified, the preparation process is more complicated, and the workload and production cost are significantly increased.
  • the bifunctional molecule of the present invention is a single protein, and the anti-CD3 full-length antibody is combined with the anti-T cell positive costimulatory molecule full-length antibody, and the T cell activation and amplification effect is better, and the protein dosage is less. It is easy to use and can be directly added as a solution without optimizing the relative proportions of the two full-length antibodies.
  • the present invention will also be able to bind and activate the first functional domain of the T cell surface CD3 molecule and be capable of binding and blocking
  • the second functional domain of the negative T cell negative costimulatory molecule is fused to the same protein peptide chain to form a bifunctional molecule, which is produced by a eukaryotic cell expression system, has a single structure, a simple purification process, high protein yield, and a stable preparation process and product;
  • the anti-CD3 monoclonal full-length antibody and the anti-T cell negative costimulatory molecule monoclonal full-length antibody are used in combination, the two antibodies need to be separately expressed and purified, the preparation process is more complicated, and the workload and production cost are significantly increased.
  • the bifunctional molecule of the present invention is a single protein, and the anti-CD3 full-length antibody is combined with the anti-T cell negative costimulatory molecule full-length antibody, and the T cell activation and amplification effect is better, and the protein dosage is less. It is easy to use and can be directly added as a solution without optimizing the relative proportions of the two full-length antibodies.
  • Figure 1-1 A. Structure of the monomeric form of anti-CD3/anti-CD28 bispecific antibody (CD3-CD28 BsAb_M); B. Dimeric form of anti-CD3/anti-CD28 bispecific antibody (CD3-CD28 BsAb_D) Structure diagram.
  • Figure 1-2 Final purified CD3-CD28 BsAb_M and CD3-CD28 BsAb_D recombinant protein was analyzed by SDS-PAGE, electrophoresis under reducing and non-reducing conditions,
  • Figure 1-3A ELISA identification of CD3-CD28 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen CD28-hFc; Does not coat the results of any antigen determination.
  • Figure 1-3B ELISA identification of CD3-CD28 BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD28-hFc; Does not coat the results of any antigen determination.
  • Figure 1-4 CIK cell expansion factor curve, peripheral blood PBMC for experimental cells, add CD3-CD28 BsAb_M, CD3-CD28 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), for a total of 14 days of culture, counting the number of cells per count divided by the number of cells on day 1, counting and comparing cell expansion folds; wherein, control group 1: 5 ug / ml Anti-CD3 and 5 ug / ml Anti- CD28 coated plate; control group 2: 100 ng/ml Anti-CD3 and 100 ng/ml Anti-CD28 were added in solution; experimental group 1: 10 ng/ml CD3-CD28 BsAb_M was added in solution; experimental group 2: added in solution state 10 ng/ml CD3-CD28 BsAb_D.
  • control group 1 5 ug / ml Anti-CD3 and 5 ug / ml Anti-
  • Figure 1-5 The ratio of CD3 + CD56 + CIK cells was determined by flow cytometry. The expanded cells were determined as shown in Figure 1-4, and the ratio of CD3 + CD56 + double positive CIK cells was determined. 1; B: control group 2; C: experimental group 1; D: experimental group 2.
  • Figure 1-6 Determination of CD8 + /CD4 + ratio in CIK cells based on flow cytometry.
  • the expanded cells described in Figures 1-4 were used to determine the ratio of CD8 + positive to CD4 + positive cells, respectively.
  • FIG 2-1 A Structural map of the monomeric form of anti-CD3/anti-T cell positive costimulatory molecule bispecific antibody (BsAb_M); B. Dimeric form of anti-CD3/anti-T cell positive costimulatory molecule bispecific A structural diagram of the antibody (BsAb_D).
  • Figure 2-2 A. Purified CD3-4-1BB BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-4-1BB BsAb_M; lane 3: non-reducing CD3-4- 1BB BsAb_M; B. Purified CD3-4-1BB BsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-4-1BB BsAb_D; lane 3: non-reducing CD3-4-1BB BsAb_D .
  • Figure 2-3A ELISA identification of CD3-4-1BB BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen 4-1BB -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 2-3B ELISA identification of CD3-4-1BB BsAb_D, the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen 4-1BB-hFc; ⁇ did not coat the results of any antigen measurement.
  • Figure 2-4 CIK cell expansion fold curve, peripheral blood PBMC for experimental cells, add CD3-4-1BB BsAb_M, CD3-4-1BB BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-4-1BB BsAb_M was added in solution; Experimental group 2: 10 ng/ml CD3-4-1BB BsAb_D was added in solution.
  • Figure 2-5 The ratio of CD8 + /CD4 + in CIK cells was determined by flow cytometry. The cells after 30 days of expansion as shown in Figure 2-4 were used to determine the ratio of CD8 + positive to CD4 + positive cells.
  • Figure 2-6 A. Purified CD3-ICOS BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-ICOS BsAb_M; lane 3: non-reducing CD3-ICOS BsAb_M; B. purification CD3-ICOS BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-ICOS BsAb_D; lane 3: non-reducing CD3-ICOS BsAb_D.
  • Figure 2-7A ELISA identification of CD3-ICOS BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen ICOS-hFc; Does not coat the results of any antigen determination.
  • Figure 2-7B ELISA identification of CD3-ICOS BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen ICOS-hFc; Does not coat the results of any antigen determination.
  • Figure 2-8 CIK cell expansion factor curve, peripheral blood PBMC as experimental cells, add CD3-ICOS BsAb_M, CD3-ICOS BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), for a total of 14 days of culture, dividing the number of cells per count by the number of cells on day 1, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 package Cell culture plate; Experimental group 1: 10 ng/ml CD3-ICOS BsAb_M was added in solution; Experimental group 2: 10 ng/ml CD3-ICOS BsAb_D was added in solution.
  • Figure 2-9 The ratio of CD3 + CD56 + CIK cells was determined by flow cytometry. The cells after 14 days of expansion as shown in Figure 2-8 were used to determine the proportion of CD3 + CD56 + double positive cells; Control group; B: experimental group 1; C: experimental group 2.
  • Figure 2-10 A. Purified CD3-OX40 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-OX40 BsAb_M; lane 3: non-reducing CD3-OX40 BsAb_M; B. purification CD3-OX40 BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-OX40 BsAb_D; lane 3: non-reducing CD3-OX40 BsAb_D.
  • Figure 2-11A ELISA identification of CD3-OX40 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen OX40-hFc; Does not coat the results of any antigen determination.
  • Figure 2-11B ELISA identification of CD3-OX40 BsAb_D, the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g / ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g / ml recombinant antigen OX40-hFc; Does not coat the results of any antigen determination.
  • Figure 2-12 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-OX40 BsAb_M, CD3-OX40 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 package Cell culture Plate; Experimental group 1: 10 ng/ml CD3-OX40 BsAb_M was added in the solution state; Experimental group 2: 10 ng/ml CD3-OX40 BsAb_D was added in the solution state.
  • Figure 2-13 Detection of killing activity of expanded CIK cells on tumor cells, using CIK cells as killing effector cells and Raji lymphoma cells as target cells after 14 and 30 days of amplification as shown in Figure 2-12, respectively.
  • Figure 2-14 A. Purified CD3-GITR BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-GITR BsAb_M; lane 3: non-reducing CD3-GITR BsAb_M; B. purification CD3-GITR BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-GITR BsAb_D; lane 3: non-reducing CD3-GITR BsAb_D.
  • Figure 2-15A ELISA identification of CD3-GITR BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen GITR-hFc; Does not coat the results of any antigen determination.
  • Figure 2-15B ELISA identification of CD3-GITR BsAb_D, the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g / ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g / ml recombinant antigen GITR-hFc; Does not coat the results of any antigen determination.
  • Figure 2-16 CIK cell expansion fold curve, peripheral blood PBMC for experimental cells, add CD3-GITR BsAb_M, CD3-GITR BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), for a total of 14 days of culture, the number of cells counted per time was divided by the number of cells on day 1, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-GITR BsAb_M in solution; experimental group 2: 10 ng / ml CD3- in solution GITR BsAb D.
  • Figure 2-17 A. Purified CD3-CD40L BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CD40L BsAb_M; lane 3: non-reducing CD3-CD40L BsAb_M; B. purification CD3-CD40L BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CD40L BsAb_D; lane 3: non-reducing CD3-CD40L BsAb_D.
  • Figure 2-18A ELISA identification of CD3-CD40L BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen CD40L-hFc; Does not coat the results of any antigen determination.
  • Figure 2-18B ELISA identification of CD3-CD40L BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD40L-hFc; Does not coat the results of any antigen determination.
  • Figure 2-19 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-CD40L BsAb_M, CD3-CD40L BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), for a total of 14 days of culture, the number of cells counted per time was divided by the number of cells on day 1, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-CD40L BsAb_M in solution; experimental group 2: 10 ng / ml CD3- in solution CD40L BsAb_D.
  • Figure 2-20 A. Purified CD3-CD27 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD3-CD27 BsAb_M; lane 3: non-reducing CD3-CD27 BsAb_M; B. purification CD3-CD27 BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CD27 BsAb_D; lane 3: non-reducing CD3-CD27 BsAb_D.
  • Figure 2-21A ELISA identification of CD3-CD27 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen CD27-hFc; Does not coat the results of any antigen determination.
  • Figure 2-21B ELISA identification of CD3-CD27 BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD27-hFc; Does not coat the results of any antigen determination.
  • Figure 2-22 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-CD27 BsAb_M, CD3-CD27 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-CD27 BsAb_M in solution; experimental group 2: 10 ng / ml CD3- in solution CD27 BsAb_D.
  • Figure 3-1 A Structural map of the anti-CD3/T cell positive costimulatory molecule ligand bispecific molecule (BsM_M) in monomeric form; B. Dimeric form of anti-CD3/T cell positive costimulatory molecule A structural diagram of a body bispecific molecule (BsM_D).
  • Figure 3-2 A. Purified CD3-4-1BBL BsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-4-1BBL BsM_M; lane 3: non-reducing CD3-4- 1BBL BsM_M; B. Purified CD3-4-1BBL BsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-4-1BBL BsM_D; lane 3: non-reducing CD3-4-1BBL BsM_D .
  • Figure 3-3A ELISA identification of CD3-4-1BBL BsM_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant protein 4-1BB. -hFc; ⁇ does not coat the results of any protein assay.
  • Figure 3-3B ELISA identification of CD3-4-1BBL BsM_D, the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g / ml recombinant protein CD3-hFc; ⁇ coated with 1 ⁇ g / ml recombinant protein 4-1BB -hFc; ⁇ does not coat the results of any protein assay.
  • Figure 3-4 CIK cell expansion fold curve, peripheral blood PBMC for experimental cells, add CD3-4-1BBL BsM_M, CD3-4-1BBL BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-4-1 BBL BsM_M was added in solution; Experimental group 2: 10 ng/ml CD3-4-1 BBL BsM_D was added in solution.
  • Figure 3-5 A. Purified CD3-B7RP-1 BsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-B7RP-1 BsM_M; lane 3: non-reducing CD3-B7RP- 1 BsM_M; B. Purified CD3-B7RP-1 BsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-B7RP-1 BsM_D; lane 3: non-reducing CD3-B7RP-1 BsM_D .
  • Figure 3-6A ELISA identification of CD3-B7RP-1 BsM_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant protein ICOS-hFc ; ⁇ does not include the results of any protein measurement.
  • Figure 3-6B ELISA identification of CD3-B7RP-1 BsM_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant protein ICOS-hFc ; ⁇ does not include the results of any protein measurement.
  • Figure 3-7 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-B7RP-1 BsM_M, CD3-B7RP-1 BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-B7RP-1 BsM_M was added in solution; Experimental group 2: 10 ng/ml CD3-B7RP-1 BsM_D was added in solution.
  • Figure 3-8 A. Purified CD3-OX40L BsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-OX40L BsM_M; lane 3: non-reducing CD3-OX40L BsM_M; B. purification CD3-OX40L BsM_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-OX40L BsM_D; lane 3: non-reducing CD3-OX40L BsM_D.
  • Figure 3-9A ELISA identification of CD3-OX40L BsM_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant protein OX40-hFc; Does not coat the results of any protein assay.
  • Figure 3-9B ELISA identification of CD3-OX40L BsM_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant protein OX40-hFc; Does not coat the results of any protein assay.
  • Figure 3-10 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-OX40L BsM_M, CD3-OX40L BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 package Cell culture plate; Experimental group 1: 10 ng/ml CD3-OX40L BsM_M was added in the solution state; Experimental group 2: 10 ng/ml CD3-OX40L BsM_D was added in the solution state.
  • Figure 3-11 A. Purified CD3-GITRL BsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-GITRL BsM_M; lane 3: non-reducing CD3-GITRL BsM_M; B. purification CD3-GITRL BsM_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-GITRL BsM_D; lane 3: non-reducing CD3-GITRL BsM_D.
  • Figure 3-12A ELISA identification of CD3-GITRL BsM_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant protein GITR-hFc; Does not coat the results of any protein assay.
  • Figure 3-12B ELISA identification of CD3-GITRL BsM_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant protein GITR-hFc; Does not coat the results of any protein assay.
  • Figure 3-13 CIK cell expansion fold curve, peripheral blood PBMC for experimental cells, add CD3-GITRL BsM_M, CD3-GITRL BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-GITRL BsM_M in solution; experimental group 2: 10 ng / ml CD3- in solution GITRL BsM_D.
  • Figure 3-14 A. Purified CD3-CD70 BsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD3-CD70 BsM_M; lane 3: non-reducing CD3-CD70 BsM_M; B. purification CD3-CD70 BsM_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CD70BsM_D; lane 3: non-reducing CD3-CD70 BsM_D.
  • Figure 3-15A ELISA identification of CD3-CD70 BsM_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant protein CD27-hFc; Does not coat the results of any protein assay.
  • Figure 3-15B ELISA identification of CD3-CD70 BsM_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant protein CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant protein CD27-hFc; Does not coat the results of any protein assay.
  • Figure 3-16 CIK cell expansion factor curve, peripheral blood PBMC as experimental cells, respectively, add CD3-CD70 BsM_M, CD3-CD70 BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody combined use (Anti-CD3/Anti-CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • CD3-CD70 BsM_M CD3-CD70 BsM_D or anti-CD3/anti-CD28 monoclonal full-length antibody combined use (Anti-CD3/Anti-CD28)
  • Anti-CD3/Anti-CD28 monoclonal full-length antibody combined use
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-CD70 BsM_M in solution; experimental group 2: 10 ng / ml CD3- in solution CD70 BsM_D.
  • Figure 4-1 A. Structural map of the monomeric form of anti-CD3/anti-T cell negative costimulatory molecule bispecific antibody (BsAb_M); B. Dimeric form of anti-CD3/anti-T cell negative costimulatory molecule bispecific A structural diagram of the antibody (BsAb_D).
  • Figure 4-2 A. Purified CD3-PD-1 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-PD-1 BsAb_M; lane 3: non-reducing CD3-PD- 1 BsAb_M; B. Purified CD3-PD-1 BsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-PD-1 BsAb_D; lane 3: non-reducing CD3-PD-1 BsAb_D .
  • Figure 4-3A ELISA identification of CD3-PD-1 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen PD-1 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-3B ELISA identification of CD3-PD-1 BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen PD-1 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-4 CIK cell expansion factor curve, peripheral blood PBMC is experimental cells, add CD3-PD-1 BsAb_M, CD3-PD-1 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-PD-1 BsAb_M was added in solution; Experimental group 2: 10 ng/ml CD3-PD-1 BsAb_D was added in solution.
  • the control group 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate
  • experimental group 1 10 ng/ml CD3-PD-1 BsAb_M was added in solution
  • Experimental group 2 10 ng
  • Figure 4-5 CD3-PD-1 bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • Control group 2 ⁇ 10 5 CIK cells cultured in the control group (Anti-CD3/Anti-CD28) of Example 4-4 for 25 days were centrifuged, and the amount of IFN- ⁇ secreted by the cells was measured by an ELISA kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D in solution for 25 days, and the same number of cells were taken and centrifuged. In the supernatant, the amount of IFN- ⁇ secreted by the cells was measured, and the relative secretion amount of IFN- ⁇ was divided by the control group.
  • Figure 4-6 A. Purified CD3-CTLA-4 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CTLA-4 BsAb_M; lane 3: non-reducing CD3-CTLA- 4 BsAb_M; B. Purified CD3-CTLA-4 BsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-CTLA-4 BsAb_D; lane 3: non-reducing CD3-CTLA-4 BsAb_D .
  • Figure 4-7A ELISA identification of CD3-CTLA-4 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen CTLA-4 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-7B ELISA identification of CD3-CTLA-4 BsAb_D, the curves in the figure represent three test results: ⁇ coating 1 ⁇ g / ml recombinant antigen CD3-hFc; ⁇ coating 1 ⁇ g / ml recombinant antigen CTLA-4 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-8 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-CTLA-4 BsAb_M, CD3-CTLA-4 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-CTLA-4 BsAb_M was added in solution; Experimental group 2: 10 ng/ml CD3-CTLA-4 BsAb_D was added in solution.
  • Figure 4-9 CD3-CTLA-4 bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • control group 2 ⁇ 10 5 CIK cells were cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of Example 4-9, and the supernatant was centrifuged, and the amount of IFN- ⁇ secreted by the cells was measured by an ELISA kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D in solution for 25 days, and the same number of cells were taken and centrifuged. In the supernatant, the amount of IFN- ⁇ secreted by the cells was measured, and the relative secretion amount of IFN- ⁇ was divided by the control group.
  • Figure 4-10 A. Purified CD3-LAG-3 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-LAG-3 BsAb_M; lane 3: non-reducing CD3-LAG- 3 BsAb_M; B. Purified CD3-LAG-3 BsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-LAG-3 BsAb_D; lane 3: non-reducing CD3-LAG-3 BsAb_D .
  • Figure 4-11A ELISA identification of CD3-LAG-3 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen LAG-3 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-11B ELISA identification of CD3-LAG-3 BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen LAG-3 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-12 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-LAG-3 BsAb_M, CD3-LAG-3 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti -CD3/Anti-CD28), total culture for 30 days, counting the number of cells per count divided by the number of cells on the first day, counting and comparing cell expansion folds, wherein the control group: 5 ug/ml Anti-CD3 and 5 ug/ml Anti-CD28 coated cell culture plate; experimental group 1: 10 ng/ml CD3-LAG-3 BsAb_M was added in solution; Experimental group 2: 10 ng/ml CD3-LAG-3 BsAb_D was added in solution.
  • Figure 4-13 CD3-LAG-3 bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • Control group 2 ⁇ 10 5 CIK cells were cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of Example 4-14, and the supernatant was centrifuged, and the amount of IFN- ⁇ secreted by the cells was measured by an ELISA kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D in solution for 25 days, and the same number of cells were taken and centrifuged. In the supernatant, the amount of IFN- ⁇ secreted by the cells was measured, and the relative secretion amount of IFN- ⁇ was divided by the control group.
  • Figure 4-14 A. Purified CD3-TIM-3 BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-TIM-3 BsAb_M; lane 3: non-reducing CD3-TIM- 3 BsAb_M; B. Purified CD3-TIM-3 BsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-TIM-3 BsAb_D; lane 3: non-reducing CD3-TIM-3 BsAb_D .
  • Figure 4-15A ELISA identification of CD3-TIM-3 BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen TIM-3 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-15B ELISA identification of CD3-TIM-3 BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen TIM-3 -hFc; ⁇ does not coat the results of any antigen measurement.
  • Figure 4-16 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-TIM-3 BsAb_M, CD3-TIM-3 BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti - CD3/Anti-CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-TIM-3 BsAb_M in solution; experimental group 2: 10 ng / ml in solution CD3-TIM-3 BsAb_D.
  • Figure 4-17 CD3-TIM-3 bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • Control group 2 ⁇ 10 5 CIK cells were cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of Example 4-19, and the supernatant was centrifuged, and the amount of IFN- ⁇ secreted by the cells was measured by an ELISA kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D in solution for 25 days, and the same number of cells were taken and centrifuged. In the supernatant, the amount of IFN- ⁇ secreted by the cells was measured, and the relative secretion amount of IFN- ⁇ was divided by the control group.
  • Figure 4-18 A. Purified CD3-TIGIT BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-TIGIT BsAb_M; lane 3: non-reducing CD3-TIGIT BsAb_M; B. purification CD3-TIGIT BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD3-TIGIT BsAb_D; lane 3: non-reducing CD3-TIGIT BsAb_D.
  • Figure 4-19A ELISA identification of CD3-TIGIT BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen TIGIT-hFc; Does not coat the results of any antigen determination.
  • Figure 4-19B ELISA identification of CD3-TIGIT BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen TIGIT-hFc; Does not coat the results of any antigen determination.
  • Figure 4-20 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-TIGIT BsAb_M, CD3-TIGIT BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-TIGIT BsAb_M in solution; experimental group 2: 10 ng / ml CD3- in solution TIGIT BsAb_D.
  • Figure 4-21 CD3-TIGIT bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • Control group 2 ⁇ 10 5 CIK cells cultured in the control group (Anti-CD3/Anti-CD28) of Example 4-24 for 25 days, centrifuged to take the supernatant, and the amount of IFN- ⁇ secreted by the cells was detected by ELISA Kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D in solution for 25 days, and the same number of cells were taken as the control group, and the supernatant was centrifuged to detect the cells. The amount of secreted IFN- ⁇ was divided by the control group to determine the relative amount of IFN- ⁇ .
  • Figure 4-22 A. Purified CD3-BTLA BsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD3-BTLA BsAb_M; lane 3: non-reducing CD3-BTLA BsAb_M; B. purification CD3-BTLA BsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: non-reducing CD3-BTLA BsAb_D; lane 3: reducing CD3-BTLA BsAb_D.
  • Figure 4-23A ELISA identification of CD3-BTLA BsAb_M.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc, ⁇ coated with 1 ⁇ g/ml recombinant antigen BTLA-hFc; Does not coat the results of any antigen determination.
  • Figure 4-23B ELISA identification of CD3-BTLA BsAb_D.
  • the curves in the figure represent three test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen BTLA-hFc; Does not coat the results of any antigen determination.
  • Figure 4-24 CIK cell expansion fold curve, peripheral blood PBMC as experimental cells, add CD3-BTLA BsAb_M, CD3-BTLA BsAb_D or anti-CD3/anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti -CD28), cultured for a total of 30 days, and the number of cells counted per time was divided by the number of cells on the first day, and the cell expansion factor was counted and compared.
  • control group 5 ug / ml Anti-CD3 and 5 ug / ml Anti-CD28 coated plate; experimental group 1: 10 ng / ml CD3-BTLA BsAb_M in solution; experimental group 2: 10 ng / ml CD3- in solution BTLA BsAb_D.
  • Figure 4-25 CD3-BTLA bispecific antibody mediated secretion of IFN- ⁇ from CIK cells.
  • Control group 2 ⁇ 10 5 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of Example 4-29 were centrifuged, and the amount of IFN- ⁇ secreted by the cells was measured by an ELISA kit. It was defined as 1; experimental group 1 and experimental group 2 were respectively added with CD3-BTLA BsAb_M and CD3-BTLA BsAb_D in solution for 25 days, and the same number of cells were taken as the control group, and the supernatant was taken by centrifugation. The amount of IFN- ⁇ secreted by the cells was divided by the control group to be the relative amount of IFN- ⁇ .
  • scFv single-chain variable fragment (Single-chain variable fragment), also known as single-chain antibody
  • V H Heavy chain variable region
  • V L Light chain variable region
  • Extracellular domain extracellular domain
  • CD3-CD28 BsAb_M Monoclonal form of anti-CD3/anti-CD28 bispecific antibody
  • CD3-CD28 BsAb_D a dimeric form of anti-CD3/anti-CD28 bispecific antibody
  • CD3-4-1BB BsAb_M Monomeric form of anti-CD3/anti-4-1BB bispecific antibody
  • CD3-4-1BB BsAb_D anti-CD3/anti-4-1BB bispecific antibody in dimeric form
  • CD3-ICOS BsAb_M Monomeric form of anti-CD3/anti-ICOS bispecific antibody
  • CD3-ICOS BsAb_D a dimeric form of anti-CD3/anti-ICOS bispecific antibody
  • CD3-OX40 BsAb_M Monoclonal form of anti-CD3/anti-OX40 bispecific antibody
  • CD3-OX40 BsAb_D a dimeric form of anti-CD3/anti-OX40 bispecific antibody
  • CD3-GITR BsAb_M Monomeric form of anti-CD3/anti-GITR bispecific antibody
  • CD3-GITR BsAb_D a dimeric form of anti-CD3/anti-GITR bispecific antibody
  • CD3-CD40L BsAb_M Monoclonal form of anti-CD3/anti-CD40L bispecific antibody
  • CD3-CD40L BsAb_D a dimeric form of anti-CD3/anti-CD40L bispecific antibody
  • CD3-CD27 BsAb_M monomeric form of anti-CD3/anti-CD27 bispecific antibody
  • CD3-CD27 BsAb_D a dimeric form of anti-CD3/anti-CD27 bispecific antibody
  • Co-stimulatory molecule co-stimulatory molecule
  • 4-1BBL T cell positive costimulatory molecule 4-1BB ligand
  • B7RP-1 T cell positive costimulatory molecule ICOS ligand
  • OX4OL T cell positive costimulatory molecule OX40 ligand
  • GITRL ligand for T cell positive costimulatory molecule
  • CD70 T cell positive costimulatory molecule CD27 ligand
  • CD3-4-1BBL BsM_M Monomeric form of anti-CD3/4-1BBL bispecific molecule
  • CD3-4-1BBL BsM_D Dimeric form of anti-CD3/4-1BBL bispecific molecule
  • CD3-B7RP-1BsM_M Monomeric form of anti-CD3/B7RP-1 bispecific molecule
  • CD3-B7RP-1BsM_D a dimeric form of anti-CD3/B7RP-1 bispecific molecule
  • CD3-OX40L BsM_M Monomeric form of anti-CD3/OX40L bispecific molecule
  • CD3-OX40L BsM_D dimeric form of anti-CD3/OX40L bispecific molecule
  • CD3-GITRL BsM_M Monomeric form of anti-CD3/GITRL bispecific molecule
  • CD3-GITRL BsM_D a dimeric form of anti-CD3/GITRL bispecific molecule
  • CD3-CD70 BsM_M Monomeric form of anti-CD3/CD70 bispecific molecule
  • CD3-CD70 BsM_D a dimeric form of anti-CD3/CD70 bispecific molecule
  • CD3-PD-1 BsAb_M Monomeric form of anti-CD3/anti-PD-1 bispecific antibody
  • CD3-PD-1 BsAb_D a dimeric form of anti-CD3/anti-PD-1 bispecific antibody
  • CD3-CTLA-4 BsAb_M Monomeric form of anti-CD3/anti-CTLA-4 bispecific antibody
  • CD3-CTLA-4 BsAb_D a dimeric form of anti-CD3/anti-CTLA-4 bispecific antibody
  • CD3-LAG-3 BsAb_M Monomeric form of anti-CD3/anti-LAG-3 bispecific antibody
  • CD3-LAG-3 BsAb_D a dimeric form of anti-CD3/anti-LAG-3 bispecific antibody
  • CD3-TIM-3 BsAb_M Monomeric form of anti-CD3/anti-TIM-3 bispecific antibody
  • CD3-TIM-3 BsAb_D a dimeric form of anti-CD3/anti-TIM-3 bispecific antibody
  • CD3-TIGIT BsAb_M Monomeric form of anti-CD3/anti-TIGIT bispecific antibody
  • CD3-TIGIT BsAb_D a dimeric form of anti-CD3/anti-TIGIT bispecific antibody
  • CD3-BTLA BsAb_M Monomeric form of anti-CD3/anti-BTLA bispecific antibody
  • CD3-BTLA BsAb_D a dimeric form of anti-CD3/anti-BTLA bispecific antibody
  • a bifunctional molecule of the invention comprising a first functional domain capable of binding to and activating a T cell surface CD3 molecule and a second functional domain capable of binding and activating a T cell surface CD28 molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating the T cell surface CD3 molecule and the CD28 molecule, thereby generating a first signal and a second signal required for T cell activation.
  • the present invention is not particularly limited to the first functional domain and the second functional domain as long as the T cell surface CD3 molecule and the CD28 molecule can be simultaneously bound and activated, thereby generating the first signal and the second signal required for T cell activation.
  • the first functional domain can be an anti-CD3 antibody and the second functional domain can be an anti-CD28 antibody.
  • the antibody can be in any form. However, regardless of the form of the antibody, the antigen binding site thereof contains a heavy chain variable region and a light chain variable region.
  • the antibody may preferably be a small molecule antibody.
  • the small molecule antibody is a small molecular weight antibody fragment, and the antigen binding site thereof includes a heavy chain variable region and a light chain variable region.
  • the small molecule antibody has a small molecular weight but retains the affinity of the parental monoclonal antibody and has the same specificity as the parental monoclonal antibody.
  • the types of the small molecule antibodies mainly include Fab antibodies, Fv antibodies, single chain antibodies (scFv), and the like.
  • Fab antibody by the complete light chain (V L variable region and a constant region C L) and heavy chain Fd segments (V H variable region and first constant domain C H 1) is formed by disulfide bonds.
  • Fv antibodies are only joined by non-covalent bonds by the variable regions of the light and heavy chains and are the minimal functional fragments of the antibody molecule that retain the intact antigen binding site.
  • a single-chain antibody (scFv) is a single-protein peptide chain molecule in which a heavy chain variable region and a light chain variable region are joined by a ligation fragment.
  • the first functional domain and the second functional domain are connected by a connection fragment.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited.
  • the C-terminus of the first functional domain can be joined to the N-terminus of the second domain.
  • the number of amino acids of the ligated fragment is preferably ⁇ 2.
  • the present invention is also not particularly limited as to the linker, as long as it does not limit the object of the present invention.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD, and the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is shown in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the structure of the bifunctional molecule is shown in Figure 1-1 as a bispecific antibody.
  • the bifunctional molecule may be in the form of a monomer or a dimer.
  • a schematic diagram of the structure of the monomeric bifunctional molecule of the present invention is shown in A of Figure 1-1, the bifunctional molecule having a first functional domain that binds to the CD3 antigen and a binding to the CD28 antigen.
  • a di-domain, the first domain is a single-chain antibody (scFv) that binds to a CD3 antigen
  • the second domain is a single-chain antibody (scFv) that binds to a CD28 antigen.
  • a schematic diagram of the structure of the dimeric form of the bifunctional molecule of the present invention is shown in B of Figure 1-1, the bifunctional molecule having two first functional domains that bind to the CD3 antigen and two CD28 antigens.
  • a second domain of binding, the first domain is a single chain antibody (scFv) that binds to a CD3 antigen
  • the second domain is a single chain antibody (scFv) that binds to a CD28 antigen.
  • the dimeric form of the difunctional molecule of the present invention has an antigen binding titer which is more than twice that of the monomeric form, and the effect of expanding T cells in vitro is more excellent.
  • the first functional domain is a single chain antibody against CD3.
  • the anti-CD3 single chain antibody includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.
  • the second domain is a single chain antibody against CD28.
  • the anti-CD28 single chain antibody includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is set forth in SEQ ID NO.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is set forth in SEQ ID NO.
  • Another bifunctional molecule of the invention comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and activating a T cell positive costimulatory molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating a T cell surface CD3 molecule and a T cell positive costimulatory molecule, thereby generating a first signal and a second signal required for T cell activation.
  • the T cell positive costimulatory molecules include, but are not limited to, human CD28, 4-1BB, ICOS, OX40, GITR, CD40L or CD27.
  • the present invention has no particular limitation on the first functional domain and the second functional domain, as long as the T cell surface CD3 molecule and the T cell positive costimulatory molecule can be simultaneously bound and activated, thereby generating the first signal and the second signal required for T cell activation.
  • the signal can be.
  • the first functional domain can be an anti-CD3 antibody and the second functional domain can be an antibody against a T cell positive costimulatory molecule.
  • the antibody can be in any form. However, regardless of the form of the antibody, the antigen binding site thereof contains a heavy chain variable region and a light chain variable region.
  • the antibody may preferably be a small molecule antibody.
  • the small molecule antibody is a small molecular weight antibody fragment, and the antigen binding site thereof includes a heavy chain variable region and a light chain variable region.
  • the small molecule antibody has a small molecular weight but retains the affinity of the parental monoclonal antibody and has the same specificity as the parental monoclonal antibody.
  • the types of the small molecule antibodies mainly include Fab antibodies, Fv antibodies, single chain antibodies (scFv), and the like.
  • Fab antibody by the complete light chain (V L variable region and a constant region C L) and heavy chain Fd segments (V H variable region and first constant domain C H 1) is formed by disulfide bonds.
  • Fv antibodies are only joined by non-covalent bonds by the variable regions of the light and heavy chains and are the minimal functional fragments of the antibody molecule that retain the intact antigen binding site.
  • a single-chain antibody is a single-protein peptide chain molecule in which a heavy chain variable region and a light chain variable region are joined by a ligation fragment.
  • the first functional domain and the second functional domain are connected by a connection fragment.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited.
  • the C-terminus of the first functional domain can be joined to the N-terminus of the second domain.
  • the number of amino acids of the ligated fragment is preferably ⁇ 2.
  • the present invention is also not particularly limited as to the linker, as long as it does not limit the object of the present invention.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the schematic structure of the bifunctional molecule is shown in Figure 2-1 and is a bispecific antibody.
  • the bifunctional molecule may be in the form of a monomer or a dimer.
  • a schematic diagram of the structure of the bifunctional molecule of the monomeric form of the present invention is shown in A of Figure 2-1, wherein the structure of the bifunctional molecule contains a first functional domain that binds to the CD3 antigen and one is positive with either T cell.
  • a second functional domain that co-stimulates a binding of a molecular antigen
  • the first functional domain being a single-chain antibody (scFv) that binds to a CD3 antigen
  • the second functional domain is a T-cell positive co-stimulatory molecule extracellular domain (Extracellular domain) a combined single chain antibody (scFv).
  • a schematic diagram of the structure of the dimeric form of the bifunctional molecule of the present invention is shown in B of Figure 2-1, wherein the structure of the bifunctional molecule contains two first functional domains that bind to the CD3 antigen and two The T cell is co-stimulation of a second domain of molecular antigen binding, the first domain is a single chain antibody (scFv) that binds to a CD3 antigen, and the second domain is a co-stimulatory molecule extracellular region with a T cell (Extracellular domain) bound single chain antibody (scFv).
  • the dimeric form of the difunctional molecule of the present invention has an antigen binding titer which is more than twice that of the monomeric form, and the effect of expanding T cells in vitro is more excellent.
  • the T cell positive costimulatory molecule may be CD28, 4-1BB, ICOS, OX40, GITR, CD40L or CD27, and the like.
  • T cell positive costimulatory molecule The amino acid sequence of the human CD28 extracellular domain is shown in SEQ ID NO. 36, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the human 4-1BB extracellular domain is set forth in SEQ ID NO. 37, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human ICOS is shown in SEQ ID NO. 38, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the human OX40 extracellular domain is shown in SEQ ID NO. 39, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human GITR is shown in SEQ ID NO. 40, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human CD40L is shown in SEQ ID NO. 41, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the human CD27 extracellular domain is shown in SEQ ID NO. 42, specifically:
  • the first functional domain is a single chain antibody against CD3.
  • the anti-CD3 single chain antibody includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.
  • the second domain is a single chain antibody against a T cell positive costimulatory molecule.
  • the single chain antibody of the anti-T cell positive costimulatory molecule includes a heavy chain variable region and a light chain variable region.
  • the single-chain antibody of the anti-T cell positive costimulatory molecule may be a single chain antibody against 4-1BB, a single chain antibody against ICOS, a single chain antibody against OX40, a single chain antibody against GITR, and a single chain against CD40L. Any of antibodies or single-chain antibodies against CD27.
  • the amino acid sequence of the heavy chain variable region of the anti-4-1BB single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-4-1BB single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-4-1BB single-chain antibody is shown in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD27 single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is SEQ ID NO. 43, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 55, SEQ ID NO. 59 or SEQ. Any of ID NO. 63 is shown.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is any one of SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 61 or SEQ ID NO. Shown. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • Another bifunctional molecule of the invention comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and activating a T cell positive costimulatory molecule.
  • the bifunctional molecule is capable of simultaneously binding and activating a T cell surface CD3 molecule and a T cell positive costimulatory molecule, thereby generating a first signal and a second signal required for T cell activation.
  • the present invention has no particular limitation on the first functional domain and the second functional domain, as long as the T cell surface CD3 molecule and the T cell positive costimulatory molecule can be simultaneously bound and activated, thereby generating the first signal and the second signal required for T cell activation.
  • the signal can be.
  • the first functional domain can be an anti-CD3 antibody and the second functional domain can be a ligand extracellular domain domain of a T cell positive costimulatory molecule.
  • the antibody can be in any form. However, regardless of the form of the antibody, the antigen-binding portion thereof contains a heavy chain variable region and a light chain variable region.
  • the antibody may preferably be a small molecule antibody.
  • the small molecule antibody is a small molecular weight antibody fragment, and the antigen binding site thereof includes a heavy chain variable region and a light chain variable region.
  • the small molecule antibody has a small molecular weight but retains the affinity of the parental monoclonal antibody and has the same specificity as the parental monoclonal antibody.
  • the types of the small molecule antibodies mainly include Fab antibodies, Fv antibodies, single chain antibodies (scFv), and the like.
  • Fab antibody by the complete light chain (V L variable region and a constant region C L) and heavy chain Fd segments (V H variable region and first constant domain C H 1) is formed by disulfide bonds.
  • Fv antibodies are only joined by non-covalent bonds by the variable regions of the light and heavy chains and are the minimal functional fragments of the antibody molecule that retain the intact antigen binding site.
  • a single-chain antibody is a single-protein peptide chain molecule in which a heavy chain variable region and a light chain variable region are joined by a ligation fragment.
  • the first functional domain and the second functional domain are connected by a connection fragment.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited.
  • the C-terminus of the first functional domain can be joined to the N-terminus of the second domain.
  • the number of amino acids of the ligated fragment is preferably ⁇ 2.
  • the present invention is also not particularly limited as to the linker, as long as it does not limit the object of the present invention.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the structure of the bifunctional molecule is shown in Figure 3-1.
  • the bifunctional molecule may be in the form of a monomer or a dimer.
  • a schematic diagram of the structure of the bifunctional molecule of the monomeric form of the present invention is shown in A of Figure 3-1, the structure of the bifunctional molecule containing a first functional domain that binds to the CD3 antigen and one positive with either T cell
  • the costimulatory molecule-bound T cells are co-stimulatory with the extracellular domain of the ligand.
  • a schematic diagram of the structure of the dimeric form of the bifunctional molecule of the present invention is shown in B of Figure 3-1, the structure of the bifunctional molecule containing two first functional domains that bind to the CD3 antigen and two T cells are co-stimulatory molecule-bound T cells that are co-stimulatory molecular ligand extracellular domain domains.
  • the dimeric form of the difunctional molecule of the present invention has an antigen binding titer which is more than twice that of the monomeric form, and the effect of expanding T cells in vitro is more excellent.
  • the T cell positive costimulatory molecule may be human 4-1BB (UniProt ID: Q07011), the amino acid sequence is shown in SEQ ID NO. 139, and the ligand is human 4-1BBL (UniProt ID: P41273). The amino acid sequence is shown in SEQ ID NO.
  • the T cell positive costimulatory molecule may be human ICOS (UniProt ID: Q9Y6W8), the amino acid sequence is shown in SEQ ID NO. 141, the ligand is human B7RP-1 (UniProt ID: O75144), and the amino acid sequence is SEQ ID. Shown in NO.142.
  • the T cell positive costimulatory molecule may be human OX40 (UniProt ID: P43489), the amino acid sequence is shown as SEQ ID NO. 143, the ligand is human OX40L (UniProt ID: P2351O), and the amino acid sequence is SEQ ID NO. 144 is shown.
  • the T cell positive costimulatory molecule may be human GITR (UniProt ID: Q9Y5U5), the amino acid sequence is shown as SEQ ID NO. 145, the ligand is human GITRL (UniProt ID: Q9UNG2), and the amino acid sequence is SEQ ID NO. 146 is shown.
  • the T cell positive costimulatory molecule may be human CD27 (UniProt ID: P26842), the amino acid sequence is shown as SEQ ID NO. 147, the ligand is human CD70 (UniProt ID: P32970), and the amino acid sequence is SEQ ID NO. 148 is shown.
  • the first functional domain is a single chain antibody against CD3.
  • the anti-CD3 single chain antibody includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.
  • the second domain is the ligand extracellular domain of a T cell positive costimulatory molecule.
  • the ligand extracellular domain of the T cell positive costimulatory molecule may be a 4-1BBL extracellular domain domain, a B7RP-1 extracellular domain domain, an OX40L extracellular domain domain, a GITRL extracellular domain domain or CD70 extracellular domain domain.
  • amino acid sequence of the 4-1BBL extracellular domain domain is set forth in SEQ ID NO.
  • the amino acid sequence of the B7RP-1 extracellular domain domain is set forth in SEQ ID NO.
  • the amino acid sequence of the OX40L extracellular domain domain is set forth in SEQ ID NO.
  • amino acid sequence of the GITRL extracellular domain domain is set forth in SEQ ID NO.
  • the amino acid sequence of the CD70 extracellular domain domain is set forth in SEQ ID NO.
  • the amino acid sequence of the monomeric form of the bifunctional molecule is as defined in SEQ ID NO. 149, SEQ ID NO. 153, SEQ ID NO. 157, SEQ ID NO. 161 or SEO ID NO. One is shown.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is as set forth in any one of SEQ ID NO. 151, SEQ ID NO. 155, SEQ ID NO. 159, SEQ ID NO. 163 or SEQ ID NO. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • Another bifunctional molecule of the invention comprising a first domain capable of binding to and activating a T cell surface CD3 molecule and a second domain capable of binding and blocking a T cell negative costimulatory molecule.
  • the bifunctional molecule is capable of binding and blocking T cell negative costimulatory molecules while binding to and activating T cell surface CD3 molecules, thereby generating a first signal and a second signal required for T cell activation.
  • the T cell negative costimulatory molecules include, but are not limited to, human PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, and BTLA, and the like.
  • the present invention has no particular limitation on the first functional domain and the second functional domain, as long as it can bind and block the T cell negative costimulatory molecule while binding and activating the T cell surface CD3 molecule, thereby generating the T cell activation required.
  • a signal and a second signal are sufficient.
  • the first functional domain can be an anti-CD3 antibody and the second functional domain can be an antibody against an anti-T cell negative costimulatory molecule.
  • the antibody can be in any form. However, regardless of the form of the antibody, the antigen binding site thereof contains a heavy chain variable region and a light chain variable region.
  • the antibody may preferably be a small molecule antibody.
  • the small molecule antibody is a small molecular weight antibody fragment, and the antigen binding site thereof includes a heavy chain variable region and a light chain variable region.
  • the small molecule antibody has a small molecular weight but retains the affinity of the parental monoclonal antibody and has the same specificity as the parental monoclonal antibody.
  • the types of the small molecule antibodies mainly include Fab antibodies, Fv antibodies, single chain antibodies (scFv), and the like.
  • Fab antibody by the complete light chain (V L variable region and a constant region C L) and heavy chain Fd segments (V H variable region and first constant domain C H 1) is formed by disulfide bonds.
  • Fv antibodies are only joined by non-covalent bonds by the variable regions of the light and heavy chains and are the minimal functional fragments of the antibody molecule that retain the intact antigen binding site.
  • a single-chain antibody is a single-protein peptide chain molecule in which a heavy chain variable region and a light chain variable region are joined by a ligation fragment.
  • the first functional domain and the second functional domain are connected by a connection fragment.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited.
  • the C-terminus of the first functional domain may be associated with the second domain N-terminal connection.
  • the number of amino acids of the ligated fragment is preferably ⁇ 2.
  • the present invention is also not particularly limited as to the linker, as long as it does not limit the object of the present invention.
  • the ligation fragment is selected from a ligation fragment in G4S or a hinge region fragment of immunoglobulin IgD.
  • the G4S is specifically GGGGS.
  • the connected segment in units of G4S includes one or more G4S units. For example, one, two, three or more G4S units may be included.
  • the first functional domain and the second functional domain are joined by a linker fragment in units of G4S, the linker fragment comprising three G4S units
  • the amino acid sequence of the ligated fragment is set forth in SEQ ID NO.
  • the hinge region fragment of the immunoglobulin IgD may be the hinge Ala90-Val170 of immunoglobulin IgD.
  • the first functional domain and the second functional domain are joined by a hinge region fragment of an immunoglobulin IgD, the immunoglobulin IgD
  • the hinge region fragment is the hinge Ala90-Val170 of immunoglobulin IgD
  • the amino acid sequence of the hinge region fragment of the immunoglobulin IgD is set forth in SEQ ID NO.
  • the linking fragments may be linked to each other to form a dimer by disulfide bonds.
  • the schematic structure of the bifunctional molecule is shown in Figure 4-1 and is a bispecific antibody.
  • the bifunctional molecule may be in the form of a monomer or a dimer.
  • a schematic diagram of the structure of the bifunctional molecule of the monomeric form of the present invention is shown in A of Figure 4-1, the structure of the bifunctional molecule containing a first functional domain that binds to the CD3 antigen and one negative with either T cell.
  • a second functional domain that co-stimulates the binding of a molecular antigen
  • the first functional domain being a single-chain antibody (scFv) that binds to a CD3 antigen
  • the second functional domain is an extracellular domain of a negative costimulatory molecule with a T cell (Extracellular domain) a combined single chain antibody (scFv).
  • a schematic diagram of the structure of the dimeric form of the bifunctional molecule of the present invention is shown in B of Figure 4-1, and the structure of the bifunctional molecule contains two first functional domains that bind to the CD3 antigen and two A second domain of T cell negative co-stimulatory molecule antigen binding, the first domain is a single chain antibody (scFv) that binds to a CD3 antigen, and the second domain is a co-stimulatory molecule extracellular region with a T cell (Extracellular domain) bound single chain antibody (scFv).
  • the dimeric form of the difunctional molecule of the present invention has an antigen binding titer which is more than twice that of the monomeric form, and the effect of expanding T cells in vitro is more excellent.
  • the T cell negative costimulatory molecule may be human PD-1, CTLA-4, LAG-3, TIM-3, TIGIT or BTLA, and the like.
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human PD-1 (Uniprot ID: Q15116) is shown in SEQ ID NO. 212, specifically:
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human CTLA-4 (Uniprot ID: P16410) is shown in SEQ ID NO. 213, specifically:
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human LAG-3 (Uniprot ID: P18627) is shown in SEQ ID NO. 214, specifically:
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human TIM-3 (Uniprot ID: Q8TDQ0) is shown in SEQ ID NO. 215, specifically:
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human TIGIT (Uniprot ID: Q495A1) is shown in SEQ ID NO. 216, specifically:
  • T cell negative costimulatory molecule The amino acid sequence of the extracellular domain of human BTLA (Uniprot ID: Q7Z6A9) is shown in SEQ ID NO. 217, specifically:
  • the first functional domain is a single chain antibody against CD3.
  • the anti-CD3 single chain antibody includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.
  • the second domain is a single chain antibody against an anti-T cell negative costimulatory molecule.
  • the single chain antibody of the anti-T cell negative costimulatory molecule includes a heavy chain variable region and a light chain variable region.
  • the single-chain antibody against the T cell negative costimulatory molecule may be a single-chain antibody against PD-1, a single-chain antibody against CTLA-4, a single-chain antibody against LAG-3, and a single-chain antibody against TIM-3 Any one of a single-chain antibody against TIGIT or a single-chain antibody against BTLA.
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the single-chain antibody against PD-1 is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • the amino acid sequence of the single-chain antibody against CTLA-4 is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-TIGIT single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-TIGIT single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-TIGIT single-chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the bifunctional molecule in monomeric form is SEQ ID NO. 218, SEQ ID NO. 222, SEQ ID NO. 226, SEQ ID NO. 230, SEQ ID NO. SEQ ID NO. 238 Show.
  • the amino acid sequence of the dimeric form of the bifunctional molecule is any one of SEQ ID NO. 220, SEQ ID NO. 224, SEQ ID NO. 228, SEQ ID NO. 232, SEQ ID NO. 236 or SEQ ID NO. Shown. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • the polynucleotide encoding the bifunctional molecule of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotide encoding the bifunctional molecule of the invention can be prepared by any suitable technique well known to those skilled in the art. Such techniques are described in the general description of the art, such as the Guide to Molecular Cloning (J. Sambrook et al., Science Press, 1995). Methods including, but not limited to, recombinant DNA techniques, chemical synthesis, and the like; for example, overlapping extension PCR.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD3 single-chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the anti-CD3 single-chain antibody is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region encoding the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-CD28 is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD28 is shown in SEQ ID NO.
  • nucleotide sequence of the ligated fragment encoding the amino acid sequence of SEQ ID NO: 17 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO: 19 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the bifunctional molecule in monomeric form is set forth in SEQ ID NO.
  • nucleotide sequence encoding the bifunctional molecule in the form of a dimer is set forth in SEQ ID NO.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD3 single-chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence encoding the anti-CD3 single-chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-4-1BB single-chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the single-chain antibody against 4-1BB is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-4-1BB is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the anti-ICOS single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-ICOS is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the single-chain antibody against OX40 is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the anti-OX40 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the single-chain antibody encoding the anti-GITR is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-GITR is set forth in SEQ ID NO.
  • nucleotide sequence encoding the anti-GITR single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region of the single-chain antibody encoding the anti-CD40L is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-CD40L is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the anti-CD40L single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-CD27 is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the anti-CD27 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 32 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 34 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the bifunctional molecule in monomeric form is SEQ ID NO. 44, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56, SEQ ID NO. 60 or SEQ ID NO. Any of the .64 is shown.
  • a nucleotide sequence encoding a bifunctional molecule in the form of a dimer is as SEQ ID NO. 46, SEQ ID NO. 50, SEQ ID NO. 54, SEQ ID NO. 58, SEQ ID NO. 62 or SEQ ID NO. Either 66 is shown.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence encoding the anti-CD3 single-chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence encoding the 4-1BBL extracellular domain domain is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the B7RP-1 extracellular domain domain is set forth in SEQ ID NO.
  • nucleotide sequence encoding the OX40L extracellular domain domain is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the GITRL extracellular domain domain is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the CD70 extracellular domain domain is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 135 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 137 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the bifunctional molecule in monomeric form is as set forth in any one of SEQ ID NO. 150, SEQ ID NO. 154, SEQ ID NO. 158, SEQ ID NO. 162 or SEQ ID NO. Show.
  • the nucleotide sequence encoding the difunctional form of the bifunctional molecule is as set forth in any one of SEQ ID NO. 152, SEQ ID NO. 156, SEQ ID NO. 160, SEQ ID NO. 164 or SEQ ID NO.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence encoding the anti-CD3 single-chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-PD-1 is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region of the single-chain antibody encoding the anti-CTLA-4 is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region of the single chain antibody encoding the anti-CTLA-4 is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-CTLA-4 is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the anti-LAG-3 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the anti-LAG-3 single-chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-TIM-3 single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-TIM-3 is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-TIM-3 is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region of the single-chain antibody encoding the anti-TIGIT is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region of the single-chain antibody encoding the anti-TIGIT is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-TIGIT is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-BTLA single chain antibody is set forth in SEQ ID NO.
  • the nucleotide sequence of the light chain variable region encoding the single-chain antibody against BTLA is set forth in SEQ ID NO.
  • the nucleotide sequence of the single-chain antibody encoding the anti-BTLA is shown in SEQ ID NO.
  • the nucleotide sequence encoding the SEQ ID NO. Show.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 3 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the bifunctional molecule in monomeric form is SEQ ID NO. 219, SEQ ID NO. 223, SEQ ID NO. 227, SEQ ID NO. 231, SEQ ID NO. 235 or SEQ ID NO. Any of the .239 is shown.
  • a nucleotide sequence encoding a bifunctional molecule in the form of a dimer is as set forth in SEQ ID NO. 221, SEQ ID NO. 225, SEQ ID NO. 229, SEQ ID NO. 233, SEQ ID NO. 237 or SEQ ID NO. Any of 241 is shown.
  • the expression vector of the present invention contains a polynucleotide encoding the bifunctional molecule. Methods well known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA techniques, DNA synthesis techniques, and the like.
  • the DNA encoding the fusion protein can be operably linked to a multiple cloning site in the vector to direct mRNA synthesis to express the protein, or for homologous recombination.
  • the expression vector is pcDNA3.1.
  • the host cell uses Chinese hamster ovary cells (CHO).
  • the method for preparing the aforementioned bifunctional molecule of the present invention comprises: constructing an expression vector containing a bifunctional molecule gene sequence, and then transforming the expression vector containing the bifunctional molecule gene sequence into a host cell to induce expression, and isolating the expression product from the expression product.
  • the expression vector is pcDNA3.1.
  • the host cell uses Chinese hamster ovary cells (CHO).
  • the bifunctional molecules of the invention can be used to prepare T cell in vitro amplification agents.
  • human peripheral blood mononuclear cells are used as experimental materials, and the structure prepared by the invention comprises the first structure capable of binding and activating CD3 molecules on the surface of T cells. a functional domain and a bifunctional molecule capable of binding and activating a second domain of the T cell surface CD28 molecule, and an anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) acting on the same donor, respectively.
  • the human blood PBMC of the source was counted after cell culture, and the amplification factor was compared.
  • the structure includes a first functional domain capable of binding and activating a T cell surface CD3 molecule and a bifunctional molecule capable of binding and activating a second functional domain of a T cell surface CD28 molecule, which can well mediate CIK (Cytokine induced Killer) cell proliferation
  • the use of the structure of the invention includes a first domain capable of binding and activating a T cell surface CD3 molecule and a bifunctional molecule capable of binding and activating a second domain of a T cell surface CD28 molecule for CIK cells
  • the proliferation effect is superior to that of the anti-CD3/anti-CD28 monoclonal full-length antibody, and the amount of protein is less.
  • the first functional domain capable of binding and activating T cell surface CD3 molecules and the second function capable of binding and activating T cell positive costimulatory molecules are included in the structure of the bifunctional molecule.
  • the bifunctional molecules in both monomeric and dimeric forms have in vitro binding activity to CD3 and positive co-stimulatory molecule recombinant antigen, and can be applied to T cell activation and expansion in vitro, wherein the dimer is better than the monomer. Effect.
  • the first functional domain capable of binding and activating T cell surface CD3 molecules and the second function capable of binding and activating T cell positive costimulatory molecules are included in the structure of the bifunctional molecule.
  • both the monomeric and dimeric forms of the bifunctional molecule have in vitro binding activity to the CD3 recombinant antigen and the corresponding positive costimulatory molecule recombinant protein, and can be applied to T cell activation and expansion in vitro, wherein the dimer is simpler. The body has a better effect.
  • the structure of the bifunctional molecule includes a first functional domain capable of binding and activating a T cell surface CD3 molecule and a second capable of binding and blocking a T cell negative costimulatory molecule
  • the bifunctional molecules in both monomeric and dimeric forms have in vitro binding activity to CD3 and the corresponding T cell negative costimulatory molecule recombinant antigen, and can be applied to T cell activation and expansion in vitro, wherein the dimer is more Monomers have a better effect.
  • the method of in vitro expansion of T cells of the invention comprises the action of the aforementioned bifunctional molecule on T cells.
  • the method can For non-therapeutic purposes.
  • human peripheral blood mononuclear cells are used as experimental materials, and the structure prepared by the present invention includes the first function capable of binding and activating CD3 molecules on the surface of T cells. Domain and bifunctional molecule capable of binding and activating a second domain of the T cell surface CD28 molecule, and anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) acting on the same donor source, respectively.
  • the human blood PBMC was counted after cell culture, and the amplification factor was compared.
  • the structure includes a first functional domain capable of binding and activating a T cell surface CD3 molecule and a bifunctional molecule capable of binding and activating a second functional domain of a T cell surface CD28 molecule, which can well mediate CIK (Cytokine induced Killer) cell proliferation
  • the use of the structure of the invention includes a first domain capable of binding and activating a T cell surface CD3 molecule and a bifunctional molecule capable of binding and activating a second domain of a T cell surface CD28 molecule for CIK cells
  • the proliferation effect is superior to that of the anti-CD3/anti-CD28 monoclonal full-length antibody, and the amount of protein is less.
  • the present invention is directed to the insufficiency of the combined application of anti-CD3 and anti-CD28 monoclonal full-length antibodies, and constructs a bifunctional molecule capable of simultaneously recognizing and activating CD3 and CD28 by genetic engineering and antibody engineering methods, the bifunctional molecule not only having the above-mentioned resistance
  • the combination of CD3 and anti-CD28 diabody has obvious advantages in preparation process and practical application. When added in the form of solution, it can achieve or even better than the effect of adding or coating the two plates together, greatly improving the effect. The efficacy of expanding T cells in vitro increases the convenience of use.
  • the first functional domain capable of binding and activating T cell surface CD3 molecules and the second function capable of binding and activating T cell positive costimulatory molecules are included in the structure of the bifunctional molecule.
  • the bifunctional molecules in both monomeric and dimeric forms have in vitro binding activity to CD3 and positive co-stimulatory molecule recombinant antigen, and can be applied to T cell activation and expansion in vitro, wherein the dimer is better than the monomer. Effect.
  • the present invention is directed to the insufficiency of the combination of anti-CD3 and anti-T cell positive costimulatory molecules, and the dual function of simultaneously identifying and activating CD3 and any T cell positive costimulatory molecule by genetic engineering and antibody engineering methods.
  • Molecules, the bifunctional molecule not only has the characteristics of the above-mentioned diabody combined use, but also has obvious advantages in the preparation process and practical application, and can be achieved even in the form of solution to achieve or even better than the combination of two antibodies or coated plates. The effect greatly improves the efficacy of in vitro activation and expansion of T cells, and increases the convenience of use.
  • the first functional domain capable of binding and activating T cell surface CD3 molecules and the second function capable of binding and activating T cell positive costimulatory molecules are included in the structure of the bifunctional molecule.
  • both the monomeric and dimeric forms of the bifunctional molecule have in vitro binding activity to the CD3 recombinant antigen and the corresponding positive costimulatory molecule recombinant protein, and can be applied to T cell activation and expansion in vitro, wherein the dimer is simpler. The body has a better effect.
  • the present invention is directed to the insufficiency of the combination of anti-CD3 and anti-T cell positive costimulatory molecules, and the dual function of simultaneously identifying and activating CD3 and any T cell positive costimulatory molecule by genetic engineering and antibody engineering methods.
  • Molecules, the bifunctional molecule not only has the characteristics of the above-mentioned diabody combined use, but also has obvious advantages in the preparation process and practical application, and can be achieved even in the form of solution to achieve or even better than the combination of two antibodies or coated plates. The effect greatly improves the efficacy of in vitro activation and expansion of T cells, and increases the convenience of use.
  • the structure of the bifunctional molecule includes a first functional domain capable of binding and activating a T cell surface CD3 molecule and a second capable of binding and blocking a T cell negative costimulatory molecule
  • the bifunctional molecules in both monomeric and dimeric forms have in vitro binding activity to CD3 and the corresponding T cell negative costimulatory molecule recombinant antigen, and can be applied to T cell activation and expansion in vitro, wherein the dimer is more Monomers have a better effect.
  • the present invention is directed to the insufficiency of the combined application of anti-CD3 and anti-T cell positive (negative) costimulatory molecule full-length antibodies, and can be used to identify and activate CD3 and recognize and block any one of the T cells by genetic engineering and antibody engineering.
  • a bifunctional molecule of a costimulatory molecule which not only has the characteristics of the combination of the above-mentioned diabody, but also has obvious advantages in the preparation process and practical application, and can be added or even better than the combination of two antibodies when added in the form of a solution. Or the effect of coating the culture plate greatly improves the efficacy of in vitro activation and expansion of T cells, and increases the convenience of use.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman And AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • CD3-CD28BsAb a bispecific antibody targeting human CD3 and CD28 proteins on the surface of T cells.
  • CD3-CD28BsAb_M The specific construction of the monomeric form of CD3-CD28BsAb_M is that the anti-CD3 scFv and anti-CD28 scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-CD28BsAb_D The specific construction of the dimeric form of CD3-CD28BsAb_D is that the anti-CD3 scFv and anti-CD28 scFv sequences are linked by Linker as an IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 12, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 13, specifically:
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 11, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-CD28 scFv is set forth in SEQ ID NO. 15, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD28 scFv is shown in SEQ ID NO. 16, specifically:
  • nucleotide sequence of the anti-CD28 scFv is shown in SEQ ID NO. 14, specifically:
  • nucleotide sequence of the monomeric form of the CD3-CD28 BsAb_M ligation fragment is set forth in SEQ ID NO. 18, specifically:
  • nucleotide sequence of the dimeric form of the CD3-CD28 BsAb_D ligation fragment is set forth in SEQ ID NO. 20, specifically:
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO. 21, specifically:
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO. 22, specifically:
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 1-1 were designed. All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hongxun Technology. Limited synthesis.
  • the signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS) 3 were used, respectively.
  • -CD28-F and pcDNA3.1-CD28-R amplify the gene sequence of anti-CD3 scFv, (GGGGS) 3 Linker, anti-CD28 scFv; for the cloning of CD3-CD28 BsAb_D, the primer pcDNA3.1-Sig is also used first.
  • -F and Sig-R amplify the signal peptide fragment, and then use the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD28-F and pcDNA3.1-CD28-R, respectively.
  • the anti-CD3 scFv, IgD hinge region, and anti-CD28 scFv gene sequences were amplified.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomer and dimer bispecific antibodies and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII. On the expression vector.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-CD28 BsAb_M is shown in SEQ ID NO. 2, specifically:
  • the nucleotide sequence of the dimeric form of CD3-CD28 BsAb_D is shown in SEQ ID NO. 4, specifically:
  • Example 1-2 Expression and purification of CD3-CD28 BsAb_M and CD3-CD28 BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • Buffer B the equilibrate
  • Buffer C the eluent collection tube needs to be pre-added 1% 1M Tris
  • the pH of the eluate was neutralized at pH 8.0, the final concentration of Tris was approximately 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-CD28 BsAb_M and CD3-CD28 BsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electrophoresis patterns under reducing and non-reducing conditions are shown in Figure 1-2.
  • CD3-CD28 BsAb_M and CD3-CD28 BsAb_D recombinant protein are >95% after purification by Protein L affinity chromatography column;
  • the theoretical molecular weight of CD3-CD28BsAb_M recombinant protein is 54.4 kDa, reduction Under the condition of non-reducing, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer, so the bispecific antibody is in monomeric form (Fig. 1-2A);
  • the theoretical molecular weight of the CD3-CD28 BsAb_D recombinant protein is 62.2 kDa.
  • the molecular weight of the protein electrophoresis band is consistent with the monomer.
  • the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 1-2B), indicating that the two protein molecules can be interconnected by disulfide bonds.
  • the bispecific antibody is in the form of a dimer.
  • CD3-CD28 BsAb_M was a monomeric form.
  • CD3-CD28 BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-CD28 BsAb_M is as shown in SEQ ID NO. 1, specifically:
  • the amino acid sequence of the dimeric form of CD3-CD28 BsAb_D is shown in SEQ ID NO. 3, specifically:
  • amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 5, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 6, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 7, specifically:
  • amino acid sequence of the anti-CD28 scFv is shown in SEQ ID NO. 8, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD28 scFv is set forth in SEQ ID NO. 9, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD28 scFv is set forth in SEQ ID NO. 10, specifically:
  • the amino acid sequence of the ligated fragment in the monomeric form of CD3-CD28 BsAb_M is set forth in SEQ ID NO. 17, specifically: GGGGSGGGGSGGGGS.
  • the amino acid sequence of the ligated fragment in the dimeric form of CD3-CD28 BsAb_D is shown in SEQ ID NO. 19, specifically:
  • Example 1-3 ELISA assay for antigen binding activity of CD3-CD28 BsAb_M and CD3-CD28 BsAb_D
  • Recombinant antigen coating human CD3-hFc and human CD28-hFc fusion protein (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml and a coating volume of 100 ⁇ l/well. The coating conditions were 1 hour at 37 ° C or 4 ° C overnight.
  • the formulation of the coating buffer (PBS) was: 3.58 g Na 2 HPO 4 , 0.24 g NaH 2 PO 4 , 0.2 g KCl, 8.2 g NaCl, 950 ml H 2 O, adjust the pH to 7.4 with 1mol / L HCl or 1mol / L NaOH, hydrate to iL;
  • Blocking After washing the plate 4 times with PBS, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Figures 1-3A and 1-3B The ELISA results are shown in Figures 1-3A and 1-3B:
  • Figure 1-3A illustrates that CD3-CD28 BsAb_M has in vitro binding activity to both recombinant antigens CD3-hFc and CD28-hFc, wherein CD28 binding activity is higher than CD3 binding activity.
  • Figure 1-3B illustrates that CD3-CD28 BsAb_D has the same in vitro binding activity as the recombinant antigens CD3-hFc and CD28-hFc, with higher CD28 binding activity.
  • Example 1-4 CD3-CD28 bispecific antibody-mediated cell proliferation of CIK (Cytokine induced killer)
  • the above-mentioned monomeric form of the bispecific antibody CD3-CD28 BsAb_M prepared in the present invention, the dimeric form of the bispecific antibody CD3-CD28 BsAb_D And anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) were applied to human blood PBMC of the same donor source, and the cells were counted after the cells were cultured, and the amplification factor was compared.
  • PBMC Separation of PBMC: Take anticoagulation, add an equal volume of medical saline, and slowly add an equal volume of lymphocyte separation solution (purchased from GE Healthcare) along the wall of the centrifuge tube to maintain a clear liquid level, 2000 rpm After centrifugation for 20 min, the middle white misty cell layer was aspirated into a new centrifuge tube, washed twice with a volume of PBS buffer, centrifuged at 1100 rpm for 10 min, and washed repeatedly with a small amount of pre-cooled X-vivo 15 serum-free medium. (purchased from Lonza) resuspended, cell counts to be used;
  • PBMC was resuspended in CIK basal medium (90% X-vivo15+10% FBS), and the cell density was adjusted to 1 ⁇ 10 6 /ml.
  • control group 1 Anti-CD3 5ug/ml and Anti-CD28 5ug/ml coated cell culture plates, full-length antibodies were purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • control group 2 added full-length antibody Anti-CD3 100ng in solution) /ml and Anti-CD28 100ng/ml
  • experimental group 1 addition of bispecific antibody CD3-CD28 BsAb_M 10ng/ml in solution
  • experimental group 2 addition of bispecific antibody CD3-CD28 BsAb_D 10ng/ in solution state Ml).
  • cytokine IFN- ⁇ 200 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • IL-1 ⁇ 2 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the incubator was cultured under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • Cell density was performed at a density of /ml. According to this method, the cells were cultured for 14 days, and finally the amplification factor of the cells was counted, and a growth curve was drawn;
  • CD3-CD28 bispecific antibody alone have different proliferative effects on CIK cells than anti-CD3/anti-CD28 singles.
  • the cloning full-length antibody was used in combination with less protein (10 ng/ml vs 100 ng/ml), and the dimeric form of CD3-CD28 BsAb_D could mediate CZ cells to expand 373-fold for two weeks with the best effect (experimental group 2).
  • the monomeric form of CD3-CD28 BsAb_M can mediate 278-fold expansion of CIK cells for two weeks, with the second effect (experimental group 1).
  • Control group name Control group 1
  • Experimental group 2 Experimental group 1
  • Experimental group 2 14-day amplification factor 224 196 278 373
  • Example 1-5 CD3-CD28 bispecific antibody-mediated phenotypic detection of CIK cell proliferation
  • control group 2 Take 1 control group of 1 cell, and the other 3 groups (control group 2, experimental group 1, experimental group 2) take 1 part of each cell, the number of each cell is 1 ⁇ 10 6 ;
  • control group 1 1.3 4 cells of control group 1 were added with 5 ul PBS, Anti-CD3-FITC, Anti-CD56-PE and Anti-CD3-FITC and Anti-CD56-PE, and the remaining 3 cells were only added with Anti-CD3- FITC and Anti-CD56-PE, incubated at 4 ° C for 1 h;
  • CD3-CD28 BsAb_M mediates the proliferation of CIK cells for 2 weeks
  • the proportion of CD3 + CD56 + double positive cells is 13.23%
  • CD3-CD28 BsAb_D mediates the proliferation of CIK cells for 2 weeks
  • CD3 The proportion of CD56 + double positive cells was 13.92%, which was not significantly different from Anti-CD3/Anti-CD28 (CD3 + CD56 + double positive ratio: 12.90% for coating; 11.40% for solution addition). It is indicated that both monomeric and dimeric forms of CD3-CD28 bispecific antibodies can be used in place of anti-CD3/anti-CD28 full-length antibodies.
  • control group 2 Take 4 cells of the control group, and the other 3 groups (control group 2, experimental group 1, experimental group 2) took 1 cell each, and the number of cells per cell was 1 ⁇ 10 6 ;
  • CD3-CD28BsAb_M mediates the proliferation of CIK cells for 2 weeks, the proportion of CD8 + positive cells is 67.60%, CD3-CD28 BsAb_D mediates the proliferation of CIK cells for 2 weeks, the proportion of CD8 + positive cells is 78.65 %, were significantly better than Anti-CD3/Anti-CD28 (CD8 + positive ratio: 48.95% for coating; 48.47% for solution), indicating that CD3-CD28 bispecific antibody is more than anti-CD3/anti-CD28
  • the combined use of antibodies is more conducive to the growth and expansion of CD8 + positive cells, wherein the dimer has a better effect than the monomer.
  • CD3-4-1BB BsAb a bispecific antibody targeting T cell surface human CD3 protein and T cell positive costimulatory molecule 4-1BB protein.
  • the specific construction scheme of the CD3-4-1BB BsAb_M in monomeric form is that the anti-CD3 scFv and the anti-4-1BB scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-4-1BB BsAb_D The specific construction scheme of the dimeric form of CD3-4-1BB BsAb_D is that the anti-CD3 scFv and the anti-4-1BB scFv sequences are linked by Linker as an IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 89, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 90, specifically:
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 88, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 92, specifically:
  • nucleotide sequence of the light chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 93, specifically:
  • nucleotide sequence of the anti-4-1BB scFv is shown in SEQ ID NO. 91, specifically:
  • nucleotide sequence of the monomeric form of the CD3-4-1BB BsAb_M ligation fragment is set forth in SEQ ID NO. 33, specifically: GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC.
  • nucleotide sequence of the dimeric form of the CD3-4-1BB BsAb_D ligation fragment is set forth in SEQ ID NO. 35, specifically:
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO. 109, specifically:
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO. 110, specifically:
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 2-1 were designed. All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hongxun Technology. Limited synthesis.
  • the signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS were used, respectively.
  • -1BB-F and pcDNA3.1-4-1BB-R amplify the gene sequence of anti-CD3 scFv, IgD hinge region, and anti-4-1BB scFv.
  • the PCR one-step cloning kit purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-4-1BB BsAb_M is shown in SEQ ID NO. 44, specifically:
  • nucleotide sequence of the dimeric form of CD3-4-1BB BsAb_D is shown in SEQ ID NO. 46, specifically:
  • Example 2-2 Expression and purification of CD3-4-1BB BsAb_M and CD3-4-1BB BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • the culture supernatant was sampled and the effluent was collected.
  • equilibrate the column with at least 1.5ml BufferA, equilibrate and then use Buffer B and Buffer C to collect the target protein eluent (the eluent collection tube needs to be pre-added 1% 1M Tris, pH8). .0 to neutralize the pH of the eluate, the final concentration of Tris was about 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-4-1BB BsAb_M and CD3-4-1BB BsAb_D recombinant proteins were analyzed by SDS-PAGE.
  • the electropherograms of the reduced and non-reduced conditions are shown in Figure 2-2.
  • the purity of CD3-4-1BB BsAb_M and CD3-4-1BB BsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column; among them, CD3-4-1BB BsAb_M recombinant protein
  • the theoretical molecular weight is 53.7kDa. Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer.
  • the bispecific antibody is in monomer form (Fig. 2-2A); CD3-4-1BB BsAb_D
  • the theoretical molecular weight of the recombinant protein is 61.5kDa. Under the reducing condition, the molecular weight of the protein is consistent with the monomer. Under the non-reducing condition, the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 2-2B), indicating two The protein molecules can be linked to each other by a disulfide bond, and thus the bispecific antibody is in the form of a dimer.
  • CD3-4-1BB BsAb_M was single.
  • CD3-4-1BB BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-4-1BB BsAb_M is as shown in SEQ ID NO. 43, specifically:
  • the amino acid sequence of the dimeric form of CD3-4-1BB BsAb_D is shown in SEQ ID NO. 45, specifically:
  • amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 68, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 69, specifically:
  • amino acid sequence of the anti-4-1BB scFv is shown in SEQ ID NO. 70, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 71, specifically:
  • amino acid sequence of the light chain variable region of the anti-4-1BB scFv is set forth in SEQ ID NO. 72, specifically:
  • the amino acid sequence of the ligated fragment of the CD3-4-1BB BsAb_D in monomeric form is shown in SEQ ID NO. 32, specifically: GGGGSGGGGSGGGGS.
  • amino acid sequence of the ligated fragment of the dimeric form of CD3-4-1BB BsAb_D is shown in SEQ ID NO. 34, specifically:
  • Example 2-3 ELISA for detection of antigen binding activity of CD3-4-1BB BsAb_M and CD3-4-1BB BsAb_D
  • Recombinant antigen coating Human CD3-hFc and human 4-1BB-hFc fusion protein (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml and a coating volume of 100 ⁇ l. /well, coating conditions were 37 ° C for 1 hour or 4 ° C overnight, coating buffer (PBS) formulation: 3.58g Na 2 HPO4, 0.24g NaH 2 PO4, 0.2g KCl, 8.2g NaCl, 950ml H 2 O, adjust the pH to 7.4 with 1mol / L HCl or 1mol / L NaOH, hydration to 1L;
  • PBS coating buffer
  • Blocking After washing the plate 4 times with PBS, add blocking solution PBSA (PBS + 2% BSA (V / W)), 200 ⁇ l / well, and block at 37 ° C for 1 hour;
  • PBSA PBS + 2% BSA (V / W)
  • Figure 2-3A shows that CD3-4-1BB BsAb_M has in vitro binding activity to both recombinant antigens CD3-hFc and 4-1BB-hFc, of which 4-1BB binding activity The binding activity is higher than CD3;
  • Figure 2-3B shows that CD3-4-1BB BsAb_D has the same in vitro binding activity as the recombinant antigens CD3-hFc and 4-1BB-hFc, with 4-1BB binding activity being higher.
  • Example 2-4 CD3-4-1BB bispecific antibody-mediated cell proliferation of CIK (Cytokine induced killer)
  • the human peripheral blood mononuclear cell (PBMC) was used as the experimental material, and the above-mentioned monomeric form of the bispecific antibody CD3-4-1BB BsAb_M and the dimeric form of the bispecific antibody CD3- prepared by the present invention were used.
  • 4-1BB BsAb_D and anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) were applied to human blood PBMC of the same donor source, and the cells were counted after the cells were cultured, and the amplification factor was compared.
  • PBMC Separation of PBMC: Take anticoagulation, add an equal volume of medical saline, and slowly add an equal volume of lymphocyte separation solution (purchased from GE Healthcare) along the wall of the centrifuge tube to maintain a clear liquid level, 2000 rpm After centrifugation for 20 min, the middle white misty cell layer was aspirated into a new centrifuge tube, washed twice with a volume of PBS buffer, centrifuged at 1100 rpm for 10 min, and washed repeatedly with a small amount of pre-cooled X-vivo 15 serum-free medium. (purchased from Lonza) resuspended, cell counts to be used;
  • CIK cell culture and expansion PBMC were resuspended in CIK basal medium (90% X-vivo15+10% FBS), and the cell density was adjusted to 1 ⁇ 10 6 /ml.
  • the following three experimental groups were designed: Group (Anti-CD3 5ug/ml and Anti-CD28 5ug/ml coated cell culture plates); experimental group 1 (addition of bispecific antibody CD3-4-1BB BsAb_M10ng/ml in solution); experimental group 2 (solution status) The bispecific antibody CD3-4-1BB BsAb_D 10 ng/ml) was added.
  • cytokine IFN- ⁇ 200 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • IL-1 ⁇ 2 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the incubator was cultured under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • Cell density was performed at a density of /ml. After culturing for 30 days according to this method, the amplification factor of the cells is finally counted, and a growth curve is drawn;
  • CD3-4-1BB bispecific antibody alone have superior proliferation effects on CIK cells than anti-CD3/anti-CD28 monoclonal full-length antibodies.
  • Anti-CD3/Anti-CD28 combined showed a large number of cell deaths, and the cell expansion fold decreased significantly; while the monomeric form of CD3-4-1BB BsAb_M or the dimeric form of CD3-4-1BB BsAb_D was added. No cell death occurred, but the rate of cell expansion was relatively slow. Therefore, the two forms of CD3-4-1BB bispecific antibody prepared by the present invention can effectively amplify and prolong the survival of CIK cells, wherein the dimeric form is more effective.
  • Example 2-5 CD3-4-1BB bispecific antibody-mediated phenotypic detection of CIK cells after proliferation. Flow cytometry of CD8 + /CD4 + positive cells
  • Example 2-4 Three groups of experimental cells cultured for 30 days as described in Example 2-4 were subjected to double staining of antibodies against Anti-CD4-FITC and Anti-CD8-PE (both purchased from Ebiosciense), and CD8 + and CD4 were detected by flow cytometry. + number of positive cells, the respective proportions are counted.
  • Example 2-6 Construction of CD3-ICOS BsAb_M and CD3-ICOS BsAb_D eukaryotic expression vector
  • CD3-ICOS BsAb a bispecific antibody targeting T cell surface human CD3 protein and T cell positive costimulatory molecule ICOS protein.
  • CD3-ICOS BsAb_M The specific construction of the monomeric form of CD3-ICOS BsAb_M is: the anti-CD3 scFv and the anti-ICOS scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-ICOS BsAb_D The specific construction of the dimeric form of CD3-ICOS BsAb_D is that the anti-CD3 scFv and anti-ICOS scFv sequences are linked by Linker as an IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-ICOS scFv is shown in SEQ ID NO. 95, specifically:
  • nucleotide sequence of the light chain variable region of the anti-ICOS scFv is shown in SEQ ID NO. 96, specifically:
  • nucleotide sequence of the anti-ICOS scFv is shown in SEQ ID NO. 94, specifically:
  • nucleotide sequence of the monomeric form of the CD3-ICOS BsAb_M ligation fragment is set forth in SEQ ID NO.
  • nucleotide sequence of the dimeric form of the CD3-ICOS BsAb_D ligation fragment is set forth in SEQ ID NO.
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • the amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 2-2 were designed, and all primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hongxun Technology. Limited synthesis.
  • the signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS) 3 were used, respectively.
  • -ICOS-F and pcDNA3.1-ICOS-R amplify the anti-CD3 scFv, (GGGGS) 3 Linker, anti-ICOS scFv gene sequence; for CD3-ICOS BsAb_D clone construction, the same primer pcDNA3.1-Sig is used first.
  • -F and Sig-R amplify the signal peptide fragment, and then use the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-ICOS-F and pcDNA3.1-ICOS-R, respectively.
  • the anti-CD3 scFv, IgD hinge region, and anti-ICOS scFv gene sequences were amplified.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomer and dimer bispecific antibodies and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII. On the expression vector.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-ICOS BsAb_M is shown in SEQ ID NO. 48, specifically:
  • nucleotide sequence of the dimeric form of CD3-ICOS BsAb_D is shown in SEQ ID NO. 50, specifically:
  • Example 2-7 Expression and purification of CD3-ICOS BsAb_M and CD3-ICOS BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • the culture supernatant was sampled and the effluent was collected.
  • equilibrate the column with at least 1.5ml BufferA, equilibrate and then use Buffer B and Buffer C to collect the target protein eluent (the eluent collection tube needs to be pre-added 1% 1M Tris, pH8). .0 to neutralize the pH of the eluate, the final concentration of Tris was about 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-ICOS BsAb_M and CD3-ICOS BsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electrophoresis patterns under reducing and non-reducing conditions are shown in Figure 2-6.
  • the purity of CD3-ICOS BsAb_M and CD3-ICOS BsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column; the theoretical molecular weight of CD3-ICOS BsAb_M recombinant protein was 53.8 kDa.
  • the protein Under reduced and non-reducing conditions, the protein exhibits a single electrophoresis band with a molecular weight consistent with the monomer, so the bispecific antibody is in monomeric form (Fig. 2-6A); the theoretical molecular weight of the CD3-ICOS BsAb_D recombinant protein is 61.7 kDa. Under the reducing condition, the molecular weight of the protein electrophoresis band is consistent with the monomer. Under the non-reducing condition, the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 2-6B), indicating that the two protein molecules can cross each other through disulfide bonds. Linked, thus the bispecific antibody is in the form of a dimer.
  • CD3-ICOS BsAb_M was a monomeric form.
  • CD3-ICOS BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-ICOS BsAb_M is as shown in SEQ ID NO. 47, specifically:
  • the amino acid sequence of the dimeric form of CD3-ICOS BsAb_D is shown in SEQ ID NO. 49, specifically:
  • amino acid sequence of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the anti-ICOS scFv is shown in SEQ ID NO. 73, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 74, specifically:
  • amino acid sequence of the light chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 75, specifically:
  • amino acid sequence of the ligated fragment in the monomeric form of CD3-ICOS BsAb_M is set forth in SEQ ID NO.
  • amino acid sequence of the ligated fragment in the dimeric form of CD3-ICOS BsAb_D is set forth in SEQ ID NO.
  • Example 2-8 ELISA for antigen binding activity of CD3-ICOS BsAb_M and CD3-ICOS BsAb_D
  • Recombinant antigen coating human CD3-hFc and human ICOS-hFc fusion protein (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml and a coating volume of 100 ⁇ l/well.
  • the coating conditions were 37 ° C for 1 hour or 4 ° C overnight, and the formulation of the coating buffer (PBS) was: 3.58 g Na 2 HPO 4 , 0.24 g NaH 2 PO 4 , 0.2 g KCl, 8.2 g NaCl, 950 ml H 2 O, Adjust the pH to 7.4 with 1mol/L HCl or 1mol/L NaOH, and hydrate to 1L;
  • Blocking After washing the plate 4 times with PBS, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Figure 2-7A shows that CD3-ICOS BsAb_M has in vitro binding activity with recombinant antigens CD3-hFc and ICOS-hFc, and ICOS binding activity is higher than CD3 binding activity.
  • Figure 2-7B illustrates that CD3-ICOS BsAb_D has the same in vitro binding activity as the recombinant antigens CD3-hFc and ICOS-hFc, with higher ICOS binding activity.
  • Example 2-9 CD3-ICOS bispecific antibody-mediated proliferation of CIK cells
  • the above-mentioned monomeric form of the bispecific antibody CD3-ICOS BsAb_M and the dimeric form of the bispecific antibody CD3-ICOS BsAb_D were prepared using human peripheral blood mononuclear cell (PBMC) as experimental material. And anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) was applied to human blood PBMC of the same donor source, and the cells were counted after the culture, and the amplification factor was compared.
  • PBMC peripheral blood mononuclear cell
  • PBMC Separation of PBMC: Take anticoagulation, add an equal volume of medical saline, and slowly add an equal volume of lymphocyte separation solution (purchased from GE Healthcare) along the wall of the centrifuge tube to maintain a clear liquid level, 2000 rpm After centrifugation for 20 min, the middle white misty cell layer was aspirated into a new centrifuge tube, washed twice with a volume of PBS buffer, centrifuged at 1100 rpm for 10 min, and washed repeatedly with a small amount of pre-cooled X-vivo 15 serum-free medium. (purchased from Lonza) resuspended, cell counts to be used;
  • CIK cell culture and expansion PBMC were resuspended in CIK basal medium (90% X-vivo15+10% FBS), and the cell density was adjusted to 1 ⁇ 10 6 /ml.
  • the following three experimental groups were designed: Group (Anti-CD3 5ug/ml and Anti-CD28 5ug/ml coated cell culture plate); experimental group 1 (addition of bispecific antibody CD3-ICOS BsAb_M 10ng/ml in solution); experimental group 2 (in solution state) The bispecific antibody CD3-ICOS BsAb_D 10 ng/ml) was added.
  • cytokine IFN- ⁇ 200 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • IL-1 ⁇ 2 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the incubator was cultured under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • Cell density was performed at a density of /ml. According to this method, the cells were cultured for 14 days, and finally the amplification factor of the cells was counted, and a growth curve was drawn;
  • the test results are shown in Table 2-3 and Figure 2-8.
  • the single- and dimeric forms of CD3-ICOS bispecific antibody alone have different proliferative effects on CIK cells than anti-CD3/anti-CD28 singles.
  • the cloning full-length antibody was used in combination with less protein (10 ng/ml vs 5 ug/ml): the dimeric form of CD3-ICOS BsAb_D could mediate CHD cells 352-fold expansion for two weeks with the best effect (experimental group 2)
  • the monomeric form of CD3-ICOS BsAb_M can mediate 298-fold expansion of CIK cells for two weeks, followed by the effect (experimental group 1);
  • Anti CD3/Anti CD28 can mediate 224-fold expansion of CIK cells for two weeks, with the weakest effect (control) group).
  • Experimental group name Control group Experimental group 1
  • Experimental group 2 14-day amplification factor 224 298 352
  • Example 2-10 CD3-ICOS bispecific antibody-mediated phenotypic detection of CIK cells after proliferation phenotype detection of CD3 + /CD56 + double positive CIK cells
  • Example 2-11 Construction of CD3-OX40 BsAb_M and CD3-OX40 BsAb_D eukaryotic expression vector
  • CD3-OX40 BsAb a bispecific antibody targeting T cell surface human CD3 protein and T cell positive costimulatory molecule OX40 protein.
  • CD3-OX40 BsAb_M in monomeric form is that the anti-CD3 scFv and anti-OX40 scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-OX40 BsAb_D in dimeric form is that the anti-CD3 scFv and anti-OX40 scFv sequences are linked by Linker as an Linker via the IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO. 98, specifically:
  • nucleotide sequence of the light chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO. 99, specifically:
  • nucleotide sequence of the anti-OX40 scFv is as shown in SEQ ID NO. 97, specifically:
  • nucleotide sequence of the monomeric form of the CD3-OX40 BsAb_M ligation fragment is set forth in SEQ ID NO.
  • nucleotide sequence of the dimeric form of the CD3-OX40 BsAb_D ligation fragment is set forth in SEQ ID NO.
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • the amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 2-4 were designed, and all primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hong. Synthetic Technology Co., Ltd. synthesis.
  • the signal peptide fragment was first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS) 3 were used, respectively.
  • -OX40-F and pcDNA3.1-OX40-R amplify the gene sequence of anti-CD3 scFv, (GGGGS) 3 Linker, anti-OX40 scFv; for the cloning of CD3-OX40 BsAb_D, the primer pcDNA3.1-Sig- is also used first.
  • F and Sig-R amplify the signal peptide fragment, and then use the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-OX40-F and pcDNA3.1-OX40-R, respectively.
  • the gene sequence of anti-CD3 scFv, IgD hinge region, and anti-OX40 scFv was added.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomer and dimer bispecific antibodies and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII. On the expression vector.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-OX40 BsAb_M is shown in SEQ ID NO. 52, specifically:
  • nucleotide sequence of the dimeric form of CD3-OX40 BsAb_D is shown in SEQ ID NO. 54, specifically:
  • Example 2-12 Expression and purification of CD3-OX40 BsAb_M and CD3-OX40 BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • Buffer B the equilibrate
  • Buffer C the eluent collection tube needs to be pre-added 1% 1M Tris
  • the pH of the eluate was neutralized at pH 8.0, the final concentration of Tris was approximately 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-OX40 BsAb_M and CD3-OX40 BsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electropherograms under reducing and non-reducing conditions are shown in Figures 2-10. It can be seen from the figure that the purity of CD3-OX40 BsAb_M and CD3-OX40 BsAb_D recombinant protein is >95% after purification by Protein L affinity chromatography column; the theoretical molecular weight of CD3-OX40 BsAb_M recombinant protein is 53.2 kDa.
  • the protein Under reduced and non-reducing conditions, the protein exhibits a single electrophoresis band with a molecular weight consistent with the monomer, so the bispecific antibody is in monomeric form (Fig. 2-10A); the theoretical molecular weight of the CD3-OX40 BsAb_D recombinant protein is 61.1 kDa. Under the reducing condition, the molecular weight of the protein electrophoresis band is consistent with the monomer. Under the non-reducing condition, the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 2-10B), indicating that the two protein molecules can cross each other through disulfide bonds. Linked, thus the bispecific antibody is in the form of a dimer.
  • CD3-OX40 BsAb_M was a monomeric form.
  • CD3-OX40 BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-OX40 BsAb_M is as shown in SEQ ID NO. 51, specifically:
  • the amino acid sequence of the dimeric form of CD3-OX40 BsAb_D is shown in SEQ ID NO. 53, specifically:
  • amino acid sequence of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the anti-OX40 scFv is shown in SEQ ID NO: 76, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO: 77, specifically:
  • amino acid sequence of the light chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO: 78, specifically:
  • amino acid sequence of the ligated fragment in the monomeric form of CD3-OX40 BsAb_M is set forth in SEQ ID NO.
  • amino acid sequence of the ligated fragment in the dimeric form of CD3-OX40 BsAb_D is set forth in SEQ ID NO.
  • Example 2-13 ELISA for antigen binding activity of CD3-OX40 BsAb_M and CD3-OX40 BsAb_D
  • Recombinant antigen coating Human CD3-hFc and human OX40-hFc fusion protein (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml and a coating volume of 100 ⁇ l/well.
  • the coating conditions were 37 ° C for 1 hour or 4 ° C overnight, and the formulation of the coating buffer (PBS) was: 3.58 g Na 2 HPO 4 , 0.24 g NaH 2 PO 4 , 0.2 g KCl, 8.2 g NaCl, 950 ml H 2 O, Adjust the pH to 7.4 with 1mol/L HCl or 1mol/L NaOH, and hydrate to 1L;
  • Blocking After washing the plate 4 times with PBS, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • FIG. 11A illustrates that CD3-OX40 BsAb_M has in vitro binding activity to both recombinant antigens CD3-hFc and OX40-hFc, with OX40 binding activity being higher than CD3 binding activity.
  • FIG. 2-11B illustrates that CD3-OX40 BsAb_D has the same in vitro binding activity as the recombinant antigens CD3-hFc and OX40-hFc, with OX40 binding activity being higher.
  • Example 2-14 CD3-OX40 bispecific antibody mediated proliferation of CIK cells
  • the above-mentioned monomeric form of the bispecific antibody CD3-OX40 BsAb_M prepared in the present invention, the dimeric form of the bispecific antibody CD3-OX40 BsAb_D And anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) were applied to human blood PBMC of the same donor source, and the cells were counted after the cells were cultured, and the amplification factor was compared.
  • PBMC Separation of PBMC: Take anticoagulation, add an equal volume of medical saline, and slowly add an equal volume of lymphocyte separation solution (purchased from GE Healthcare) along the wall of the centrifuge tube to maintain a clear liquid level, 2000 rpm After centrifugation for 20 min, the middle white misty cell layer was aspirated into a new centrifuge tube, washed twice with a volume of PBS buffer, centrifuged at 1100 rpm for 10 min, and washed repeatedly with a small amount of pre-cooled X-vivo 15 serum-free medium. (purchased from Lonza) resuspended, cell counts to be used;
  • CIK cell culture and expansion PBMC were resuspended in CIK basal medium (90% X-vivo15+10% FBS), and the cell density was adjusted to 1 ⁇ 10 6 /ml.
  • the following three experimental groups were designed: Group (Anti-CD3 5ug/ml and Anti-CD28 5ug/ml coated cell culture plates, full-length antibodies were purchased from Wujiang Nearshore Protein Technology Co., Ltd.); experimental group 1 (addition of bispecific antibody CD3- in solution) OX40 BsAb_M 10 ng/ml); experimental group 2 (bispecific antibody CD3-OX40 BsAb_D 10 ng/ml was added in solution).
  • cytokine IFN- ⁇ 200 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • IL-1 ⁇ 2 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the incubator was cultured under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • Cell density was performed at a density of /ml. After culturing for 30 days according to this method, the amplification factor of the cells was finally counted, and a growth curve was drawn.
  • CD3-OX40 bispecific antibody alone were superior to anti-CD3/anti-CD28 monoclonal full-length antibody in combination with anti-CD3/anti-CD28 monoclonal antibody: culture After 18 days, Anti-CD3/Anti-CD28 combined showed a large number of cell deaths, and the cell expansion fold decreased significantly; while the monomeric form of CD3-OX40 BsAb_M or the dimeric form of CD3-OX40 BsAb_D showed no cell death. Only the cell amplification rate is relatively slow, so the two forms of CD3-OX40 bispecific antibody prepared by the invention can effectively amplify and prolong the survival of CIK cells, wherein the dimer is slightly better than the monomer.
  • Example 2-15 Detection of tumor cell killing activity by CIK cells supplemented with CD3-OX40 bispecific antibody
  • the results are shown in Figure 2-13.
  • the killing efficiency of CIK cells supplemented with CD3-OX40 bispecific antibody to Raji cells was superior to that of anti-CD3/anti-CD28 monoclonal full-length antibody.
  • the CD3-OX40 BsAb_D in the form of a polymer has a killing efficiency of about 32%, and the effect is optimal (group 3); the addition of the monomeric form of CD3-OX40 BsAb_M has a killing efficiency of about 25%, and the effect is second (group 2); Combined with CD3/Anti CD28, the killing efficiency is about 22%, and the effect is the weakest (Group 1).
  • the CIK cell killing activity of the CD3-OX40 bispecific antibody was further increased: the addition of the dimeric form of CD3-OX40 BsAb_D, the killing efficiency was about 40% (group 6); the addition of the monomeric form of CD3-OX40 BsAb_M, the killing efficiency was about 35% (Group 5); in contrast, the ability of Anti CD3/Anti CD28 in combination with cultured CIK cells to kill Raji tumor cells has been greatly reduced, and the killing efficiency is only about 10% (Group 4). .
  • the formula for calculating the killing efficiency is:
  • Example 2-16 Construction of CD3-GITR BsAb_M and CD3-GITR BsAb_D eukaryotic expression vector
  • CD3-GITR BsAb a bispecific antibody targeting T cell surface human CD3 protein and T cell positive costimulatory molecule GITR protein.
  • CD3-GITR BsAb_M The specific construction of the monomeric form of CD3-GITR BsAb_M is that the anti-CD3 scFv and the anti-GITR scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-GITR BsAb_D The specific construction of the dimeric form of CD3-GITR BsAb_D is that the anti-CD3 scFv and anti-GITR scFv sequences are linked by Linker as an IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-GITR scFv is shown in SEQ ID NO. 101, specifically:
  • nucleotide sequence of the light chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 102, specifically:
  • nucleotide sequence of the anti-GITR scFv is shown in SEQ ID NO. 100, specifically:
  • the nucleotide sequence of the monomeric form of the CD3-GITR BsAb_M ligation fragment is SEQ ID NO.
  • nucleotide sequence of the dimeric form of the CD3-GITR BsAb_D ligation fragment is set forth in SEQ ID NO.
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • the amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO.
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 2-5 were designed, and all primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hongxun Technology. Limited synthesis.
  • the signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS) 3 were used, respectively.
  • -GITR-F and pcDNA3.1-GITR-R amplify the gene sequence of anti-CD3 scFv, (GGGGS) 3 Linker, anti-GITR scFv; for the cloning of CD3-GITR BsAb_D, the primer pcDNA3.1-Sig is also used first.
  • -F and Sig-R amplify the signal peptide fragment, and then use the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-GITR-F and pcDNA3.1-GITR-R, respectively.
  • the anti-CD3 scFv, IgD hinge region, and anti-GITR scFv gene sequences were amplified.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomer and dimer bispecific antibodies and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII. On the expression vector.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-GITR BsAb_M is shown in SEQ ID NO. 56, specifically:
  • nucleotide sequence of the dimeric form of CD3-GITR BsAb_D is shown in SEQ ID NO. 58, specifically:
  • Example 2-17 Expression and purification of CD3-GITR BsAb_M and CD3-GITR BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • Buffer B the equilibrate
  • Buffer C the eluent collection tube needs to be pre-added 1% 1M Tris
  • the pH of the eluate was neutralized at pH 8.0, the final concentration of Tris was approximately 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-GITR BsAb_M and CD3-GITR BsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electropherograms under reducing and non-reducing conditions are shown in Figure 2-14.
  • the purity of CD3-GITR BsAb_M and CD3-GITR BsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column; the theoretical molecular weight of CD3-GITR BsAb_M recombinant protein was 53.2 kDa.
  • the protein Under reduced and non-reducing conditions, the protein exhibits a single electrophoresis band with a molecular weight consistent with the monomer, so the bispecific antibody is in monomeric form (Fig. 2-14A); the theoretical molecular weight of the CD3-GITR BsAb_D recombinant protein is 61.1 kDa. Under the reducing condition, the molecular weight of the protein electrophoresis band is consistent with the monomer. Under the non-reducing condition, the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 2-14B), indicating that the two protein molecules can cross each other through disulfide bonds. Linked, thus the bispecific antibody is in the form of a dimer.
  • CD3-GITR BsAb_M was a monomeric form.
  • CD3-GITR BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-GITR BsAb_M is as shown in SEQ ID NO. 55, specifically:
  • amino acid sequence of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the anti-GITR scFv is shown in SEQ ID NO. 79, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 80, specifically:
  • amino acid sequence of the light chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 81, specifically:
  • the amino acid sequence of the ligated fragment in the monomeric form of CD3-GITR BsAb_M is set forth in SEQ ID NO.
  • the amino acid sequence of the ligated fragment in the dimeric form of CD3-GITR BsAb_D is set forth in SEQ ID NO.
  • Example 2-18 ELISA assay for antigen binding activity of CD3-GITR BsAb_M and CD3-GITR BsAb_D
  • Recombinant antigen coating human CD3-hFc and human GITR-hFc fusion protein (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml and a coating volume of 100 ⁇ l/well.
  • the coating conditions were 37 ° C for 1 hour or 4 ° C overnight, and the formulation of the coating buffer (PBS) was: 3.58 g Na 2 HPO 4 , 0.24 g NaH 2 PO 4 , 0.2 g KCl, 8.2 g NaCl, 950 ml H 2 O, Adjust the pH to 7.4 with 1mol/L HCl or 1mol/L NaOH, and hydrate to 1L;
  • Blocking After washing the plate 4 times with PBS, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Figure 2-15A illustrates that CD3-GITR BsAb_M has in vitro binding activity to both recombinant antigens CD3-hFc and GITR-hFc, with GITR binding activity being higher than CD3 binding activity.
  • Figure 2-15B illustrates that CD3-GITR BsAb_D has the same in vitro binding activity as the recombinant antigens CD3-hFc and GITR-hFc, with higher GITR binding activity.
  • Example 2-19 CD3-GITR bispecific antibody mediated proliferation of CIK cells
  • the above-mentioned monomeric form of the bispecific antibody CD3-GITR BsAb_M prepared by the present invention, the dimeric form of the bispecific antibody CD3-GITR BsAb_D And anti-CD3/anti-CD28 monoclonal full-length antibody (Anti-CD3/Anti-CD28) were applied to human blood PBMC of the same donor source, and the cells were counted after the cells were cultured, and the amplification factor was compared.
  • PBMC Separation of PBMC: Take anticoagulation, add an equal volume of medical saline, and slowly add an equal volume of lymphocyte separation solution (purchased from GE Healthcare) along the wall of the centrifuge tube to maintain a clear liquid level, 2000 rpm After centrifugation for 20 min, the middle white misty cell layer was aspirated into a new centrifuge tube, washed twice with a volume of PBS buffer, centrifuged at 1100 rpm for 10 min, and washed repeatedly with a small amount of pre-cooled X-vivo 15 serum-free medium. (purchased from Lonza Company) resuspended, cell count pending;
  • CIK cell culture and expansion PBMC were resuspended in CIK basal medium (90% X-vivo15+10% FBS), and the cell density was adjusted to 1 ⁇ 10 6 /ml.
  • the following three experimental groups were designed: Group (Anti-CD35ug/ml and Anti-CD285ug/ml coated cell culture plates, full-length antibodies were purchased from Wujiang Nearshore Protein Technology Co., Ltd.); experimental group 1 (addition of bispecific antibody CD3-GITR BsAb_M in solution) 10 ng/ml); experimental group 2 (bispecific antibody CD3-GITR BsAb_D 10 ng/ml was added in solution).
  • cytokine IFN- ⁇ 200 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • IL-1 ⁇ 2 ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • the incubator was cultured under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • Cell density was performed at a density of /ml. According to this method, the cells were cultured for 14 days, and finally the amplification factor of the cells was counted, and a growth curve was drawn;
  • CD3-GITR bispecific antibody alone have a slightly better proliferation effect on CIK cells than anti-CD3/anti-CD28 monoclonals.
  • Experimental group name Control group
  • Experimental group 2 14-day amplification factor 224 248 287
  • Example 2-20 Construction of CD3-CD40L BsAb_M and CD3-CD40L BsAb_D eukaryotic expression vector
  • CD3-CD40L BsAb a bispecific antibody targeting T cell surface human CD3 protein and T cell positive costimulatory molecule CD40L protein.
  • CD3-CD40L BsAb_M The specific construction of the monomeric form of CD3-CD40L BsAb_M is that the anti-CD3 scFv and the anti-CD40L scFv sequences are linked by a (GGGGS) 3 Linker.
  • CD3-CD40L BsAb_D The specific construction of the dimeric form of CD3-CD40L BsAb_D is that the anti-CD3 scFv and anti-CD40L scFv sequences are linked by Linker as the Linker via the IgD hinge region.
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-CD40L scFv is shown in SEQ ID NO. 104, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD40L scFv is shown in SEQ ID NO. 105, specifically:
  • nucleotide sequence of the anti-CD40L scFv is as shown in SEQ ID NO. 103, specifically:
  • nucleotide sequence of the monomeric form of the CD3-CD40L BsAb_M ligation fragment is set forth in SEQ ID NO.
  • nucleotide sequence of the dimeric form of the CD3-CD40L BsAb_D ligation fragment is set forth in SEQ ID NO.
  • a signal peptide expressing the secreted expression of the antibody was selected for use in this example.
  • amino acid sequence of the secretory expression signal peptide is set forth in SEQ ID NO.
  • the nucleotide sequence of the secreted expression signal peptide is shown in SEQ ID NO.
  • the mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used.
  • primers as shown in Table 2-7 were designed, and all primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the desired gene template was amplified by Suzhou Hongxun Technology. Limited synthesis.
  • the signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the primers Sig-CD3-F and CD3-R, CD3-(GGGGS) 3 were used, respectively.
  • -CD40L-F and pcDNA3.1-CD40L-R amplify the gene sequence of anti-CD3scFv, (GGGGS) 3 Linker, anti-CD40L scFv; for the cloning of CD3-CD40L BsAb_D, the primer pcDNA3.1-Sig- is also used first.
  • F and Sig-R amplify the signal peptide fragment, and then use the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD40L-F and pcDNA3.1-CD40L-R, respectively.
  • the gene sequence of anti-CD3 scFv, IgD hinge region, and anti-CD40L scFv was added.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomer and dimer bispecific antibodies and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII. On the expression vector.
  • the target vector was transformed into E. coli DH5 ⁇ , and positive clones were identified by colony PCR.
  • the recombinants (recombinant plasmids) identified as positive were sequenced and identified.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD3-CD40L BsAb_M is shown in SEQ ID NO. 60, specifically:
  • nucleotide sequence of the dimeric form of CD3-CD40L BsAb_D is shown in SEQ ID NO. 62, specifically:
  • Example 2-21 Expression and purification of CD3-CD40L BsAb_M and CD3-CD40L BsAb_D
  • CHO-S cells purchased from Thermo Fisher Scientific have a pass density of 0.5-0.6 ⁇ 10 6 /ml 1 day before transfection;
  • the diluted transfection reagent is added to the diluted recombinant plasmid, mixed uniformly, and formulated into a transfection complex;
  • Protein purification column Protein L affinity chromatography column (purchased from GE Healthcare, column volume 1.0 ml)
  • Buffer A PBS, pH 7.4
  • Buffer B 0.1M Glycine, pH 3.0
  • Buffer C 0.1M Glycine, pH 2.7
  • the AKTA explorer 100 protein purification system purchased from GE Healthcare
  • the Protein L affinity chromatography column was pretreated with Buffer A, and the culture supernatant was sampled and the effluent was collected.
  • Buffer A the Protein L affinity chromatography column
  • Buffer B the equilibrate
  • Buffer C the eluent collection tube needs to be pre-added 1% 1M Tris
  • the pH of the eluate was neutralized at pH 8.0, the final concentration of Tris was approximately 10 mM), and finally concentrated and dialyzed into buffer PBS.
  • CD3-CD40L BsAb_M and CD3-CD40L BsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electrophoresis patterns under reducing and non-reducing conditions are shown in Figure 2-17.
  • the purity of CD3-CD40L BsAb_M and CD3-CD40L BsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column; the theoretical molecular weight of CD3-CD40L BsAb_M recombinant protein was 53.2 kDa.
  • the protein Under reduced and non-reducing conditions, the protein exhibits a single electrophoresis band with a molecular weight consistent with the monomer, so the bispecific antibody is in monomeric form (Fig. 2-17A); the theoretical molecular weight of the CD3-CD40L BsAb_D recombinant protein is 61.2 kDa. Under the reducing condition, the molecular weight of the protein electrophoresis band is consistent with the monomer. Under the non-reducing condition, the molecular weight of the electrophoresis band is consistent with the dimer (Fig. 2-17B), indicating that the two protein molecules can cross each other through disulfide bonds. Linked, thus the bispecific antibody is in the form of a dimer.
  • CD3-CD40L BsAb_M was a monomeric form.
  • CD3-CD40L BsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD3-CD40L BsAb_M is as shown in SEQ ID NO. 59, specifically:
  • amino acid sequence of the dimeric form of CD3-CD40L BsAb_D is set forth in SEQ ID NO. 61, specifically:
  • amino acid sequence of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the anti-CD40L scFv is shown in SEQ ID NO. 82, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD40L scFv is set forth in SEQ ID NO. 83, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD40L scFv is set forth in SEQ ID NO. 84, specifically:
  • the amino acid sequence of the ligated fragment in the monomeric form of CD3-CD40L BsAb_M is set forth in SEQ ID NO.
  • amino acid sequence of the ligated fragment in the dimeric form of CD3-CD40L BsAb_D is set forth in SEQ ID NO.
  • Example 2-22 ELISA assay for antigen binding activity of CD3-CD40L BsAb_M and CD3-CD40L BsAb_D

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Abstract

提供一种双功能分子及其应用。所述双功能分子包括第一功能域和第二功能域，所述第一功能域和第二功能域能够同时结合T细胞，从而产生T细胞活化所需的第一信号和第二信号。所述双功能分子为重组蛋白肽链，可采用真核细胞表达系统生产。

Description

一种双功能分子及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种双功能分子及其应用。
背景技术
T淋巴细胞(T lymphocyte)来源于胸腺(Thymus)，故称T细胞。成熟T细胞存在于外周免疫器官的胸腺依赖区，在适应性细胞免疫应答中占据核心地位，同时在胸腺依赖性抗原诱导的体液免疫应答中亦发挥重要的辅助作用。根据功能的不同，T细胞可分为细胞毒性T细胞(Cytotoxic T lymphocyte，CTL)、辅助T细胞(Helper T cell，Th)和调节性T细胞(Regulatory T cell，Treg)。其中CTL表达CD8，是适应性细胞免疫的主要效应细胞，其主要功能是特异性识别靶细胞表面的内源性抗原肽/MHC I类分子复合物，自身活化后可分泌穿孔素(Peforin)、颗粒酶(Granzyme)、颗粒溶素(Granulysin)等物质直接杀伤靶细胞(肿瘤细胞或寄生病原体感染的细胞)，也可通过Fas/FasL信号途径诱导靶细胞凋亡；而Th均表达CD4，通过分泌不同种类的细胞因子以及与其它细胞之间直接的相互作用，调节CTL的细胞活性，间接参与细胞免疫；此外，Treg可通过直接接触抑制靶细胞活化以及分泌IL-10、TGFβ等细胞因子对细胞免疫应答进行负调控，在免疫耐受、自身免疫病、感染性疾病及肿瘤等多种疾病中发挥重要的作用。
CD8阳性T细胞的完全活化与高效扩增是其有效杀伤靶细胞的基础，依赖于双信号传递途径的作用：其中抗原递呈细胞(Antigen presenting cell，APC)表面的MHC I/内源性抗原肽复合物特异性识别T细胞表达的TCR/CD3复合物，导致CD3与共受体CD8的胞质段相互作用，激活与胞质段尾部相连的蛋白质酪氨酸激酶，使CD3胞质区免疫受体酪氨酸激酶激活模体(Immunoreceptor tyrosine-based activation motif，ITAM)中的酪氨酸磷酸化，启动信号传导分子级联反应，激活转录因子，使得T细胞初步活化，这是T细胞活化的第一信号；同时，T细胞表面的共刺激分子(Co-stimulatory molecule，例如CD28、4-1BB、ICOS、OX40、GITR、CD40L、CD27、CTLA-4、PD-1、LAG-3、TIM-3、TIGIT、BTLA等)可与APC细胞表面的共刺激分子配体(例如CD80、CD86、4-1BBL、B7RP-1、OX40L、GITRL、CD40、CD70、PD-L1、PD-L2、HVEM等)相互作用，产生T细胞活化的第二信号(共刺激信号)：其中CD28、4-1BB、ICOS、OX40、GITR、CD40L和CD27等属于正共刺激分子，与相应配体(CD80、CD86、4-1BBL、B7RP-1、OX40L、GITRL、CD40、CD70等)相互作用所产生的第二信号(正共刺激信号)可导致T细胞的完全活化；而CTLA-4，PD-1、LAG-3、TIM-3、TIGIT和BTLA等属于负共刺激分子，与相应配体(CD80、CD86、PD-L1、PD-L2、Galectin-9、HVEM等)相互作用所产生的第二信号(负共刺激信号)主要是下调和终止T细胞的活化。
目前针对T细胞活化的第一信号途径，已见报道通过基因工程设计并构建了一系列的抗CD3单克隆全长抗体(Beverley PC等人，Eur J Immunol，11：329-334，1981；Lanzavecchia A等人，Eur J Immunol，17：105-111，1987；Yannelli JR等人，J Immunol Methods，130：91-100，1990)。已有的实验数据表明，该类单克隆抗体能够特异性识别T细胞表面的CD3分子，产生T细胞活化的第一信号。然而，仅有第一信号传递途径非但不能有效激活T细胞，反而会导致T细胞失能甚至产生激活诱导的T细胞死亡(Activation induced cell death，AICD)。为了克服CD3单克隆全长抗体的这一缺点，人们设计并构建了抗CD28、抗4-1BB以及抗ICOS等正共刺激分子的激活型单克隆全长抗体(US Patent 20100168400A1；US Patent 20100183621A1；US Patent 009193789B2)或者抗PD-1、抗CTLA-4以及抗LAG-3等负共刺激分子的阻断型单克隆全长抗体(World Patent 2013173223A1；US Patent 007452535B2；US Patent 2015116539A1)，通过与抗CD3全长抗体联合使用，可以为T细胞提供完整的双 信号活化途径。然而，两种单克隆全长抗体联合使用的方式在具体应用上仍存在一些不足，如明显增加了重组抗体表达与纯化的工作量与生产成本，实际应用于T细胞体外活化扩增时必须优化两种全长抗体的相对比例。此外，两个全长抗体联合使用时，为促进受体活化，需要添加较高浓度的抗体溶液或将抗体包被到培养板或微球上以增强其对受体的活化效果。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种双功能分子及其应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域。
优选地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和CD28分子，从而产生T细胞活化所需的第一信号和第二信号。
优选地，所述第一功能域为抗CD3的抗体，所述第二功能域为抗CD28的抗体。
优选地，所述抗体为小分子抗体。
优选地，所述抗体选自Fab抗体、Fv抗体或单链抗体(scFv)。
优选地，所述第一功能域和所述第二功能域通过连接片段连接。所述连接片段的氨基酸数量可为≥2个。
优选地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO：17所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.19所示。所述连接片段可通过二硫键相互连接形成二聚体。
优选地，所述第一功能域的C末端与所述第二结构域的N末端连接。
优选地，所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗CD28的单链抗体，所述单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.6所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.7所示。所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示。所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10所示。
本发明一些实施例中，列举了抗CD3的单链抗体的氨基酸序列如SEQ ID NO.5所示。抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8所示。
本发明一些实施例中，还列举了单体形式的双功能分子的氨基酸序列如SEQ ID NO.1所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.3所示。
本发明的第二方面，提供另一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
优选地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激 分子，从而产生T细胞活化所需的第一信号和第二信号。
优选地，所述第一功能域为抗CD3的抗体，所述第二功能域为抗T细胞正共刺激分子的抗体。
优选地，所述抗体为小分子抗体。
优选地，所述抗体选自Fab抗体、Fv抗体或单链抗体(scFv)。
优选地，所述第一功能域和所述第二功能域通过连接片段连接。所述连接片段的氨基酸数量可为≥2个。
优选地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.32所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.34所示。所述连接片段可通过二硫键相互连接形成二聚体。
优选地，所述第一功能域的C末端与所述第二结构域的N末端连接。
优选地，所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗T细胞正共刺激分子的单链抗体，所述单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.68所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
优选地，所述抗T细胞正共刺激分子的单链抗体可以是抗4-1BB的单链抗体、抗ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一。
优选地，所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.71所示。所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.72所示。
优选地，所述抗ICOS的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.74所示。所述抗ICOS的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.75所示。
优选地，所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.77所示。所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.78所示。
优选地，所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.80所示。所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.81所示。
优选地，所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.83所示。所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.84所示。
优选地，所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.86所示。所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.87所示。
本发明一些实施例中，列举了所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.67所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.70所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.73所示。所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.76所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.79所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.82所示。所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.85所示。
本发明一些实施例中，还列举了单体形式的双功能分子的氨基酸序列如SEQ ID NO.43、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.55、SEQ ID NO.59或SEQ ID NO.63之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.45、SEQ ID NO.49、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.61或SEQ ID NO.65之任一所示。
本发明的第三方面，提供另一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
优选地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
优选地，所述第一功能域为抗CD3的抗体，所述第二功能域为T细胞正共刺激分子的配体胞外区结构域。
优选地，所述抗体为小分子抗体。
优选地，所述抗体选自Fab抗体、Fv抗体或单链抗体(scFv)。
优选地，所述第一功能域和所述第二功能域通过连接片段连接。所述连接片段的氨基酸数量可为≥2个。
优选地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.135所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.137所示。所述连接片段可通过二硫键相互连接形成二聚体。
优选地，所述第一功能域的C末端与所述第二结构域的N末端连接。
优选地，所述第一功能域为抗CD3的单链抗体，所述抗CD3的单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.170所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.171所示。
本发明一些实施例中，列举了所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.169所示。
优选地，所述T细胞正共刺激分子的配体胞外区结构域选自4-1BBL胞外区结构域、B7RP-1胞外区结构域、OX40L胞外区结构域、GITRL胞外区结构域或CD70胞外区结构域之任一。
优选地，所述4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.172所示。
优选地，所述B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.173所示。
优选地，所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.174所示。
优选地，所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.175所示。
优选地，所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.176所示。
本发明一些实施例中，还列举了单体形式的双功能分子的氨基酸序列如SEQ ID NO.149、SEQ ID NO.153、SEQ ID NO.157、SEQ ID NO.161或SEQ ID NO.165之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.151、SEQ ID NO.155、SEQ ID NO.159、SEQ ID NO.163或SEQ ID NO.167之任一所示。
本发明的第四方面，提供另外一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻断T细胞负共刺激分子的第二功能域。
优选地，所述双功能分子能够在结合并激活T细胞表面CD3分子的同时结合并阻断T细胞负共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
优选地，所述第一功能域为抗CD3的抗体，所述第二功能域为抗T细胞负共刺激分子的抗体。
优选地，所述抗体为小分子抗体。
优选地，所述抗体选自Fab抗体、Fv抗体或单链抗体(scFv)。
优选地，所述第一功能域和所述第二功能域通过连接片段连接。所述连接片段的氨基酸数量可为≥2个。
优选地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.208所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.210所示。所述连接片段可通过二硫键相互连接形成二聚体。
优选地，所述第一功能域的C末端与所述第二结构域的N末端连接。
优选地，所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗T细胞负共刺激分子的单链抗体，所述单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.243所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
优选地，所述抗T细胞负共刺激分子的单链抗体可以是抗PD-1的单链抗体、抗CTLA-4的单链抗体、抗LAG-3的单链抗体、抗TIM-3的单链抗体、抗TIGIT的单链抗体或抗BTLA的单链抗体之任一。
优选地，所述抗PD-1的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.246所示。所述抗PD-1的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.247所示。
优选地，所述抗CTLA-4的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.249所示。所述抗CTLA-4的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.250所示。
优选地，所述抗LAG-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.252所示。所述抗LAG-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.253所示。
优选地，所述抗TIM-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.255所示。所述抗TIM-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.256所示。
优选地，所述抗TIGIT的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.258所示。所述抗TIGIT的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.259所示。
优选地，所述抗BTLA的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.261所示。所述抗BTLA的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.262所示。
本发明一些实施例中，列举了所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.242所示。所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.245所示。所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.248所示。所述抗LAG-3的单链抗体的氨基酸序列如 SEQ ID NO.251所示。所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.254所示。所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.257所示。所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.260所示。
在本案的较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.218、SEQ ID NO.222、SEQ ID NO.226、SEQ ID NO.230、SEQ ID NO.234或SEQ ID NO.238之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.220、SEQ ID NO.224、SEQ ID NO.228、SEQ ID NO.232、SEQ ID NO.236或SEQ ID NO.240之任一所示。
本发明的第五方面，提供一种多核苷酸，其编码前述双功能分子。
本发明的第六方面，提供一种表达载体，其含有前述多核苷酸。
本发明的第七方面，提供一种宿主细胞，其被前述表达载体所转化。
本发明的第八方面，提供一种制备前述双功能分子的方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。
本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary cell，CHO)。
本发明的第九方面，提供前述双功能分子用于制备T细胞体外扩增剂的用途。
本发明的第十方面，提供一种T细胞体外扩增剂，含有前述双功能分子。
本发明的第十一方面，公开了一种体外扩增T细胞的方法，包括步骤：将前述双功能分子作用于T细胞。所述方法可以是非治疗目的的。
与现有技术相比，本发明具有如下有益效果：
(1)本发明将能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域融合于同一蛋白肽链形成双功能分子，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；而抗CD3单克隆全长抗体与抗CD28单克隆全长抗体如果联合使用，两个抗体需分别表达纯化，制备工艺更复杂，工作量和生产成本显著增加。本发明所述的双功能分子为单一蛋白，相对于抗CD3全长抗体与抗CD28全长抗体联合使用，对T细胞的体外扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
(2)本发明还将能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域融合于同一蛋白肽链形成双功能分子，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；而抗CD3单克隆全长抗体与抗T细胞正共刺激分子单克隆全长抗体如果联合使用，两个抗体需分别表达纯化，制备工艺更复杂，工作量和生产成本显著增加。本发明所述的双功能分子为单一蛋白，相对于抗CD3全长抗体与抗T细胞正共刺激分子全长抗体联合使用，对T细胞的体外活化与扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
(3)本发明还将能够结合并激活T细胞表面CD3分子的第一功能域和T细胞正共刺激分子的配体胞外结构域融合于同一蛋白肽链形成双功能分子，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；而抗CD3单克隆全长抗体与抗T细胞正共刺激分子单克隆全长抗体如果联合使用，两个抗体需分别表达纯化，制备工艺更复杂，工作量和生产成本显著增加。本发明所述的双功能分子为单一蛋白，相对于抗CD3全长抗体与抗T细胞正共刺激分子全长抗体联合使用，对T细胞的体外活化与扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
(4)本发明还将能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻 断T细胞负共刺激分子的第二功能域融合于同一蛋白肽链形成双功能分子，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；而抗CD3单克隆全长抗体与抗T细胞负共刺激分子单克隆全长抗体如果联合使用，两个抗体需分别表达纯化，制备工艺更复杂，工作量和生产成本显著增加。本发明所述的双功能分子为单一蛋白，相对于抗CD3全长抗体与抗T细胞负共刺激分子全长抗体联合使用，对T细胞的体外活化与扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
附图说明
图1-1：A.单体形式抗CD3/抗CD28双特异性抗体(CD3-CD28 BsAb_M)的结构图；B.二聚体形式抗CD3/抗CD28双特异性抗体(CD3-CD28 BsAb_D)的结构图。
图1-2：最终纯化的CD3-CD28 BsAb_M和CD3-CD28 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图，A.纯化的CD3-CD28 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD28 BsAb_M；泳道3：非还原性CD3-CD28 BsAb_M；B.纯化的CD3-CD28 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD28 BsAb_D；泳道3：非还原性CD3-CD28 BsAb_D。
图1-3A：CD3-CD28 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CD28-hFc；▲不包被任何抗原的测定结果。
图1-3B：CD3-CD28 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CD28-hFc；▲不包被任何抗原的测定结果。
图1-4：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD28 BsAb_M、CD3-CD28 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数；其中，对照组1：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；对照组2：溶液状态下添加100ng/ml Anti-CD3和100ng/ml Anti-CD28；实验组1：溶液状态下添加10ng/ml CD3-CD28 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD28 BsAb_D。
图1-5：基于流式细胞分析方法测定CD3+CD56+CIK细胞比例，取图1-4所述扩增后细胞，分别测定CD3+CD56+双阳性CIK细胞比例；其中，A：对照组1；B：对照组2；C：实验组1；D：实验组2。
图1-6：基于流式细胞分析方法测定CIK细胞CD8+/CD4+比例。取图1-4所述扩增后细胞，分别测定CD8+阳性与CD4+阳性的细胞比例。其中，A：对照组1；B：对照组2；C：实验组1；D：实验组2。
图2-1：A.单体形式抗CD3/抗T细胞正共刺激分子双特异性抗体(BsAb_M)的结构图；B.二聚体形式抗CD3/抗T细胞正共刺激分子双特异性抗体(BsAb_D)的结构图。
图2-2：A.纯化的CD3-4-1BB BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BB BsAb_M；泳道3：非还原性CD3-4-1BB BsAb_M；B.纯化的CD3-4-1BB BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BB BsAb_D；泳道3：非还原性CD3-4-1BB BsAb_D。
图2-3A：CD3-4-1BB BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原4-1BB-hFc；▲不包被任何抗原的测定结果。
图2-3B：CD3-4-1BB BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果： ■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原4-1BB-hFc；▲不包被任何抗原的测定结果。
图2-4：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-4-1BB BsAb_M、CD3-4-1BB BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-4-1BB BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-4-1BB BsAb_D。
图2-5：基于流式细胞分析方法测定CIK细胞CD8+/CD4+比例，取图2-4所述扩增30天后的细胞，分别测定CD8+阳性与CD4+阳性的细胞比例；其中，A：对照组；B：实验组1；C：实验组2。
图2-6：A.纯化的CD3-ICOS BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-ICOS BsAb_M；泳道3：非还原性CD3-ICOS BsAb_M；B.纯化的CD3-ICOS BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-ICOS BsAb_D；泳道3：非还原性CD3-ICOS BsAb_D。
图2-7A：CD3-ICOS BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原ICOS-hFc；▲不包被任何抗原的测定结果。
图2-7B：CD3-ICOS BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原ICOS-hFc；▲不包被任何抗原的测定结果。
图2-8：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-ICOS BsAb_M、CD3-ICOS BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-ICOS BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-ICOS BsAb_D。
图2-9：基于流式细胞分析方法测定CD3+CD56+CIK细胞比例，取图2-8所述扩增14天后的细胞，分别测定CD3+CD56+双阳性的细胞比例；其中，A：对照组；B：实验组1；C：实验组2。
图2-10：A.纯化的CD3-OX40 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40 BsAb_M；泳道3：非还原性CD3-OX40 BsAb_M；B.纯化的CD3-OX40 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40 BsAb_D；泳道3：非还原性CD3-OX40 BsAb_D。
图2-11A：CD3-OX40 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原OX40-hFc；▲不包被任何抗原的测定结果。
图2-11B：CD3-OX40 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原OX40-hFc；▲不包被任何抗原的测定结果。
图2-12：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-OX40 BsAb_M、CD3-OX40 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养 板；实验组1：溶液状态下添加10ng/ml CD3-OX40 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-OX40 BsAb_D。
图2-13：扩增后的CIK细胞对肿瘤细胞的杀伤活性检测，分别取图2-12所述扩增14天和30天后的CIK细胞作为杀伤效应细胞，以Raji淋巴瘤细胞作为靶细胞，分别检测CIK细胞对Raji细胞的杀伤效率，效应细胞：靶细胞(E∶T比)＝1∶1，杀伤时间：3h。
图2-14：A.纯化的CD3-GITR BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITR BsAb_M；泳道3：非还原性CD3-GITR BsAb_M；B.纯化的CD3-GITR BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITR BsAb_D；泳道3：非还原性CD3-GITR BsAb_D。
图2-15A：CD3-GITR BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原GITR-hFc；▲不包被任何抗原的测定结果。
图2-15B：CD3-GITR BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原GITR-hFc；▲不包被任何抗原的测定结果。
图2-16：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-GITR BsAb_M、CD3-GITR BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-GITR BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-GITR BsAb D。
图2-17：A.纯化的CD3-CD40L BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD40L BsAb_M；泳道3：非还原性CD3-CD40L BsAb_M；B.纯化的CD3-CD40L BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD40L BsAb_D；泳道3：非还原性CD3-CD40L BsAb_D。
图2-18A：CD3-CD40L BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CD40L-hFc；▲不包被任何抗原的测定结果。
图2-18B：CD3-CD40L BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CD40L-hFc；▲不包被任何抗原的测定结果。
图2-19：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD40L BsAb_M、CD3-CD40L BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-CD40L BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD40L BsAb_D。
图2-20：A.纯化的CD3-CD27 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD27 BsAb_M；泳道3：非还原性CD3-CD27 BsAb_M；B.纯化的CD3-CD27 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD27 BsAb_D；泳道3：非还原性CD3-CD27 BsAb_D。
图2-21A：CD3-CD27 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CD27-hFc；▲不包被任何抗原的测定结果。
图2-21B：CD3-CD27 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CD27-hFc；▲不包被任何抗原的测定结果。
图2-22：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD27 BsAb_M、CD3-CD27 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-CD27 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD27 BsAb_D。
图3-1：A.单体形式的抗CD3/T细胞正共刺激分子配体双特异性分子(BsM_M)的结构图；B.二聚体形式的抗CD3/T细胞正共刺激分子配体双特异性分子(BsM_D)的结构图。
图3-2：A.纯化的CD3-4-1BBL BsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BBL BsM_M；泳道3：非还原性CD3-4-1BBL BsM_M；B.纯化的CD3-4-1BBL BsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BBL BsM_D；泳道3：非还原性CD3-4-1BBL BsM_D。
图3-3A：CD3-4-1BBL BsM_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc，●包被1μg/ml重组蛋白4-1BB-hFc；▲不包被任何蛋白的测定结果。
图3-3B：CD3-4-1BBL BsM_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc；●包被1μg/ml重组蛋白4-1BB-hFc；▲不包被任何蛋白的测定结果。
图3-4：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-4-1BBL BsM_M、CD3-4-1BBL BsM_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-4-1BBL BsM_M；实验组2：溶液状态下添加10ng/ml CD3-4-1BBL BsM_D。
图3-5：A.纯化的CD3-B7RP-1 BsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-B7RP-1 BsM_M；泳道3：非还原性CD3-B7RP-1 BsM_M；B.纯化的CD3-B7RP-1 BsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-B7RP-1 BsM_D；泳道3：非还原性CD3-B7RP-1 BsM_D。
图3-6A：CD3-B7RP-1 BsM_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc，●包被1μg/ml重组蛋白ICOS-hFc；▲不包被任何蛋白的测定结果。
图3-6B：CD3-B7RP-1 BsM_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc；●包被1μg/ml重组蛋白ICOS-hFc；▲不包被任何蛋白的测定结果。
图3-7：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-B7RP-1 BsM_M、CD3-B7RP-1 BsM_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-B7RP-1 BsM_M；实验组2：溶液状态下添加10ng/ml CD3-B7RP-1 BsM_D。
图3-8：A.纯化的CD3-OX40L BsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40L BsM_M；泳道3：非还原性CD3-OX40L BsM_M；B.纯化的CD3-OX40L BsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40L BsM_D；泳道3：非还原性CD3-OX40L BsM_D。
图3-9A：CD3-OX40L BsM_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc，●包被1μg/ml重组蛋白OX40-hFc；▲不包被任何蛋白的测定结果。
图3-9B：CD3-OX40L BsM_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc；●包被1μg/ml重组蛋白OX40-hFc；▲不包被任何蛋白的测定结果。
图3-10：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-OX40L BsM_M、CD3-OX40L BsM_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-OX40L BsM_M；实验组2：溶液状态下添加10ng/ml CD3-OX40L BsM_D。
图3-11：A.纯化的CD3-GITRL BsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITRL BsM_M；泳道3：非还原性CD3-GITRL BsM_M；B.纯化的CD3-GITRL BsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITRL BsM_D；泳道3：非还原性CD3-GITRL BsM_D。
图3-12A：CD3-GITRL BsM_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc，●包被1μg/ml重组蛋白GITR-hFc；▲不包被任何蛋白的测定结果。
图3-12B：CD3-GITRL BsM_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc；●包被1μg/ml重组蛋白GITR-hFc；▲不包被任何蛋白的测定结果。
图3-13：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-GITRL BsM_M、CD3-GITRL BsM_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-GITRL BsM_M；实验组2：溶液状态下添加10ng/ml CD3-GITRL BsM_D。
图3-14：A.纯化的CD3-CD70 BsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD70 BsM_M；泳道3：非还原性CD3-CD70 BsM_M；B.纯化的CD3-CD70 BsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD70BsM_D；泳道3：非还原性CD3-CD70 BsM_D。
图3-15A：CD3-CD70 BsM_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc，●包被1μg/ml重组蛋白CD27-hFc；▲不包被任何蛋白的测定结果。
图3-15B：CD3-CD70 BsM_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组蛋白CD3-hFc；●包被1μg/ml重组蛋白CD27-hFc；▲不包被任何蛋白的测定结果。
图3-16：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD70 BsM_M、CD3-CD70 BsM_D或抗CD3/抗CD28单克隆全长抗体联合使用 (Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-CD70 BsM_M；实验组2：溶液状态下添加10ng/ml CD3-CD70 BsM_D。
图4-1：A.单体形式抗CD3/抗T细胞负共刺激分子双特异性抗体(BsAb_M)的结构图；B.二聚体形式抗CD3/抗T细胞负共刺激分子双特异性抗体(BsAb_D)的结构图。
图4-2：A.纯化的CD3-PD-1 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-PD-1 BsAb_M；泳道3：非还原性CD3-PD-1 BsAb_M；B.纯化的CD3-PD-1 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-PD-1 BsAb_D；泳道3：非还原性CD3-PD-1 BsAb_D。
图4-3A：CD3-PD-1 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原PD-1-hFc；▲不包被任何抗原的测定结果。
图4-3B：CD3-PD-1 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原PD-1-hFc；▲不包被任何抗原的测定结果。
图4-4：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-PD-1 BsAb_M、CD3-PD-1 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-PD-1 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-PD-1 BsAb_D。
图4-5：CD3-PD-1双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-4中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISA Kit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D培养25天的CIK细胞，取与对照组相同数量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
图4-6：A.纯化的CD3-CTLA-4 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CTLA-4 BsAb_M；泳道3：非还原性CD3-CTLA-4 BsAb_M；B.纯化的CD3-CTLA-4 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CTLA-4 BsAb_D；泳道3：非还原性CD3-CTLA-4 BsAb_D。
图4-7A：CD3-CTLA-4 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CTLA-4-hFc；▲不包被任何抗原的测定结果。
图4-7B：CD3-CTLA-4 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CTLA-4-hFc；▲不包被任何抗原的测定结果。
图4-8：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CTLA-4 BsAb_M、CD3-CTLA-4 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-CTLA-4 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CTLA-4 BsAb_D。
图4-9：CD3-CTLA-4双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-9 中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISA Kit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D培养25天的CIK细胞，取与对照组相同数量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
图4-10：A.纯化的CD3-LAG-3 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-LAG-3 BsAb_M；泳道3：非还原性CD3-LAG-3 BsAb_M；B.纯化的CD3-LAG-3 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-LAG-3 BsAb_D；泳道3：非还原性CD3-LAG-3 BsAb_D。
图4-11A：CD3-LAG-3 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原LAG-3-hFc；▲不包被任何抗原的测定结果。
图4-11B：CD3-LAG-3 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原LAG-3-hFc；▲不包被任何抗原的测定结果。
图4-12：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-LAG-3 BsAb_M、CD3-LAG-3 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-LAG-3 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-LAG-3 BsAb_D。
图4-13：CD3-LAG-3双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-14中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISA Kit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D培养25天的CIK细胞，取与对照组相同数量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
图4-14：A.纯化的CD3-TIM-3 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-TIM-3 BsAb_M；泳道3：非还原性CD3-TIM-3 BsAb_M；B.纯化的CD3-TIM-3 BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-TIM-3 BsAb_D；泳道3：非还原性CD3-TIM-3 BsAb_D。
图4-15A：CD3-TIM-3 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原TIM-3-hFc；▲不包被任何抗原的测定结果。
图4-15B：CD3-TIM-3 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原TIM-3-hFc；▲不包被任何抗原的测定结果。
图4-16：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-TIM-3 BsAb_M、CD3-TIM-3 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-TIM-3 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-TIM-3 BsAb_D。
图4-17：CD3-TIM-3双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-19中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISA  Kit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D培养25天的CIK细胞，取与对照组相同数量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
图4-18：A.纯化的CD3-TIGIT BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-TIGIT BsAb_M；泳道3：非还原性CD3-TIGIT BsAb_M；B.纯化的CD3-TIGIT BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-TIGIT BsAb_D；泳道3：非还原性CD3-TIGIT BsAb_D。
图4-19A：CD3-TIGIT BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原TIGIT-hFc；▲不包被任何抗原的测定结果。
图4-19B：CD3-TIGIT BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原TIGIT-hFc；▲不包被任何抗原的测定结果。
图4-20：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-TIGIT BsAb_M、CD3-TIGIT BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-TIGIT BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-TIGIT BsAb_D。
图4-21：CD3-TIGIT双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-24中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISAKit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D培养25天的CIK细胞，取与对照组相同数量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
图4-22：A.纯化的CD3-BTLA BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-BTLA BsAb_M；泳道3：非还原性CD3-BTLA BsAb_M；B.纯化的CD3-BTLA BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：非还原性CD3-BTLA BsAb_D；泳道3：还原性CD3-BTLA BsAb_D。
图4-23A：CD3-BTLA BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原BTLA-hFc；▲不包被任何抗原的测定结果。
图4-23B：CD3-BTLA BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原BTLA-hFc；▲不包被任何抗原的测定结果。
图4-24：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-BTLA BsAb_M、CD3-BTLA BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-BTLA BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-BTLA BsAb_D。
图4-25：CD3-BTLA双特异抗体介导的CIK细胞IFN-γ分泌。对照组：取实施例4-29中对照组(Anti-CD3/Anti-CD28)培养25天的CIK细胞2×105个，离心取上清，通过ELISA Kit检测细胞分泌的IFN-γ数量，将其定义为1；实验组1和实验组2分别为溶液状态下添加CD3-BTLA BsAb_M和CD3-BTLA BsAb_D培养25天的CIK细胞，取与对照组相同数 量的细胞，离心取上清，检测细胞分泌的IFN-γ数量，除以对照组即为IFN-γ的相对分泌量。
具体实施方式
一、术语和缩略语：
BsAb：双特异性抗体(Bi-specific Antibody)
Fab：抗原结合片段(Fragement of antigen binding)
Fv：可变区片段(Variable fragment)
scFv：单链可变区片段(Single-chain variable fragment)，又称为单链抗体
VH：重链可变区(Heavy chain variable region)
VL：轻链可变区(Light chain variable region)
Linker：连接片段
Extracellular domain：胞外区
CD3-CD28 BsAb_M：单体形式的抗CD3/抗CD28双特异性抗体
CD3-CD28 BsAb_D：二聚体形式的抗CD3/抗CD28双特异性抗体
CD3-4-1BB BsAb_M：单体形式的抗CD3/抗4-1BB双特异性抗体
CD3-4-1BB BsAb_D：二聚体形式的抗CD3/抗4-1BB双特异性抗体
CD3-ICOS BsAb_M：单体形式的抗CD3/抗ICOS双特异性抗体
CD3-ICOS BsAb_D：二聚体形式的抗CD3/抗ICOS双特异性抗体
CD3-OX40 BsAb_M：单体形式的抗CD3/抗OX40双特异性抗体
CD3-OX40 BsAb_D：二聚体形式的抗CD3/抗OX40双特异性抗体
CD3-GITR BsAb_M：单体形式的抗CD3/抗GITR双特异性抗体
CD3-GITR BsAb_D：二聚体形式的抗CD3/抗GITR双特异性抗体
CD3-CD40L BsAb_M：单体形式的抗CD3/抗CD40L双特异性抗体
CD3-CD40L BsAb_D：二聚体形式的抗CD3/抗CD40L双特异性抗体
CD3-CD27 BsAb_M：单体形式的抗CD3/抗CD27双特异性抗体
CD3-CD27 BsAb_D：二聚体形式的抗CD3/抗CD27双特异性抗体
BsM：双特异性分子(Bi-specific Molecule)
Co-stimulatory molecule：共刺激分子
4-1BBL：T细胞正共刺激分子4-1BB的配体
B7RP-1：T细胞正共刺激分子ICOS的配体
OX4OL：T细胞正共刺激分子OX40的配体
GITRL：T细胞正共刺激分子GITR的配体
CD70：T细胞正共刺激分子CD27的配体
CD3-4-1BBL BsM_M：单体形式的抗CD3/4-1BBL双特异性分子
CD3-4-1BBL BsM_D：二聚体形式的抗CD3/4-1BBL双特异性分子
CD3-B7RP-1BsM_M：单体形式的抗CD3/B7RP-1双特异性分子
CD3-B7RP-1BsM_D：二聚体形式的抗CD3/B7RP-1双特异性分子
CD3-OX40L BsM_M：单体形式的抗CD3/OX40L双特异性分子
CD3-OX40L BsM_D：二聚体形式的抗CD3/OX40L双特异性分子
CD3-GITRL BsM_M：单体形式的抗CD3/GITRL双特异性分子
CD3-GITRL BsM_D：二聚体形式的抗CD3/GITRL双特异性分子
CD3-CD70 BsM_M：单体形式的抗CD3/CD70双特异性分子
CD3-CD70 BsM_D：二聚体形式的抗CD3/CD70双特异性分子
CD3-PD-1 BsAb_M：单体形式的抗CD3/抗PD-1双特异性抗体
CD3-PD-1 BsAb_D：二聚体形式的抗CD3/抗PD-1双特异性抗体
CD3-CTLA-4 BsAb_M：单体形式的抗CD3/抗CTLA-4双特异性抗体
CD3-CTLA-4 BsAb_D：二聚体形式的抗CD3/抗CTLA-4双特异性抗体
CD3-LAG-3 BsAb_M：单体形式的抗CD3/抗LAG-3双特异性抗体
CD3-LAG-3 BsAb_D：二聚体形式的抗CD3/抗LAG-3双特异性抗体
CD3-TIM-3 BsAb_M：单体形式的抗CD3/抗TIM-3双特异性抗体
CD3-TIM-3 BsAb_D：二聚体形式的抗CD3/抗TIM-3双特异性抗体
CD3-TIGIT BsAb_M：单体形式的抗CD3/抗TIGIT双特异性抗体
CD3-TIGIT BsAb_D：二聚体形式的抗CD3/抗TIGIT双特异性抗体
CD3-BTLA BsAb_M：单体形式的抗CD3/抗BTLA双特异性抗体
CD3-BTLA BsAb_D：二聚体形式的抗CD3/抗BTLA双特异性抗体
二、双功能分子
本发明的一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域。
进一步地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和CD28分子，从而产生T细胞活化所需的第一信号和第二信号。
本发明对于第一功能域和第二功能域并无特殊限制，只要能够同时结合并激活T细胞表面CD3分子和CD28分子，从而产生T细胞活化所需的第一信号和第二信号即可。例如，所述第一功能域可以是抗CD3的抗体，所述第二功能域可以是抗CD28的抗体。所述抗体可以是任意形式。但无论是何种形式的抗体，其抗原结合部位均含有重链可变区和轻链可变区。所述抗体优选地可以是小分子抗体。所述小分子抗体是分子量较小的抗体片段，其抗原结合部位包括重链可变区和轻链可变区。所述小分子抗体的分子量虽小但保持了亲本单抗的亲和力，具有亲本单抗一样的特异性。所述小分子抗体的种类主要包括Fab抗体、Fv抗体、单链抗体(scFv)等。Fab抗体由完整的轻链(可变区VL和恒定区CL)和重链Fd段(可变区VH和第一恒定区CH1)通过二硫键连接形成。Fv抗体仅由轻链和重链的可变区通过非共价键连接，是抗体分子保留完整抗原结合部位的最小功能片段。单链抗体(scFv)是重链可变区和轻链可变区通过连接片段连接而成的单一蛋白肽链分子。
所述第一功能域和所述第二功能域通过连接片段连接。本发明对于连接顺序没有特殊要求，只要不限制本发明的目的即可。例如，所述第一功能域的C末端可以与所述第二结构域的N末端连接。所述连接片段的氨基酸数量优选≥2个。本发明对于连接片段也没有特殊的限制，只要是不限制本发明的目的即可。
进一步地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.17所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.19所示。所述连接片段可通过二硫键相互连接形成二聚体。
在本发明的较佳实施例中，所述双功能分子的结构示意图如图1-1所示，为双特异性抗体。所述双功能分子可以是单体形式也可以是二聚体形式。本发明的单体形式的双功能分子的结构示意图如图1-1中A所示，所述双功能分子的结构中含有一个与CD3抗原结合的第一功能域和一个与CD28抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与CD28抗原结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的结构示意图如图1-1中B所示，所述双功能分子的结构中含有两个与CD3抗原结合的第一功能域和两个与CD28抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与CD28抗原结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的抗原结合效价是单体形式的二倍以上，体外扩增T细胞的效果更优。
具体地，所述第一功能域为抗CD3的单链抗体。所述抗CD3的单链抗体包括重链可变区和轻链可变区。所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.6所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.7所示。进一步地，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.5所示。所述第二功能域为抗CD28的单链抗体。所述抗CD28的单链抗体包括重链可变区和轻链可变区。所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示。所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10所示。所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8所示。
在本案的较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.1所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.3所示。但不限于本发明较佳案例中所列举的具体形式。
本发明的另一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
进一步地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。所述T细胞正共刺激分子包括但不限于人类CD28、4-1BB、ICOS、OX40、GITR、CD40L或CD27。
本发明对于第一功能域和第二功能域并无特殊限制，只要能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号即可。例如，所述第一功能域可以是抗CD3的抗体，所述第二功能域可以是抗T细胞正共刺激分子的抗体。所述抗体可以是任意形式。但无论是何种形式的抗体，其抗原结合部位均含有重链可变区和轻链可变区。所述抗体优选地可以是小分子抗体。所述小分子抗体是分子量较小的抗体片段，其抗原结合部位包括重链可变区和轻链可变区。所述小分子抗体的分子量虽小但保持了亲本单抗的亲和力，具有亲本单抗一样的特异性。所述小分子抗体的种类主要包括Fab抗体、Fv抗体、单链抗体(scFv)等。Fab抗体由完整的轻链(可变区VL和恒定区CL)和重链Fd段(可变区VH和第一恒定区CH1)通过二硫键连接形成。Fv抗体仅由轻链和重链的可变区通过非共价键连接，是抗体分子保留完整抗原结合部位的最小功能片段。单链抗体(scFv)是重链可变区和轻链可变区通过连接片段连接而成的单一蛋白肽链分子。
所述第一功能域和所述第二功能域通过连接片段连接。本发明对于连接顺序没有特殊要求，只要不限制本发明的目的即可。例如，所述第一功能域的C末端可以与所述第二结构域的N末端连接。所述连接片段的氨基酸数量优选≥2个。本发明对于连接片段也没有特殊的限制，只要是不限制本发明的目的即可。
进一步地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。 例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.32所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.34所示。所述连接片段可通过二硫键相互连接形成二聚体。
在本发明的较佳实施例中，所述双功能分子的结构示意图如图2-1所示，为双特异性抗体。所述双功能分子可以是单体形式也可以是二聚体形式。本发明的单体形式的双功能分子的结构示意图如图2-1中A所示，所述双功能分子的结构中含有一个与CD3抗原结合的第一功能域和一个与任一T细胞正共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞正共刺激分子胞外区(Extracellular domain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的结构示意图如图2-1中B所示，所述双功能分子的结构中含有两个与CD3抗原结合的第一功能域和两个与任一T细胞正共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞正共刺激分子胞外区(Extracellular domain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的抗原结合效价是单体形式的二倍以上，体外扩增T细胞的效果更优。
所述T细胞正共刺激分子可以是CD28、4-1BB、ICOS、OX40、GITR、CD40L或CD27等。
T细胞正共刺激分子人类CD28胞外区的氨基酸序列如SEQ ID NO.36所示，具体为：
Figure PCTCN2017096592-appb-000001
T细胞正共刺激分子人类4-1BB胞外区的氨基酸序列如SEQ ID NO.37所示，具体为：
Figure PCTCN2017096592-appb-000002
T细胞正共刺激分子人类ICOS胞外区的氨基酸序列如SEQ ID NO.38所示，具体为：
Figure PCTCN2017096592-appb-000003
T细胞正共刺激分子人类OX40胞外区的氨基酸序列如SEQ ID NO.39所示，具体为：
Figure PCTCN2017096592-appb-000004
T细胞正共刺激分子人类GITR胞外区的氨基酸序列如SEQ ID NO.40所示，具体为：
Figure PCTCN2017096592-appb-000005
T细胞正共刺激分子人类CD40L胞外区的氨基酸序列如SEQ ID NO.41所示，具体为：
Figure PCTCN2017096592-appb-000006
Figure PCTCN2017096592-appb-000007
T细胞正共刺激分子人类CD27胞外区的氨基酸序列如SEQ ID NO.42所示，具体为：
Figure PCTCN2017096592-appb-000008
具体地，所述第一功能域为抗CD3的单链抗体。所述抗CD3的单链抗体包括重链可变区和轻链可变区。所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.68所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.69所示。进一步地，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.67所示。
所述第二功能域为抗T细胞正共刺激分子的单链抗体。所述抗T细胞正共刺激分子的单链抗体包括重链可变区和轻链可变区。
所述抗T细胞正共刺激分子的单链抗体可以是抗4-1BB的单链抗体、抗ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一。
所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.71所示。所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.72所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.70所示。
所述抗ICOS的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.74所示。所述抗ICOS的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.75所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.73所示。
所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.77所示。所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.78所示。所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.76所示。
所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.80所示。所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.81所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.79所示。
所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.83所示。所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.84所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.82所示。
所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.86所示。所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.87所示。所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.85所示。
在本案的较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.43、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.55、SEQ ID NO.59或SEQ ID NO.63之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.45、SEQ ID NO.49、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.61或SEQ ID NO.65之任一所示。但不限于本发明较佳案例中所列举的具体形式。
本发明的另一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
进一步地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
本发明对于第一功能域和第二功能域并无特殊限制，只要能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号即可。例如，所述第一功能域可以是抗CD3的抗体，所述第二功能域可以是T细胞正共刺激分子的配体胞外区结构域。所述抗体可以是任意形式。但无论是何种形式的抗体，其抗原结合部 位均含有重链可变区和轻链可变区。所述抗体优选地可以是小分子抗体。所述小分子抗体是分子量较小的抗体片段，其抗原结合部位包括重链可变区和轻链可变区。所述小分子抗体的分子量虽小但保持了亲本单抗的亲和力，具有亲本单抗一样的特异性。所述小分子抗体的种类主要包括Fab抗体、Fv抗体、单链抗体(scFv)等。Fab抗体由完整的轻链(可变区VL和恒定区CL)和重链Fd段(可变区VH和第一恒定区CH1)通过二硫键连接形成。Fv抗体仅由轻链和重链的可变区通过非共价键连接，是抗体分子保留完整抗原结合部位的最小功能片段。单链抗体(scFv)是重链可变区和轻链可变区通过连接片段连接而成的单一蛋白肽链分子。
所述第一功能域和所述第二功能域通过连接片段连接。本发明对于连接顺序没有特殊要求，只要不限制本发明的目的即可。例如，所述第一功能域的C末端可以与所述第二结构域的N末端连接。所述连接片段的氨基酸数量优选≥2个。本发明对于连接片段也没有特殊的限制，只要是不限制本发明的目的即可。
进一步地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.135所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.137所示。所述连接片段可通过二硫键相互连接形成二聚体。
在本发明的较佳实施例中，所述双功能分子的结构示意图如图3-1所示。所述双功能分子可以是单体形式也可以是二聚体形式。本发明的单体形式的双功能分子的结构示意图如图3-1中A所示，所述双功能分子的结构中含有一个与CD3抗原结合的第一功能域和一个与任一T细胞正共刺激分子结合的T细胞正共刺激分子配体胞外区结构域。本发明的二聚体形式的双功能分子的结构示意图如图3-1中B所示，所述双功能分子的结构中含有两个与CD3抗原结合的第一功能域和两个与任一T细胞正共刺激分子结合的T细胞正共刺激分子配体胞外区结构域。本发明的二聚体形式的双功能分子的抗原结合效价是单体形式的二倍以上，体外扩增T细胞的效果更优。
进一步地，所述T细胞正共刺激分子可以是人类4-1BB(UniProt ID：Q07011)，氨基酸序列如SEQ ID NO.139所示，其配体为人类4-1BBL(UniProt ID：P41273)，氨基酸序列如SEQ ID NO.140所示。
SEQ ID NO.139：
Figure PCTCN2017096592-appb-000009
SEQ ID NO.140：
Figure PCTCN2017096592-appb-000010
Figure PCTCN2017096592-appb-000011
所述T细胞正共刺激分子可以是人类ICOS(UniProt ID：Q9Y6W8)，氨基酸序列如SEQ ID NO.141所示，其配体为人类B7RP-1(UniProt ID：O75144)，氨基酸序列如SEQ ID NO.142所示。
SEQ ID NO.141：
Figure PCTCN2017096592-appb-000012
SEQ ID NO.142：
Figure PCTCN2017096592-appb-000013
所述T细胞正共刺激分子可以是人类OX40(UniProt ID：P43489)，氨基酸序列如SEQ ID NO.143所示，其配体为人类OX40L(UniProt ID：P2351O)，氨基酸序列如SEQ ID NO.144所示。
SEQ ID NO.143：
Figure PCTCN2017096592-appb-000014
SEQ ID NO.144：
Figure PCTCN2017096592-appb-000015
所述T细胞正共刺激分子可以是人类GITR(UniProt ID：Q9Y5U5)，氨基酸序列如SEQ ID NO.145所示，其配体为人类GITRL(UniProt ID：Q9UNG2)，氨基酸序列如SEQ ID NO.146所示。
SEQ ID NO.145：
Figure PCTCN2017096592-appb-000016
SEQ ID NO.146：
Figure PCTCN2017096592-appb-000017
所述T细胞正共刺激分子可以是人类CD27(UniProt ID：P26842)，氨基酸序列如SEQ ID NO.147所示，其配体为人类CD70(UniProt ID：P32970)，氨基酸序列如SEQ ID NO.148所示。
SEQ ID NO.147：
Figure PCTCN2017096592-appb-000018
SEQ ID NO.148：
Figure PCTCN2017096592-appb-000019
具体地，所述第一功能域为抗CD3的单链抗体。所述抗CD3的单链抗体包括重链可变区和轻链可变区。所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.170所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.171所示。进一步地，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.169所示。
所述第二功能域为T细胞正共刺激分子的配体胞外区结构域。
所述T细胞正共刺激分子的配体胞外区结构域可以是4-1BBL胞外区结构域、B7RP-1胞外区结构域、OX40L胞外区结构域、GITRL胞外区结构域或CD70胞外区结构域。
所述4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.172所示。
所述B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.173所示。
所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.174所示。
所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.175所示。
所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.176所示。
在本案的较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.149、SEQ ID NO.153、SEQ ID NO.157、SEQ ID NO.161或SEO ID NO.165之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.151、SEQ ID NO.155、SEQ ID NO.159、SEQ ID NO.163或SEQ ID NO.167之任一所示。但不限于本发明较佳案例中所列举的具体形式。
本发明的另一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻断T细胞负共刺激分子的第二功能域。
进一步地，所述双功能分子能够在结合并激活T细胞表面CD3分子的同时结合并阻断T细胞负共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。所述T细胞负共刺激分子包括但不限于人类PD-1、CTLA-4、LAG-3、TIM-3、TIGIT和BTLA等。
本发明对于第一功能域和第二功能域并无特殊限制，只要能够在结合并激活T细胞表面CD3分子的同时结合并阻断T细胞负共刺激分子，从而产生T细胞活化所需的第一信号和第二信号即可。例如，所述第一功能域可以是抗CD3的抗体，所述第二功能域可以是抗T细胞负共刺激分子的抗体。所述抗体可以是任意形式。但无论是何种形式的抗体，其抗原结合部位均含有重链可变区和轻链可变区。所述抗体优选地可以是小分子抗体。所述小分子抗体是分子量较小的抗体片段，其抗原结合部位包括重链可变区和轻链可变区。所述小分子抗体的分子量虽小但保持了亲本单抗的亲和力，具有亲本单抗一样的特异性。所述小分子抗体的种类主要包括Fab抗体、Fv抗体、单链抗体(scFv)等。Fab抗体由完整的轻链(可变区VL和恒定区CL)和重链Fd段(可变区VH和第一恒定区CH1)通过二硫键连接形成。Fv抗体仅由轻链和重链的可变区通过非共价键连接，是抗体分子保留完整抗原结合部位的最小功能片段。单链抗体(scFv)是重链可变区和轻链可变区通过连接片段连接而成的单一蛋白肽链分子。
所述第一功能域和所述第二功能域通过连接片段连接。本发明对于连接顺序没有特殊要求，只要不限制本发明的目的即可。例如，所述第一功能域的C末端可以与所述第二结构域 的N末端连接。所述连接片段的氨基酸数量优选≥2个。本发明对于连接片段也没有特殊的限制，只要是不限制本发明的目的即可。
进一步地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.208所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.210所示。所述连接片段可通过二硫键相互连接形成二聚体。
在本发明的一些较佳实施例中，所述双功能分子的结构示意图如图4-1所示，为双特异性抗体。所述双功能分子可以是单体形式也可以是二聚体形式。本发明的单体形式的双功能分子的结构示意图如图4-1中A所示，所述双功能分子的结构中含有一个与CD3抗原结合的第一功能域和一个与任一T细胞负共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞负共刺激分子胞外区(Extracellular domain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的结构示意图如图4-1中B所示，所述双功能分子的结构中含有两个与CD3抗原结合的第一功能域和两个与任一T细胞负共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞负共刺激分子胞外区(Extracellular domain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的抗原结合效价是单体形式的二倍以上，体外扩增T细胞的效果更优。
所述T细胞负共刺激分子可以是人类PD-1、CTLA-4、LAG-3、TIM-3、TIGIT或BTLA等。
T细胞负共刺激分子人类PD-1(Uniprot ID：Q15116)胞外区的氨基酸序列如SEQ ID NO.212所示，具体为：
Figure PCTCN2017096592-appb-000020
T细胞负共刺激分子人类CTLA-4(Uniprot ID：P16410)胞外区的氨基酸序列如SEQ ID NO.213所示，具体为：
Figure PCTCN2017096592-appb-000021
T细胞负共刺激分子人类LAG-3(Uniprot ID：P18627)胞外区的氨基酸序列如SEQ ID NO.214所示，具体为：
Figure PCTCN2017096592-appb-000022
Figure PCTCN2017096592-appb-000023
T细胞负共刺激分子人类TIM-3(Uniprot ID：Q8TDQ0)胞外区的氨基酸序列如SEQ ID NO.215所示，具体为：
Figure PCTCN2017096592-appb-000024
T细胞负共刺激分子人类TIGIT(Uniprot ID：Q495A1)胞外区的氨基酸序列如SEQ ID NO.216所示，具体为：
Figure PCTCN2017096592-appb-000025
T细胞负共刺激分子人类BTLA(Uniprot ID：Q7Z6A9)胞外区的氨基酸序列如SEQ ID NO.217所示，具体为：
Figure PCTCN2017096592-appb-000026
具体地，所述第一功能域为抗CD3的单链抗体。所述抗CD3的单链抗体包括重链可变区和轻链可变区。所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.243所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.244所示。进一步地，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.242所示。
所述第二功能域为抗T细胞负共刺激分子的单链抗体。所述抗T细胞负共刺激分子的单链抗体包括重链可变区和轻链可变区。
所述抗T细胞负共刺激分子的单链抗体可以是抗PD-1的单链抗体、抗CTLA-4的单链抗体、抗LAG-3的单链抗体、抗TIM-3的单链抗体、抗TIGIT的单链抗体或抗BTLA的单链抗体之任一。
所述抗PD-1的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.246所示。所述抗PD-1的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.247所示。所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.245所示。
所述抗CTLA-4的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.249所示。所述抗CTLA-4的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.250所示。所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.248所示。
所述抗LAG-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.252所示。所述抗LAG-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.253所示。所述抗LAG-3的单链抗体的氨基酸序列如SEQ ID NO.251所示。
所述抗TIM-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.255所示。所述抗TIM-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.256所示。所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.254所示。
所述抗TIGIT的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.258所示。所述抗TIGIT的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.259所示。所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.257所示。
所述抗BTLA的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.261所示。所述抗BTLA的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.262所示。所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.260所示。
在本案的一些较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.218、SEQ ID NO.222、SEQ ID NO.226、SEQ ID NO.230、SEQ ID NO.234或SEQ ID NO.238之任一所 示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.220、SEQ ID NO.224、SEQ ID NO.228、SEQ ID NO.232、SEQ ID NO.236或SEQ ID NO.240之任一所示。但不限于本发明较佳案例中所列举的具体形式。
三、编码双功能分子的多核苷酸
本发明的编码所述双功能分子的多核苷酸，可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。
本发明的编码所述双功能分子的多核苷酸，可以通过本领域技术人员熟知的任何适当的技术制备。所述技术见于本领域的一般描述，如《分子克隆实验指南》(J.萨姆布鲁克等，科学出版社，1995)。包括但不限于重组DNA技术、化学合成等方法；例如采用重叠延伸PCR法。
在本发明的一些较佳实施例中，编码所述抗CD3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.12所示。
编码所述抗CD3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.13所示。
编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.11所示。
编码所述抗CD28的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.15所示。
编码所述抗CD28的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.16所示。
编码所述抗CD28的单链抗体的核苷酸序列如SEQ ID NO.14所示。
编码氨基酸序列如SEQ ID NO：17所示的连接片段的核苷酸序列如SEQ ID NO.18所示。
编码氨基酸序列如SEQ ID NO：19所示的连接片段的核苷酸序列如SEQ ID NO.20所示。
进一步地，编码单体形式的双功能分子的核苷酸序列如SEQ ID NO.2所示。编码二聚体形式的双功能分子的核苷酸序列如SEQ ID NO.4。
在本发明的另一些较佳实施例中，编码所述抗CD3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.89所示。编码所述抗CD3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.90所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.88所示。
编码所述抗4-1BB的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.92所示。编码所述抗4-1BB的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.93所示。编码所述抗4-1BB的单链抗体的核苷酸序列如SEQ ID NO.91所示。
编码所述抗ICOS的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.95所示。编码所述抗ICOS的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.96所示。编码所述抗ICOS的单链抗体的核苷酸序列如SEQ ID NO.94所示。
编码所述抗OX40的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.98所示。编码所述抗OX40的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.99所示。编码所述抗OX40单链抗体的核苷酸序列如SEQ ID NO.97所示。
编码所述抗GITR的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.101所示。编码所述抗GITR的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.102所示。编码所述抗GITR单链抗体的核苷酸序列如SEQ ID NO.100所示。
编码所述抗CD40L的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.104所示。编码所述抗CD40L的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.105所示。编码所述抗CD40L单链抗体的核苷酸序列如SEQ ID NO.103所示。
编码所述抗CD27的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.107所示。编码所述抗CD27的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.108所示。编码所述抗CD27单链抗体的核苷酸序列如SEQ ID NO.106所示。
编码氨基酸序列如SEQ ID NO.32所示的连接片段的核苷酸序列如SEQ ID NO.33所示。
编码氨基酸序列如SEQ ID NO.34所示的连接片段的核苷酸序列如SEQ ID NO.35所示。
进一步地，编码单体形式的双功能分子的核苷酸序列如SEQ ID NO.44、SEQ ID NO.48、SEQ ID NO.52、SEQ ID NO.56、SEQ ID NO.60或SEQ ID NO.64之任一所示。编码二聚体形式的双功能分子的核苷酸序列如如SEQ ID NO.46、SEQ ID NO.50、SEQ ID NO.54、SEQ ID NO.58、SEQ ID NO.62或SEQ ID NO.66之任一所示。
在本发明的另一些较佳实施例中，编码所述抗CD3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.178所示。编码所述抗CD3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.179所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.177所示。
编码所述4-1BBL胞外区结构域的核苷酸序列如SEQ ID NO.180所示。
编码所述B7RP-1胞外区结构域的核苷酸序列如SEQ ID NO.181所示。
编码所述OX40L胞外区结构域的核苷酸序列如SEQ ID NO.182所示。
编码所述GITRL胞外区结构域的核苷酸序列如SEQ ID NO.183所示。
编码所述CD70胞外区结构域的核苷酸序列如SEQ ID NO.184所示。
编码氨基酸序列如SEQ ID NO.135所示的连接片段的核苷酸序列如SEQ ID NO.136所示。
编码氨基酸序列如SEQ ID NO.137所示的连接片段的核苷酸序列如SEQ ID NO.138所示。
进一步地，编码单体形式的双功能分子的核苷酸序列如SEQ ID NO.150、SEQ ID NO.154、SEQ ID NO.158、SEQ ID NO.162或SEQ ID NO.166之任一所示。编码二聚体形式的双功能分子的核苷酸序列如SEQ ID NO.152、SEQ ID NO.156、SEQ ID NO.160、SEQ ID NO.164或SEQ ID NO.168之任一所示。
在本发明的另一些较佳实施例中，编码所述抗CD3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.264所示。编码所述抗CD3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.265所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.263所示。
编码所述抗PD-1的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.267所示。编码所述抗PD-1的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.268所示。编码所述抗PD-1的单链抗体的核苷酸序列如SEQ ID NO.266所示。
编码所述抗CTLA-4的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.270所示。编码所述抗CTLA-4的单链抗体的轻链可变区的核苷酸列如SEQ ID NO.271所示。编码所述抗CTLA-4的单链抗体的核苷酸序列如SEQ ID NO.269所示。
编码所述抗LAG-3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.273所示。编码所述抗LAG-3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.274所示。编码所述抗LAG-3的单链抗体的核苷酸序列如SEQ ID NO.272所示。
编码所述抗TIM-3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.276所示。编码所述抗TIM-3的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.277所示。编码所述抗TIM-3的单链抗体的核苷酸序列如SEQ ID NO.275所示。
编码所述抗TIGIT的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.279所示。编码所述抗TIGIT的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.280所示。编码所述抗TIGIT的单链抗体的核苷酸序列如SEQ ID NO.278所示。
编码所述抗BTLA的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.282所示。编码所述抗BTLA的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.283所示。编码所述抗BTLA的单链抗体的核苷酸序列如SEQ ID NO.281所示。
编码氨基酸序列如SEQ ID NO.1所示的连接片段的核苷酸序列如SEQ ID NO.209所 示。
编码氨基酸序列如SEQ ID NO.3所示的连接片段的核苷酸序列如SEQ ID NO.211所示。
进一步地，编码单体形式的双功能分子的核苷酸序列如SEQ ID NO.219、SEQ ID NO.223、SEQ ID NO.227、SEQ ID NO.231、SEQ ID NO.235或SEQ ID NO.239之任一所示。编码二聚体形式的双功能分子的核苷酸序列如如SEQ ID NO.221、SEQ ID NO.225、SEQ ID NO.229、SEQ ID NO.233、SEQ ID NO.237或SEQ ID NO.241之任一所示。
四、表达载体
本发明的所述表达载体含有编码所述双功能分子的多核苷酸。本领域的技术人员熟知的方法能用于构建所述表达载体。这些方法包括重组DNA技术、DNA合成技术等。可将编码所述融合蛋白的DNA有效连接到载体中的多克隆位点上，以指导mRNA合成进而表达蛋白，或者用于同源重组。本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary cell，CHO)。
五、制备双功能分子的方法
本发明的制备前述双功能分子的方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary cell，CHO)。
六、双功能分子的用途
本发明的双功能分子可用于制备T细胞体外扩增剂。
本发明一些较佳实施例中，以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。结果，所述结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子能够很好地介导CIK(Cytokine induced killer)细胞增殖，且使用本发明的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子对于CIK细胞的增殖效果优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3和正共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3重组抗原和相应正共刺激分子重组蛋白的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻断T细胞负共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3和相应T细胞负共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
七、体外扩增T细胞的方法
本发明的体外扩增T细胞的方法，包括将前述双功能分子作用于T细胞。所述方法可 以是非治疗目的的。
本发明一些较佳实施例中，以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。结果，所述结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子能够很好地介导CIK(Cytokine induced killer)细胞增殖，且使用本发明的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞表面CD28分子的第二功能域的双功能分子对于CIK细胞的增殖效果优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少。
本发明针对抗CD3和抗CD28单克隆全长抗体联合应用的不足之处，通过基因工程和抗体工程的方法构建能够同时识别并激活CD3和CD28的双功能分子，该双功能分子不仅具有上述抗CD3和抗CD28双抗体联合使用的特性，同时在制备工艺和实际应用方面具有明显的优势，以溶液形式添加时能够达到甚至优于两个抗体联合添加或包被培养板的效果，大大提高了体外扩增T细胞的功效，增加了使用的方便性。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3和正共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明针对抗CD3和抗T细胞正共刺激分子全长抗体联合应用的不足之处，通过基因工程和抗体工程的方法构建能够同时识别并激活CD3和任何一个T细胞正共刺激分子的双功能分子，该双功能分子不仅具有上述双抗体联合使用的特性，同时在制备工艺和实际应用方面具有明显的优势，以溶液形式添加时能够达到甚至优于两个抗体联合添加或包被培养板的效果，大大提高了体外活化与扩增T细胞的功效，增加了使用的方便性。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3重组抗原和相应正共刺激分子重组蛋白的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明针对抗CD3和抗T细胞正共刺激分子全长抗体联合应用的不足之处，通过基因工程和抗体工程的方法构建能够同时识别并激活CD3和任何一个T细胞正共刺激分子的双功能分子，该双功能分子不仅具有上述双抗体联合使用的特性，同时在制备工艺和实际应用方面具有明显的优势，以溶液形式添加时能够达到甚至优于两个抗体联合添加或包被培养板的效果，大大提高了体外活化与扩增T细胞的功效，增加了使用的方便性。
本发明另一些较佳实施例中，通过试验证明当双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻断T细胞负共刺激分子的第二功能域时，单体和二聚体形式的双功能分子均具有与CD3和相应T细胞负共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明针对抗CD3和抗T细胞正(负)共刺激分子全长抗体联合应用的不足之处，通过基因工程和抗体工程的方法构建能够识别并激活CD3和识别并阻断任何一个T细胞负共刺激分子的双功能分子，该双功能分子不仅具有上述双抗体联合使用的特性，同时在制备工艺和实际应用方面具有明显的优势，以溶液形式添加时能够达到甚至优于两个抗体联合添加或包被培养板的效果，大大提高了体外活化与扩增T细胞的功效，增加了使用的方便性。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1-1 CD3-CD28BsAb_M和CD3-CD28BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3和CD28蛋白为靶点的双特异性抗体被命名为CD3-CD28BsAb。
一、CD3-CD28BsAb_M和CD3-CD28BsAb_D构建方案设计
单体形式的CD3-CD28BsAb_M具体构建方案为：抗CD3scFv和抗CD28scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-CD28BsAb_D具体构建方案为：抗CD3scFv和抗CD28scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3scFv，抗CD28scFv及IgD铰链区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.12所示，具体为：
Figure PCTCN2017096592-appb-000027
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.13所示，具体为：
Figure PCTCN2017096592-appb-000028
Figure PCTCN2017096592-appb-000029
抗CD3 scFv的核苷酸序列如SEQ ID NO.11所示，具体为：
Figure PCTCN2017096592-appb-000030
抗CD28 scFv的重链可变区的核苷酸序列如SEQ ID NO.15所示，具体为：
Figure PCTCN2017096592-appb-000031
抗CD28 scFv的轻链可变区的核苷酸序列如SEQ ID NO.16所示，具体为：
Figure PCTCN2017096592-appb-000032
抗CD28 scFv的核苷酸序列如SEQ ID NO.14所示，具体为：
Figure PCTCN2017096592-appb-000033
单体形式的CD3-CD28 BsAb_M连接片段的核苷酸序列如SEQ ID NO.18所示，具体为：
Figure PCTCN2017096592-appb-000034
二聚体形式的CD3-CD28 BsAb_D连接片段的核苷酸序列如SEQ ID NO.20所示，具体为：
Figure PCTCN2017096592-appb-000035
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.21所示，具体为：
Figure PCTCN2017096592-appb-000036
该分泌表达信号肽的核苷酸序列如SEQ ID NO.22所示，具体为：
Figure PCTCN2017096592-appb-000037
二、CD3-CD28BsAb_M和CD3-CD28BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表1-1所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD28 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD28-F和pcDNA3.1-CD28-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗CD28 scFv的基因序列；针对CD3-CD28 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD28-F和pcDNA3.1-CD28-R扩增出抗CD3 scFv、IgD铰链区、抗CD28 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000038
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD28 BsAb_M和二聚体形式的CD3-CD28 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD28 BsAb_M的核苷酸序列如SEQ ID NO.2所示，具体为：
Figure PCTCN2017096592-appb-000039
Figure PCTCN2017096592-appb-000040
二聚体形式的CD3-CD28 BsAb_D的核苷酸序列如SEQ ID NO.4所示，具体为：
Figure PCTCN2017096592-appb-000041
Figure PCTCN2017096592-appb-000042
表1-1.双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000043
实施例1-2：CD3-CD28 BsAb_M和CD3-CD28 BsAb_D的表达与纯化
一、CD3-CD28 BsAb_M和CD3-CD28 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD28 BsAb_M和CD3-CD28 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例1-1中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD28 BsAb_M和CD3-CD28BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD28 BsAb_M和CD3-CD28 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图1-2所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-CD28 BsAb_M和CD3-CD28 BsAb_D重组蛋白的纯度均＞95％；其中CD3-CD28BsAb_M重组蛋白的理论分子量为54.4kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图1-2A)；CD3-CD28 BsAb_D重组蛋白的理论分子量为62.2kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图1-2B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD28 BsAb_M为单体形式，CD3-CD28 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD28 BsAb_M的氨基酸序列如SEQ ID NO.1所示，具体为：
Figure PCTCN2017096592-appb-000044
二聚体形式的CD3-CD28 BsAb_D的氨基酸序列如SEQ ID NO.3所示，具体为：
Figure PCTCN2017096592-appb-000045
抗CD3 scFv的氨基酸序列如SEQ ID NO.5所示，具体为：
Figure PCTCN2017096592-appb-000046
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.6所示，具体为：
Figure PCTCN2017096592-appb-000047
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.7所示，具体为：
Figure PCTCN2017096592-appb-000048
抗CD28 scFv的氨基酸序列如SEQ ID NO.8所示，具体为：
Figure PCTCN2017096592-appb-000049
抗CD28 scFv的重链可变区的氨基酸序列如SEQ ID NO.9所示，具体为：
Figure PCTCN2017096592-appb-000050
抗CD28 scFv的轻链可变区的氨基酸序列如SEQ ID NO.10所示，具体为：
Figure PCTCN2017096592-appb-000051
单体形式的CD3-CD28 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.17所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD3-CD28 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.19所示，具体为：
Figure PCTCN2017096592-appb-000052
实施例1-3：ELISA检测CD3-CD28 BsAb_M和CD3-CD28 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CD28-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至iL；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD28 BsAb_M或CD3-CD28 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释 HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图1-3A和图1-3B所示：图1-3A说明CD3-CD28 BsAb_M与重组抗原CD3-hFc和CD28-hFc均具有体外结合活性，其中CD28结合活性较CD3结合活性更高；图1-3B说明CD3-CD28 BsAb_D与重组抗原CD3-hFc和CD28-hFc同样具有体外结合活性，其中CD28结合活性更高。
实施例1-4：CD3-CD28双特异抗体介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CD28 BsAb_M、二聚体形式的双特异抗体CD3-CD28 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下实验组：对照组1(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；对照组2(溶液状态下添加全长抗体Anti-CD3 100ng/ml和Anti-CD28 100ng/ml)；实验组1(溶液状态下添加双特异性抗体CD3-CD28 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD28 BsAb_D 10ng/ml)。此外，四组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表1-2和图1-4所示，单体和二聚体形式的CD3-CD28双特异性抗体单一使用对CIK细胞的增殖效果均不同程度地优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 100ng/ml)，其中二聚体形式的CD3-CD28 BsAb_D可以介导CIK细胞两周扩增373倍，效果最优(实验组2)；单体形式的CD3-CD28 BsAb_M可以介导CIK细胞两周扩增278倍，效果次之(实验组1)。
表1-2 CIK细胞扩增倍数
实验组名 对照组1 对照组2 实验组1 实验组2
14天扩增倍数 224 196 278 373
实施例1-5：CD3-CD28双特异抗体介导的CIK细胞增殖后表型检测
1.CD3+CD56+双阳性CIK细胞的流式检测
取实施例1-4所述培养14天后的4组实验细胞，分别进行Anti-CD3-FITC和Anti-CD56-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD3+CD56+双阳性细胞比例。
流式检测步骤：
1.1取对照组1细胞4份，其他3组(对照组2、实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
1.2 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
1.3对照组1的4份细胞分别添加5ul PBS、Anti-CD3-FITC、Anti-CD56-PE以及Anti-CD3-FITC和Anti-CD56-PE同时添加，其余3份细胞只同时添加Anti-CD3-FITC和Anti-CD56-PE，4℃孵育1h；
1.4所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图1-5所示：其中CD3-CD28 BsAb_M介导CIK细胞增殖2周后，CD3+CD56+双阳性的细胞比例为13.23％，CD3-CD28 BsAb_D介导CIK细胞增殖2周后，CD3+CD56+双阳性的细胞比例为13.92％，与Anti-CD3/Anti-CD28联用(CD3+CD56+双阳性比例：包被使用为12.90％；溶液添加为11.40％)的效果无明显差别，说明单体和二聚体形式CD3-CD28双特异抗体均可用于替代抗CD3/抗CD28两种全长抗体联合使用。
2.CD8+/CD4+阳性细胞的流式检测
取实施例1-4所述培养14天后的4组实验细胞，分别进行Anti-CD4-FITC和Anti-CD8-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD8+和CD4+阳性细胞数目，统计各自比例。
流式检测步骤：
2.1取对照组1细胞4份，其他3组(对照组2、实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
2.2 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
2.3对照组1的4份细胞分别添加5ul PBS、Anti-CD4-FITC、Anti-CD8-PE以及Anti-CD4-FITC和Anti-CD8-PE同时添加，其余3份细胞只同时添加Anti-CD4-FITC和Anti-CD8-PE，4℃孵育1h；
2.4所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图1-6所示：CD3-CD28BsAb_M介导CIK细胞增殖2周后，CD8+阳性细胞比例为67.60％，CD3-CD28 BsAb_D介导CIK细胞增殖2周后，CD8+阳性细胞比例为78.65％，均明显优于Anti-CD3/Anti-CD28联用(CD8+阳性比例：包被使用为48.95％；溶液添加为48.47％)，说明CD3-CD28双特异抗体比抗CD3/抗CD28全长抗体联合使用更有利于CD8+阳性细胞的生长扩增，其中二聚体较单体具有更好的效果。
实施例2-1 CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子4-1BB蛋白为靶点的双特异性抗体被命名为CD3-4-1BB BsAb。
一、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D构建方案设计
单体形式的CD3-4-1BB BsAb_M具体构建方案为：抗CD3 scFv和抗4-1BB scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-4-1BB BsAb_D具体构建方案为：抗CD3 scFv和抗4-1BB scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗4-1BB scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89所示，具体为：
Figure PCTCN2017096592-appb-000053
Figure PCTCN2017096592-appb-000054
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.90所示，具体为：
Figure PCTCN2017096592-appb-000055
抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示，具体为：
Figure PCTCN2017096592-appb-000056
抗4-1BB scFv的重链可变区的核苷酸序列如SEQ ID NO.92所示，具体为：
Figure PCTCN2017096592-appb-000057
抗4-1BB scFv的轻链可变区的核苷酸序列如SEQ ID NO.93所示，具体为：
Figure PCTCN2017096592-appb-000058
抗4-1BB scFv的核苷酸序列如SEQ ID NO.91所示，具体为：
Figure PCTCN2017096592-appb-000059
Figure PCTCN2017096592-appb-000060
单体形式的CD3-4-1BB BsAb_M连接片段的核苷酸序列如SEQ ID NO.33所示，具体为：GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。
二聚体形式的CD3-4-1BB BsAb_D连接片段的核苷酸序列如SEQ ID NO.35所示，具体为：
Figure PCTCN2017096592-appb-000061
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.109所示，具体为：
Figure PCTCN2017096592-appb-000062
该分泌表达信号肽的核苷酸序列如SEQ ID NO.110所示，具体为：
Figure PCTCN2017096592-appb-000063
二、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-1所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-4-1BB BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-4-1BB-F和pcDNA3.1-4-1BB-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗4-1BB scFv的基因序列；针对CD3-4-1BB BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-4-1BB-F和pcDNA3.1-4-1BB-R扩增出抗CD3 scFv、IgD铰链区、抗4-1BB scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000064
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-4-1BB BsAb_M和二聚体形式的CD3-4-1BB BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-4-1BB BsAb_M的核苷酸序列如SEQ ID NO.44所示，具体为：
Figure PCTCN2017096592-appb-000065
具体地，二聚体形式的CD3-4-1BB BsAb_D的核苷酸序列如SEQ ID NO.46所示，具体为：
Figure PCTCN2017096592-appb-000066
Figure PCTCN2017096592-appb-000067
表2-1.CD3-4-1BB双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000068
实施例2-2：CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的表达与纯化
一、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D)需准备 两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-1中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml BufferA平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-2所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D重组蛋白的纯度均＞95％；其中CD3-4-1BB BsAb_M重组蛋白的理论分子量为53.7kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-2A)；CD3-4-1BB BsAb_D重组蛋白的理论分子量为61.5kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-2B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-4-1BB BsAb_M为单体形式，CD3-4-1BB BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-4-1BB BsAb_M的氨基酸序列如SEQ ID NO.43所示，具体为：
Figure PCTCN2017096592-appb-000069
二聚体形式的CD3-4-1BB BsAb_D的氨基酸序列如SEQ ID NO.45所示，具体为：
Figure PCTCN2017096592-appb-000070
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示，具体为：
Figure PCTCN2017096592-appb-000071
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示，具体为：
Figure PCTCN2017096592-appb-000072
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示，具体为：
Figure PCTCN2017096592-appb-000073
抗4-1BB scFv的氨基酸序列如SEQ ID NO.70所示，具体为：
Figure PCTCN2017096592-appb-000074
抗4-1BB scFv的重链可变区的氨基酸序列如SEQ ID NO.71所示，具体为：
Figure PCTCN2017096592-appb-000075
抗4-1BB scFv的轻链可变区的氨基酸序列如SEQ ID NO.72所示，具体为：
Figure PCTCN2017096592-appb-000076
单体形式的CD3-4-1BB BsAb_D中连接片段的氨基酸序列如SEQ ID NO.32所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD3-4-1BB BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示，具体为：
Figure PCTCN2017096592-appb-000077
实施例2-3：ELISA检测CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类4-1BB-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-4-1BB BsAb_M或CD3-4-1BB BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-3A和图2-3B所示：图2-3A说明CD3-4-1BB BsAb_M与重组抗原CD3-hFc和4-1BB-hFc均具有体外结合活性，其中4-1BB结合活性较CD3结合活性更高；图2-3B说明CD3-4-1BB BsAb_D与重组抗原CD3-hFc和4-1BB-hFc同样具有体外结合活性，其中4-1BB结合活性更高。
实施例2-4：CD3-4-1BB双特异抗体介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-4-1BB BsAb_M、二聚体形式的双特异抗体CD3-4-1BB BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-4-1BB BsAb_M10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-4-1BB BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图2-4所示，单体和二聚体形式的CD3-4-1BB双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-4-1BB BsAb_M或二聚体形式的CD3-4-1BB BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-4-1BB双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例2-5：CD3-4-1BB双特异抗体介导的CIK细胞增殖后表型检测CD8+/CD4+阳性细胞 的流式检测
取实施例2-4所述培养30天后的3组实验细胞，分别进行Anti-CD4-FITC和Anti-CD8-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD8+和CD4+阳性细胞数目，统计各自比例。
流式检测步骤：
1.取对照组细胞4份，其他2组(实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
2. 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
3.对照组的4份细胞分别添加5ul PBS、Anti-CD4-FITC、Anti-CD8-PE以及Anti-CD4-FITC和Anti-CD8-PE同时添加，其余2份细胞只同时添加Anti-CD4-FITC和Anti-CD8-PE，4℃孵育1h；
4.所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图2-5所示：CD3-4-1BB BsAb_D介导CIK细胞增殖30天后，CD8+阳性细胞比例达到88.17％，CD3-4-1BB BsAb_M介导CIK细胞增殖30天后，CD8+阳性细胞比例为78.02％，均明显优于Anti-CD3/Anti-CD28联合(CD8+阳性细胞比例：包被使用48.47％)，说明两种形式的CD3-4-1BB双特异抗体比抗CD3/抗CD28单克隆全长抗体联合使用更有利于CD8+阳性细胞的生长扩增，其中二聚体较单体具有更好的效果。
实施例2-6：CD3-ICOS BsAb_M和CD3-ICOS BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子ICOS蛋白为靶点的双特异性抗体被命名为CD3-ICOS BsAb。
一、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D构建方案设计
单体形式的CD3-ICOS BsAb_M具体构建方案为：抗CD3 scFv和抗ICOS scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-ICOS BsAb_D具体构建方案为：抗CD3 scFv和抗ICOS scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗ICOS scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.90所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示。
具体地，抗ICOS scFv的重链可变区的核苷酸序列如SEQ ID NO.95所示，具体为：
Figure PCTCN2017096592-appb-000078
具体地，抗ICOS scFv的轻链可变区的核苷酸序列如SEQ ID NO.96所示，具体为：
Figure PCTCN2017096592-appb-000079
具体地，抗ICOS scFv的核苷酸序列如SEQ ID NO.94所示，具体为：
Figure PCTCN2017096592-appb-000080
单体形式的CD3-ICOS BsAb_M连接片段的核苷酸序列如SEQ ID NO.33所示。
二聚体形式的CD3-ICOS BsAb_D连接片段的核苷酸序列如SEQ ID NO.35所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.109所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.110所示。
二、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-2所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-ICOS BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗ICOS scFv的基因序列；针对CD3-ICOS BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD3 scFv、IgD铰链区、抗ICOS scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000081
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-ICOS BsAb_M和二聚体形式的CD3-ICOS BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-ICOS BsAb_M的核苷酸序列如SEQ ID NO.48所示，具体为：
Figure PCTCN2017096592-appb-000082
Figure PCTCN2017096592-appb-000083
具体地，二聚体形式的CD3-ICOS BsAb_D的核苷酸序列如SEQ ID NO.50所示，具体为：
Figure PCTCN2017096592-appb-000084
Figure PCTCN2017096592-appb-000085
表2-2.CD3-ICOS双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000086
实施例2-7：CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的表达与纯化
一、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-ICOS BsAb_M和CD3-ICOS BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-6中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测
二、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml BufferA平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-ICOS BsAb_M和CD3-ICOS BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-6所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-ICOS BsAb_M和CD3-ICOS BsAb_D重组蛋白的纯度均＞95％；其中CD3-ICOS BsAb_M重组蛋白的理论分子量为53.8kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-6A)；CD3-ICOS BsAb_D重组蛋白的理论分子量为61.7kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-6B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-ICOS BsAb_M为单体形式，CD3-ICOS BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-ICOS BsAb_M的氨基酸序列如SEQ ID NO.47所示，具体为：
Figure PCTCN2017096592-appb-000087
二聚体形式的CD3-ICOS BsAb_D的氨基酸序列如SEQ ID NO.49所示，具体为：
Figure PCTCN2017096592-appb-000088
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
抗ICOS scFv的氨基酸序列如SEQ ID NO.73所示，具体为：
Figure PCTCN2017096592-appb-000089
Figure PCTCN2017096592-appb-000090
抗ICOS scFv的重链可变区的氨基酸序列如SEQ ID NO.74所示，具体为：
Figure PCTCN2017096592-appb-000091
抗ICOS scFv的轻链可变区的氨基酸序列如SEQ ID NO.75所示，具体为：
Figure PCTCN2017096592-appb-000092
单体形式的CD3-ICOS BsAb_M中连接片段的氨基酸序列如SEQ ID NO.32所示。
二聚体形式的CD3-ICOS BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示。
实施例2-8：ELISA检测CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类ICOS-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-ICOS BsAb_M或CD3-ICOS BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-7A和图2-7B所示：图2-7A说明CD3-ICOS BsAb_M与重组抗原CD3-hFc和ICOS-hFc均具有体外结合活性，其中ICOS结合活性较CD3结合活性更高；图2-7B说明CD3-ICOS BsAb_D与重组抗原CD3-hFc和ICOS-hFc同样具有体外结合活性，其中ICOS结合活性更高。
实施例2-9：CD3-ICOS双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-ICOS BsAb_M、二聚体形式的双特异抗体CD3-ICOS BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬， 调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-ICOS BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-ICOS BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表2-3和图2-8所示，单体和二聚体形式的CD3-ICOS双特异性抗体单一使用对CIK细胞的增殖效果均不同程度地优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-ICOS BsAb_D可以介导CIK细胞两周扩增352倍，效果最优(实验组2)；单体形式的CD3-ICOS BsAb_M可以介导CIK细胞两周扩增298倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表2-3 CIK细胞扩增倍数
实验组名 对照组 实验组1 实验组2
14天扩增倍数 224 298 352
实施例2-10：CD3-ICOS双特异抗体介导的CIK细胞增殖后表型检测CD3+/CD56+双阳性CIK细胞的流式检测
取实施例2-9所述培养14天后的3组实验细胞，分别进行Anti-CD3-FITC和Anti-CD56-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD3+CD56+双阳性细胞比例。
流式检测步骤：
1.1取对照组细胞4份，其他2组(实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
1.2 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
1.3对照组的4份细胞分别添加5ul PBS、Anti-CD3-FITC、Anti-CD56-PE以及Anti-CD3-FITC和Anti-CD56-PE同时添加，其余2份细胞只同时添加Anti-CD3-FITC和Anti-CD56-PE，4℃孵育1h；
1.4所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图2-9所示：其中Anti-CD3/Anti-CD28联用介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为12.9％(图2-9A)；单体形式的CD3-ICOS BsAb_M介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为24.18％(图2-9B)；二聚体形式的CD3-ICOS BsAb_D介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为39.71％(图2-9C)，说明单体和二聚体形式CD3-ICOS双特异抗体均可用于替代抗CD3/抗CD28两种全长抗体联合使用，且介导的CIK细胞在增殖后具有更高的CD3+CD56+双阳性比例，其中二聚体较单体具有更好的效果。
实施例2-11：CD3-OX40 BsAb_M和CD3-OX40 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子OX40蛋白为靶点的双特异性抗体被命名为CD3-OX40 BsAb。
一、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D构建方案设计
单体形式的CD3-OX40 BsAb_M具体构建方案为：抗CD3 scFv和抗OX40 scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-OX40 BsAb_D具体构建方案为：抗CD3 scFv和抗OX40 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗OX40 scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.90所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示。
具体地，抗OX40 scFv的重链可变区的核苷酸序列如SEQ ID NO.98所示，具体为：
Figure PCTCN2017096592-appb-000093
具体地，抗OX40 scFv的轻链可变区的核苷酸序列如SEQ ID NO.99所示，具体为：
Figure PCTCN2017096592-appb-000094
具体地，抗OX40 scFv的核苷酸序列如SEQ ID NO.97所示，具体为：
Figure PCTCN2017096592-appb-000095
单体形式的CD3-OX40 BsAb_M连接片段的核苷酸序列如SEQ ID NO.33所示。
二聚体形式的CD3-OX40 BsAb_D连接片段的核苷酸序列如SEQ ID NO.35所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.109所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.110所示。
二、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-4所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿 讯科技有限公司合成。
针对CD3-OX40 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-OX40-F和pcDNA3.1-OX40-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗OX40scFv的基因序列；针对CD3-OX40 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-OX40-F和pcDNA3.1-OX40-R扩增出抗CD3 scFv、IgD铰链区、抗OX40 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000096
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-OX40 BsAb_M和二聚体形式的CD3-OX40 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-OX40 BsAb_M的核苷酸序列如SEQ ID NO.52所示，具体为：
Figure PCTCN2017096592-appb-000097
具体地，二聚体形式的CD3-OX40 BsAb_D的核苷酸序列如SEQ ID NO.54所示，具体为：
Figure PCTCN2017096592-appb-000098
表2-4.CD3-OX40双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000099
实施例2-12：CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的表达与纯化
一、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-OX40 BsAb_M和CD3-OX40 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-11中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-OX40 BsAb_M和CD3-OX40 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-10所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-OX40 BsAb_M和CD3-OX40 BsAb_D重组蛋白的纯度均＞95％；其中CD3-OX40 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-10A)；CD3-OX40 BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-10B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-OX40 BsAb_M为单体形式，CD3-OX40 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-OX40 BsAb_M的氨基酸序列如SEQ ID NO.51所示，具体为：
Figure PCTCN2017096592-appb-000100
Figure PCTCN2017096592-appb-000101
二聚体形式的CD3-OX40 BsAb_D的氨基酸序列如SEQ ID NO.53所示，具体为：
Figure PCTCN2017096592-appb-000102
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
抗OX40 scFv的氨基酸序列如SEQ ID NO：76所示，具体为：
Figure PCTCN2017096592-appb-000103
抗OX40 scFv的重链可变区的氨基酸序列如SEQ ID NO：77所示，具体为：
Figure PCTCN2017096592-appb-000104
抗OX40 scFv的轻链可变区的氨基酸序列如SEQ ID NO：78所示，具体为：
Figure PCTCN2017096592-appb-000105
单体形式的CD3-OX40 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.32所示。
二聚体形式的CD3-OX40 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示。
实施例2-13：ELISA检测CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类OX40-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-OX40 BsAb_M或CD3-OX40 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-11A和图2-11B所示：图2-11A说明CD3-OX40 BsAb_M与重组抗原CD3-hFc和OX40-hFc均具有体外结合活性，其中OX40结合活性较CD3结合活性更高；图2-11B说明CD3-OX40 BsAb_D与重组抗原CD3-hFc和OX40-hFc同样具有体外结合活性，其中OX40结合活性更高。
实施例2-14：CD3-OX40双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-OX40 BsAb_M、二聚体形式的双特异抗体CD3-OX40 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-OX40 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-OX40 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线。
检测结果如图2-12所示，单体和二聚体形式的CD3-OX40双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-OX40 BsAb_M或二聚体形式的CD3-OX40 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓，因此本发明制备的两种形式的CD3-OX40双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体较单体效果稍好。
实施例2-15：添加CD3-OX40双特异抗体的CIK细胞对肿瘤细胞杀伤活性检测
分别取实施例2-14所述培养14天或30天后的3组实验细胞作为杀伤效应细胞，以CCL-86 Raji淋巴瘤细胞(购自ATCC)作为靶细胞，将两种细胞混合培养，检测CIK细胞对Raji细胞的杀伤活性。
CIK细胞对Raji细胞的杀伤效率检测：
在96孔板中进行细胞杀伤实验，反应体积为100uL，设置以下实验组别：组1(5ug/ml Anti-CD3/Anti-CD28联用培养14天后的CIK细胞)、组2(10ng/ml CD3-OX40 BsAb_M培养14天后的CIK细胞)、组3(10ng/ml CD3-OX40 BsAb_D培养14天后的CIK细胞)、组4(5ug/ml Anti-CD3/Anti-CD28联用培养30天后的CIK细胞)、组5(10ng/ml CD3-OX40 BsAb_M培养30天后的CIK细胞)、组6(10ng/ml CD3-OX40 BsAb_D培养30天后的CIK细胞)。分别取上述6组CIK细胞1×105个，加入Raji细胞1×105个(CIK效应细胞：Raji靶细胞(E∶T比)为1∶1)，37℃共培养3h后，每孔添加10ul的CCK8，37℃继续反应2-3h，随后用酶标仪测OD450值，按照以下公式计算细胞杀伤效率，每组实验重复检测3次；同时以未添加任何抗体进行培养的CIK细胞对Raji细胞的杀伤效率作为空白对照。
结果如图2-13显示，培养14天后，添加CD3-OX40双特异抗体的CIK细胞对Raji细胞的杀伤效率均不同程度地优于抗CD3/抗CD28单克隆全长抗体联合使用：其中添加二聚体形式的CD3-OX40 BsAb_D，杀伤效率约为32％，效果最优(组3)；添加单体形式的CD3-OX40 BsAb_M，杀伤效率约为25％，效果次之(组2)；Anti CD3/Anti CD28联用，杀伤效率约为22％，效果最弱(组1)。培养30天后，添加CD3-OX40双特异抗体的CIK细胞杀伤活性进一步提高：其中添加二聚体形式的CD3-OX40 BsAb_D，杀伤效率约为40％(组6)；添加单体形式的CD3-OX40 BsAb_M，杀伤效率约为35％(组5)；与之相比，Anti CD3/Anti CD28联用培养的CIK细胞杀伤Raji肿瘤细胞的能力已大幅下降，杀伤效率仅约为10％(组4)。
其中，杀伤效率的计算公式为：
Figure PCTCN2017096592-appb-000106
实施例2-16：CD3-GITR BsAb_M和CD3-GITR BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子GITR蛋白为靶点的双特异性抗体被命名为CD3-GITR BsAb。
一、CD3-GITR BsAb_M和CD3-GITR BsAb_D构建方案设计
单体形式的CD3-GITR BsAb_M具体构建方案为：抗CD3 scFv和抗GITR scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-GITR BsAb_D具体构建方案为：抗CD3 scFv和抗GITR scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗GITR scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.90所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示。
具体地，抗GITR scFv的重链可变区的核苷酸序列如SEQ ID NO.101所示，具体为：
Figure PCTCN2017096592-appb-000107
具体地，抗GITR scFv的轻链可变区的核苷酸序列如SEQ ID NO.102所示，具体为：
Figure PCTCN2017096592-appb-000108
Figure PCTCN2017096592-appb-000109
具体地，抗GITR scFv的核苷酸序列如SEQ ID NO.100所示，具体为：
Figure PCTCN2017096592-appb-000110
单体形式的CD3-GITR BsAb_M连接片段的核苷酸序列如SEQ ID NO.33。
二聚体形式的CD3-GITR BsAb_D连接片段的核苷酸序列如SEQ ID NO.35。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.109所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.110所示。
二、CD3-GITR BsAb_M和CD3-GITR BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-5所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-GITR BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-GITR-F和pcDNA3.1-GITR-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗GITR scFv的基因序列；针对CD3-GITR BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-GITR-F和pcDNA3.1-GITR-R扩增出抗CD3 scFv、IgD铰链区、抗GITR scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000111
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-GITR BsAb_M和二聚体形式的CD3-GITR BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-GITR BsAb_M的核苷酸序列如SEQ ID NO.56所示，具体为：
Figure PCTCN2017096592-appb-000112
Figure PCTCN2017096592-appb-000113
具体地，二聚体形式的CD3-GITR BsAb_D的核苷酸序列如SEQ ID NO.58所示，具体为：
Figure PCTCN2017096592-appb-000114
Figure PCTCN2017096592-appb-000115
表2-5.CD3-GITR双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000116
实施例2-17：CD3-GITR BsAb_M和CD3-GITR BsAb_D的表达与纯化
一、CD3-GITR BsAb_M和CD3-GITR BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-GITR BsAb_M和CD3-GITR BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-16中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-GITR BsAb_M和CD3-GITR BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-GITR BsAb_M和CD3-GITR BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-14所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-GITR BsAb_M和CD3-GITR BsAb_D重组蛋白的纯度均＞95％；其中CD3-GITR BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-14A)；CD3-GITR BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-14B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-GITR BsAb_M为单体形式，CD3-GITR BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-GITR BsAb_M的氨基酸序列如SEQ ID NO.55所示，具体为：
Figure PCTCN2017096592-appb-000117
二聚体形式的CD3-GITR BsAb_D的氨基酸序列如SEQ ID NO.57所示，具体为：
Figure PCTCN2017096592-appb-000118
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
抗GITR scFv的氨基酸序列如SEQ ID NO.79所示，具体为：
Figure PCTCN2017096592-appb-000119
抗GITR scFv的重链可变区的氨基酸序列如SEQ ID NO.80所示，具体为：
Figure PCTCN2017096592-appb-000120
抗GITR scFv的轻链可变区的氨基酸序列如SEQ ID NO.81所示，具体为：
Figure PCTCN2017096592-appb-000121
单体形式的CD3-GITR BsAb_M中连接片段的氨基酸序列如SEQ ID NO.32所示。
二聚体形式的CD3-GITR BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示。
实施例2-18：ELISA检测CD3-GITR BsAb_M和CD3-GITR BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类GITR-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-GITR BsAb_M或CD3-GITR BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-15A和图2-15B所示：图2-15A说明CD3-GITR BsAb_M与重组抗原CD3-hFc和GITR-hFc均具有体外结合活性，其中GITR结合活性较CD3结合活性更高；图2-15B说明CD3-GITR BsAb_D与重组抗原CD3-hFc和GITR-hFc同样具有体外结合活性，其中GITR结合活性更高。
实施例2-19：CD3-GITR双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-GITR BsAb_M、二聚体形式的双特异抗体CD3-GITR BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza 公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD35ug/ml和Anti-CD285ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-GITR BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-GITR BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表2-6和图2-16所示，单体和二聚体形式的CD3-GITR双特异性抗体单一使用对CIK细胞的增殖效果均稍优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-GITR BsAb_D可以介导CIK细胞两周扩增287倍，效果最优(实验组2)；单体形式的CD3-GITR BsAb_M可以介导CIK细胞两周扩增248倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表2-6 CIK细胞扩增倍数
实验组名 对照组 实验组1 实验组2
14天扩增倍数 224 248 287
实施例2-20：CD3-CD40L BsAb_M和CD3-CD40L BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子CD40L蛋白为靶点的双特异性抗体被命名为CD3-CD40L BsAb。
一、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D构建方案设计
单体形式的CD3-CD40L BsAb_M具体构建方案为：抗CD3 scFv和抗CD40L scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-CD40L BsAb_D具体构建方案为：抗CD3 scFv和抗CD40L scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗CD40L scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.80所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示。
具体地，抗CD40L scFv的重链可变区的核苷酸序列如SEQ ID NO.104所示，具体为：
Figure PCTCN2017096592-appb-000122
具体地，抗CD40L scFv的轻链可变区的核苷酸序列如SEQ ID NO.105所示，具体为：
Figure PCTCN2017096592-appb-000123
Figure PCTCN2017096592-appb-000124
具体地，抗CD40L scFv的核苷酸序列如SEQ ID NO.103所示，具体为：
Figure PCTCN2017096592-appb-000125
单体形式的CD3-CD40L BsAb_M连接片段的核苷酸序列如SEQ ID NO.33。
二聚体形式的CD3-CD40L BsAb_D连接片段的核苷酸序列如SEQ ID NO.35。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-7所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD40L BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD3scFv、(GGGGS)3Linker、抗CD40L scFv的基因序列；针对CD3-CD40L BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD3 scFv、IgD铰链区、抗CD40L scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000126
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD40L BsAb_M和二聚体形式的CD3-CD40L BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD40L BsAb_M的核苷酸序列如SEQ ID NO.60所示，具体为：
Figure PCTCN2017096592-appb-000127
Figure PCTCN2017096592-appb-000128
具体地，二聚体形式的CD3-CD40L BsAb_D的核苷酸序列如SEQ ID NO.62所示，具体为：
Figure PCTCN2017096592-appb-000129
Figure PCTCN2017096592-appb-000130
表2-7.CD3-CD40L双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000131
实施例2-21：CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的表达与纯化
一、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD40L BsAb_M和CD3-CD40L BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-20中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD40L BsAb_M和CD3-CD40L BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-17所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-CD40L BsAb_M和CD3-CD40L BsAb_D重组蛋白的纯度均＞95％；其中CD3-CD40L BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-17A)；CD3-CD40L BsAb_D重组蛋白的理论分子量为61.2kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-17B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD40L BsAb_M为单体形式，CD3-CD40L BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD40L BsAb_M的氨基酸序列如SEQ ID NO.59所示，具体为：
Figure PCTCN2017096592-appb-000132
二聚体形式的CD3-CD40L BsAb_D的氨基酸序列如SEQ ID NO.61所示，具体为：
Figure PCTCN2017096592-appb-000133
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
抗CD40L scFv的氨基酸序列如SEQ ID NO.82所示，具体为：
Figure PCTCN2017096592-appb-000134
抗CD40L scFv的重链可变区的氨基酸序列如SEQ ID NO.83所示，具体为：
Figure PCTCN2017096592-appb-000135
抗CD40L scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示，具体为：
Figure PCTCN2017096592-appb-000136
单体形式的CD3-CD40L BsAb_M中连接片段的氨基酸序列如SEQ ID NO.32所示。
二聚体形式的CD3-CD40L BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示。
实施例2-22：ELISA检测CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CD40L-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD40L BsAb_M或CD3-CD40L BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-18A和图2-18B所示：图2-18A说明CD3-CD40LBsAb_M与重组抗原CD3-hFc和CD40L-hFc均具有体外结合活性，其中CD40L结合活性较CD3结合活性更高；图2-18B说明CD3-CD40L BsAb_D与重组抗原CD3-hFc和CD40L-hFc同样具有体外结合活性，其中CD40L结合活性更高。
实施例2-23：CD3-CD40L双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CD40L BsAb_M、二聚体形式的双特异抗体CD3-CD40L BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤， 1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD285ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-CD40L BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD40L BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表2-8和图2-19所示，单体和二聚体形式的CD3-CD40L双特异性抗体单一使用对CIK细胞的增殖效果均显著地优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-CD40L BsAb_D可以介导CIK细胞两周扩增367倍，效果最优(实验组2)；单体形式的CD3-CD40L BsAb_M可以介导CIK细胞两周扩增301倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表2-8 CIK细胞扩增倍数
实验组名 对照组1 实验组1 实验组2
14天扩增倍数 224 301 367
实施例2-24：CD3-CD27 BsAb_M和CD3-CD27 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子CD27蛋白为靶点的双特异性抗体被命名为CD3-CD27 BsAb。
一、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D构建方案设计
单体形式的CD3-CD27 BsAb_M具体构建方案为：抗CD3 scFv和抗CD27 scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-CD27 BsAb_D具体构建方案为：抗CD3 scFv和抗CD27 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗CD27 scFv及IgD铰链区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.89。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.90。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.88所示。
具体地，抗CD27 scFv的重链可变区的核苷酸序列如SEQ ID NO.107所示，具体为：
Figure PCTCN2017096592-appb-000137
具体地，抗CD27 scFv的轻链可变区的核苷酸序列如SEQ ID NO.108所示，具体为：
Figure PCTCN2017096592-appb-000138
Figure PCTCN2017096592-appb-000139
具体地，抗CD27 scFv的核苷酸序列如SEQ ID NO.106所示，具体为：
Figure PCTCN2017096592-appb-000140
单体形式的CD3-CD27 BsAb_M连接片段的核苷酸序列如SEQ ID NO.33所示。
二聚体形式的CD3-CD27 BsAb_D连接片段的核苷酸序列如SEQ ID NO.35所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.109所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.110所示。
二、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2-9所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD27 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD27-F和pcDNA3.1-CD27-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗CD27 scFv的基因序列；针对CD3-CD27 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD27-F和pcDNA3.1-CD27-R扩增出抗CD3 scFv、IgD铰链区、抗CD27 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000141
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD27 BsAb_M和二聚体形式的CD3-CD27 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD27 BsAb_M的核苷酸序列如SEQ ID NO.64所示，具体为：
Figure PCTCN2017096592-appb-000142
具体地，二聚体形式的CD3-CD27 BsAb_D的核苷酸序列如SEQ ID NO.66所示，具体为：
Figure PCTCN2017096592-appb-000143
Figure PCTCN2017096592-appb-000144
表2-9.CD3-CD27双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000145
实施例2-25：CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的表达与纯化
一、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD27 BsAb_M和CD3-CD27 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-24中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD27 BsAb_M和CD3-CD27 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-20所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-CD27 BsAb_M和CD3-CD27 BsAb_D重组蛋白的纯度均＞95％；其中CD3-CD27 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2-20A)；CD3-CD27 BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2-20B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD27 BsAb_M为单体形式，CD3-CD27 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD27 BsAb_M的氨基酸序列如SEQ ID NO.63所示，具体为：
Figure PCTCN2017096592-appb-000146
二聚体形式的CD3-CD27 BsAb_D的氨基酸序列如SEQ ID NO.65所示，具体为：
Figure PCTCN2017096592-appb-000147
抗CD3 scFv的氨基酸序列如SEQ ID NO.67所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.68所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.69所示。
抗CD27 scFv的氨基酸序列如SEQ ID NO.85所示，具体为：
Figure PCTCN2017096592-appb-000148
抗CD27 scFv的重链可变区的氨基酸序列如SEQ ID NO.86所示，具体为：
Figure PCTCN2017096592-appb-000149
抗CD27 scFv的轻链可变区的氨基酸序列如SEQ ID NO.87所示，具体为：
Figure PCTCN2017096592-appb-000150
单体形式的CD3-CD27 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.32所示。
二聚体形式的CD3-CD27 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.34所示。
实施例2-26：ELISA检测CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CD27-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD27 BsAb_M或CD3-CD27 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图2-21A和图2-21B所示：图2-21A说明CD3-CD27 BsAb_M与重组抗原CD3-hFc和CD27-hFc均具有体外结合活性，其中CD27结合活性较CD3结合活性更高；图2-21B说明CD3-CD27 BsAb_D与重组抗原CD3-hFc和CD27-hFc同样具有体外结合活性，其中CD27结合活性更高。
实施例2-27：CD3-CD27双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CD27 BsAb_M、二聚体形式的双特异抗体CD3-CD27 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤， 1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD35ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-CD27 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD27 BsAb_D 10ng/ml)。此外，三组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图2-22所示，单体和二聚体形式的CD3-CD27双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-CD27 BsAb_M或二聚体形式的CD3-CD27 BsAb_D均未出现细胞死亡，细胞扩增速度未见明显下降。因此本发明制备的两种形式的CD3-CD27双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式的效果更好。
实施例3-1：CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D真核表达载体的构建
在本发明中，融合抗T细胞表面人类CD3蛋白scFv结构域和T细胞正共刺激分子配体4-1BBL胞外区结构域的双特异性分子被命名为CD3-4-1BBL BsM。
一、CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D构建方案设计
单体形式的CD3-4-1BBL BsM_M具体构建方案为：抗CD3 scFv和4-1BBL胞外区序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-4-1BBL BsM_D具体构建方案为：抗CD3 scFv和4-1BBL胞外区序列之间通过IgD铰链区作为Linker相连。
为使双特异性分子在哺乳细胞中进行表达，针对抗CD3 scFv，4-1BBL胞外区及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.178所示，具体为：
Figure PCTCN2017096592-appb-000151
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.179所示，具体为：
Figure PCTCN2017096592-appb-000152
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.177所示，具体为：
Figure PCTCN2017096592-appb-000153
具体地，4-1BBL胞外区的核苷酸序列如SEQ ID NO.180所示，具体为：
Figure PCTCN2017096592-appb-000154
单体形式的CD3-4-1BBL BsM_M连接片段的核苷酸序列如SEQ ID NO.136所示，具体为：GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。
二聚体形式的CD3-4-1BBL BsM_D连接片段的核苷酸序列如SEQ ID NO.138所示，具体为：
Figure PCTCN2017096592-appb-000155
为使双特异性分子在CHO-S细胞中表达并成功分泌到培养基中，选择了分泌型表达信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.185所示，具体为：
Figure PCTCN2017096592-appb-000156
该分泌表达信号肽的核苷酸序列如SEQ ID NO.186所示，具体为：
Figure PCTCN2017096592-appb-000157
二、CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D真核表达载体构建
本发明双特异性分子的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性分子，分别设计了如表 3-1所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-4-1BBL BsM_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-4-1BBL-F和pcDNA3.1-4-1BBL-R扩增出抗CD3 scFv、(GGGGS)3Linker、4-1BBL胞外区的基因序列；针对CD3-4-1BBL BsM_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-4-1BBL-F和pcDNA3.1-4-1BBL-R扩增出抗CD3 scFv、IgD铰链区、4-1BBL胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000158
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性分子全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-4-1BBL BsM_M和二聚体形式的CD3-4-1BBL BsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-4-1BBL BsM_M的核苷酸序列如SEQ ID NO.150所示，具体为：
Figure PCTCN2017096592-appb-000159
具体地，二聚体形式的CD3-4-1BBL BsM_D的核苷酸序列如SEQ ID NO.152所示，具体为：
Figure PCTCN2017096592-appb-000160
表3-1.CD3-4-1BBL双特异性分子基因克隆中使用的引物
Figure PCTCN2017096592-appb-000161
Figure PCTCN2017096592-appb-000162
实施例3-2：CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D的表达与纯化
一、CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例3-1中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml BufferA平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图3-2所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D重组蛋白的纯度均＞95％；其中CD3-4-1BBL BsM_M重组蛋白的理论分子量为48.8kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性分子为单体形式(图3-2A)；CD3-4-1BBL BsM_D重组蛋白的理论分子量为56.6kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图3-2B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性分子为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-4-1BBL BsM_M为单体形式，CD3-4-1BBL BsM_D为二聚体形式。
因此，可得知，单体形式的CD3-4-1BBL BsM_M的氨基酸序列如SEQ ID NO.149所示，具体为：
Figure PCTCN2017096592-appb-000163
二聚体形式的CD3-4-1BBL BsM_D的氨基酸序列如SEQ ID NO.151所示，具体为：
Figure PCTCN2017096592-appb-000164
抗CD3 scFv的氨基酸序列如SEQ ID NO.169所示，具体为：
Figure PCTCN2017096592-appb-000165
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.170所示，具体为：
Figure PCTCN2017096592-appb-000166
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.171所示，具体为：
Figure PCTCN2017096592-appb-000167
4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.172所示，具体为：
Figure PCTCN2017096592-appb-000168
单体形式的CD3-4-1BBL BsM_M中连接片段的氨基酸序列如SEQ ID NO.135所示，具体为：
Figure PCTCN2017096592-appb-000169
二聚体形式的CD3-4-1BBL BsM_D中连接片段的氨基酸序列如SEQ ID NO.137所示，具体为：
Figure PCTCN2017096592-appb-000170
Figure PCTCN2017096592-appb-000171
实施例3-3：ELISA检测CD3-4-1BBL BsM_M和CD3-4-1BBL BsM_D的CD3抗原及正共刺激分子4-1BB结合活性
ELISA操作步骤：
1.重组蛋白包被：人类CD3-hFc与人类4-1BB-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，蛋白浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-4-1BBL BsM_M或CD3-4-1BBL BsM_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3-3A和图3-3B所示：图3-3A说明CD3-4-1BBL BsM_M与抗原CD3-hFc和T细胞正共刺激分子4-1BB-hFc均具有体外结合活性，其中4-1BB结合活性较CD3结合活性更高；图3-3B说明CD3-4-1BBL BsM_D与抗原CD3-hFc和T细胞正共刺激分子4-1BB-hFc同样具有体外结合活性，其中4-1BB结合活性更高。
实施例3-4：CD3-4-1BBL双特异性分子介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异性分子CD3-4-1BBL BsM_M、二聚体形式的双特异性分子CD3-4-1BBL BsM_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD35ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性分子CD3-4-1BBL BsM_M10ng/ml)；实验组2(溶液状态下添加双特异性分子CD3-4-1BBL BsM_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图3-4所示，单体和二聚体形式的CD3-4-1BBL双特异性分子单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的 CD3-4-1BBL BsM_M或二聚体形式的CD3-4-1BBL BsM_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-4-1BBL双特异性分子均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例3-5：CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D真核表达载体的构建
在本发明中，融合抗T细胞表面人类CD3蛋白scFv结构域和T细胞正共刺激分子配体B7RP-1胞外区结构域的双特异性分子被命名为CD3-B7RP-1 BsM。
一、CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D构建方案设计
单体形式的CD3-B7RP-1 BsM_M具体构建方案为：抗CD3 scFv和B7RP-1胞外区序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-B7RP-1 BsM_D具体构建方案为：抗CD3 scFv和B7RP-1胞外区序列之间通过IgD铰链区作为Linker相连。
为使双特异性分子在哺乳细胞中进行表达，针对抗CD3 scFv，B7RP-1胞外区及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.178所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.179所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.177所示。
具体地，B7RP-1胞外区的核苷酸序列如SEQ ID NO.181所示，具体为：
Figure PCTCN2017096592-appb-000172
单体形式的CD3-B7RP-1 BsM_M连接片段的核苷酸序列如SEQ ID NO.136所示。
二聚体形式的CD3-B7RP-1 BsM_D连接片段的核苷酸序列如SEQ ID NO.138所示。
为使双特异性分子在CHO-S细胞中表达并成功分泌到培养基中，选择了分泌型表达信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.185所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.186所示。
二、CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D真核表达载体构建
本发明双特异性分子的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性分子，分别设计了如表3-2所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-B7RP-1 BsM_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-B7RP-1-F和pcDNA3.1-B7RP-1-R扩增出抗CD3 scFv、(GGGGS)3Linker、B7RP-1胞外区的基因序列； 针对CD3-B7RP-1 BsM_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-B7RP-1-F和pcDNA3.1-B7RP-1-R扩增出抗CD3 scFv、IgD铰链区、B7RP-1胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000173
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性分子全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-B7RP-1 BsM_M和二聚体形式的CD3-B7RP-1 BsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-B7RP-1 BsM_M的核苷酸序列如SEQ ID NO.154所示，具体为：
Figure PCTCN2017096592-appb-000174
具体地，二聚体形式的CD3-B7RP-1 BsM_D的核苷酸序列如SEQ ID NO.156所示，具体为：
Figure PCTCN2017096592-appb-000175
Figure PCTCN2017096592-appb-000176
表3-2.CD3-B7RP-1双特异性分子基因克隆中使用的引物
Figure PCTCN2017096592-appb-000177
实施例3-6：CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D的表达与纯化
一、CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例3-5中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图3-5所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D重组蛋白的纯度均＞95％；其中CD3-B7RP-1 BsM_M重组蛋白的理论分子量为53.7kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，由于B7RP-1胞外区结构域存在翻译后N-糖基化修饰，因此实际分子量与理论值相比偏大，该双特异性分子为糖基化的单体形式(图3-5A)；CD3-B7RP-1 BsM_D重组蛋白的理论分子量为61.6kDa，还原条件下该蛋白电泳条带所呈现分子量与糖基化的单体一致，非还原条件下电泳条带所呈现分子量与糖基化的二聚体一致(图3-5B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性分子为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-B7RP-1 BsM_M为单体形式，CD3-B7RP-1 BsM_D为二聚体形式。
因此，可得知，单体形式的CD3-B7RP-1 BsM_M的氨基酸序列如SEQ ID NO.153所示，具体为：
Figure PCTCN2017096592-appb-000178
Figure PCTCN2017096592-appb-000179
二聚体形式的CD3-B7RP-1 BsM_D的氨基酸序列如SEQ ID NO.155所示，具体为：
Figure PCTCN2017096592-appb-000180
抗CD3 scFv的氨基酸序列如SEQ ID NO.169所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.170所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.171所示。
B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.173所示，具体为：
Figure PCTCN2017096592-appb-000181
单体形式的CD3-4-1BBL BsM_M中连接片段的氨基酸序列如SEQ ID NO.135所示。
二聚体形式的CD3-4-1BBL BsM_D中连接片段的氨基酸序列如SEQ ID NO.137所示。
实施例3-7：ELISA检测CD3-B7RP-1 BsM_M和CD3-B7RP-1 BsM_D的CD3抗原及正共刺激分子ICOS结合活性
ELISA操作步骤：
1.重组蛋白包被：人类CD3-hFc与人类ICOS-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，蛋白浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-B7RP-1 BsM_M或CD3-B7RP-1 BsM_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3-6A和图3-6B所示：图3-6A说明CD3-B7RP-1 BsM_M与抗原CD3-hFc和T细胞正共刺激分子ICOS-hFc均具有体外结合活性，其中CD3结合活性较ICOS结合活性更高；图3-6B说明CD3-B7RP-1 BsM_D与抗原CD3-hFc和T细胞正共刺激分子ICOS-hFc同样具有体外结合活性，其中CD3结合活性更高。
实施例3-8：CD3-B7RP-1双特异性分子介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异性分子CD3-B7RP-1 BsM_M、二聚体形式的双特异性分子CD3-B7RP-1 BsM_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性分子CD3-B7RP-1 BsM_M 10ng/ml)；实验组2(溶液状态下添加双特异性分子CD3-B7RP-1 BsM_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图3-7所示，单体和二聚体形式的CD3-B7RP-1双特异性分子单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-B7RP-1 BsM_M或二聚体形式的CD3-B7RP-1 BsM_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-B7RP-1双特异性分子均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例3-9：CD3-OX40L BsM_M和CD3-OX40L BsM_D真核表达载体的构建
在本发明中，融合抗T细胞表面人类CD3蛋白scFv结构域和T细胞正共刺激分子配体OX40L胞外区结构域的双特异性分子被命名为CD3-OX40L BsM。
一、CD3-OX40L BsM_M和CD3-OX40L BsM_D构建方案设计
单体形式的CD3-OX40L BsM_M具体构建方案为：抗CD3 scFv和OX40L胞外区序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-OX40L BsM_D具体构建方案为：抗CD3 scFv和OX40L胞外区序列之间通过IgD铰链区作为Linker相连。
为使双特异性分子在哺乳细胞中进行表达，针对抗CD3 scFv，OX40L胞外区及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.178所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.179所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.177所示。
具体地，OX40L胞外区的核苷酸序列如SEQ ID NO.182所示，具体为：
Figure PCTCN2017096592-appb-000182
Figure PCTCN2017096592-appb-000183
单体形式的CD3-OX40L BsM_M连接片段的核苷酸序列如SEQ ID NO.136所示。
二聚体形式的CD3-OX40L BsM_D连接片段的核苷酸序列如SEQ ID NO.138所示。
为使双特异性分子在CHO-S细胞中表达并成功分泌到培养基中，选择了分泌型表达信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.185所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.186所示。
二、CD3-OX40L BsM_M和CD3-OX40L BsM_D真核表达载体构建
本发明双特异性分子的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性分子，分别设计了如表3-3所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-OX40L BsM_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-OX40L-F和pcDNA3.1-OX40L-R扩增出抗CD3 scFv、(GGGGS)3Linker、OX40L胞外区的基因序列；针对CD3-OX40L BsM_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-OX40L-F和pcDNA3.1-OX40L-R扩增出抗CD3 scFv、IgD铰链区、OX40L胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000184
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性分子全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-OX40L BsM_M和二聚体形式的CD3-OX40L BsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-OX40L BsM_M的核苷酸序列如SEQ ID NO.158所示，具体为：
Figure PCTCN2017096592-appb-000185
Figure PCTCN2017096592-appb-000186
具体地，二聚体形式的CD3-OX40L BsM_D的核苷酸序列如SEQ ID NO.160所示，具体为：
Figure PCTCN2017096592-appb-000187
表3-3.CD3-OX40L双特异性分子基因克隆中使用的引物
Figure PCTCN2017096592-appb-000188
Figure PCTCN2017096592-appb-000189
实施例3-10：CD3-OX40L BsM_M和CD3-OX40L BsM_D的表达与纯化
一、CD3-OX40L BsM_M和CD3-OX40L BsM_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-OX40L BsM_M和CD3-OX40L BsM_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例3-9中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-OX40L BsM_M和CD3-OX40L BsM_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-OX40L BsM_M和CD3-OX40L BsM_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图3-8所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-OX40L BsM_M和CD3-OX40L BsM_D重组蛋白的纯度均＞95％；其中CD3-OX40L BsM_M重组蛋白的理论分子量为42.7kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，由于OX40L胞外区结构域存在翻译后N-糖基化修饰，因此实际分子量与理论值相比偏大，该双特异性分子为糖基化的单体形式(图3-8A)；CD3-OX40L BsM_D重组蛋白的理论分子量为50.6kDa，还原条件下该蛋白电泳条带所呈现分子量与糖基化的单体一致，非还原条件下电泳条带所呈现分子量与糖基化的二聚体一致(图3-8B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性分子为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-OX40L BsM_M为单体形式，CD3-OX40L BsM_D为二聚体形式。
因此，可得知，单体形式的CD3-OX40L BsM_M的氨基酸序列如SEQ ID NO.157所示，具体为：
Figure PCTCN2017096592-appb-000190
Figure PCTCN2017096592-appb-000191
二聚体形式的CD3-OX40L BsM_D的氨基酸序列如SEQ ID NO.159所示，具体为：
Figure PCTCN2017096592-appb-000192
抗CD3 scFv的氨基酸序列如SEQ ID NO.169所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.170所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.171所示。
OX40L胞外区结构域的氨基酸序列如SEQ ID NO.174所示，具体为：
Figure PCTCN2017096592-appb-000193
单体形式的CD3-4-1BBL BsM_M中连接片段的氨基酸序列如SEQ ID NO.135所示。
二聚体形式的CD3-4-1BBL BsM_D中连接片段的氨基酸序列如SEQ ID NO.137所示。
实施例3-11：ELISA检测CD3-OX40L BsM_M和CD3-OX40L BsM_D的CD3抗原及正共刺激分子OX40结合活性
ELISA操作步骤：
1.重组蛋白包被：人类CD3-hFc与人类OX40-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，蛋白浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-OX40L BsM_M或CD3-OX40L BsM_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3-9A和图3-9B所示：图3-9A说明CD3-OX40L BsM_M与抗原CD3-hFc和T细胞正共刺激分子OX40-hFc均具有体外结合活性，其中OX40结合活性较CD3结合活性更高；图3-9B说明CD3-OX40L BsM_D与抗原CD3-hFc和T细胞正共刺激分子 OX40-hFc同样具有体外结合活性，其中OX40结合活性更高。
实施例3-12：CD3-OX40L双特异性分子介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异性分子CD3-OX40L BsM_M、二聚体形式的双特异性分子CD3-OX40L BsM_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性分子CD3-OX40L BsM_M 10ng/ml)；实验组2(溶液状态下添加双特异性分子CD3-OX40L BsM_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图3-10所示，单体和二聚体形式的CD3-OX40L双特异性分子单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-OX40L BsM_M或二聚体形式的CD3-OX40L BsM_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-OX40L双特异性分子均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例3-13：CD3-GITRL BsM_M和CD3-GITRL BsM_D真核表达载体的构建
在本发明中，融合抗T细胞表面人类CD3蛋白scFv结构域和T细胞正共刺激分子配体GITRL胞外区结构域的双特异性分子被命名为CD3-GITRL BsM。
一、CD3-GITRL BsM_M和CD3-GITRL BsM_D构建方案设计
单体形式的CD3-GITRL BsM_M具体构建方案为：抗CD3 scFv和GITRL胞外区序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-GITRL BsM_D具体构建方案为：抗CD3 scFv和GITRL胞外区序列之间通过IgD铰链区作为Linker相连。
为使双特异性分子在哺乳细胞中进行表达，针对抗CD3 scFv，GITRL胞外区及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.178所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.179所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.177所示。
具体地，GITRL胞外区的核苷酸序列如SEQ ID NO.183所示，具体为：
Figure PCTCN2017096592-appb-000194
Figure PCTCN2017096592-appb-000195
单体形式的CD3-GITRL BsM_M连接片段的核苷酸序列如SEQ ID NO.136所示。
二聚体形式的CD3-GITRL BsM_D连接片段的核苷酸序列如SEQ ID NO.138所示。
为使双特异性分子在CHO-S细胞中表达并成功分泌到培养基中，选择了分泌型表达信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.185所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.186所示。
二、CD3-GITRL BsM_M和CD3-GITRL BsM_D真核表达载体构建
本发明双特异性分子的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性分子，分别设计了如表3-4所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-GITRL BsM_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-GITRL-F和pcDNA3.1-GITRL-R扩增出抗CD3 scFv、(GGGGS)3Linker、GITRL胞外区的基因序列；针对CD3-GITRL BsM_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-GITRL-F和pcDNA3.1-GITRL-R扩增出抗CD3 scFv、IgD铰链区、GITRL胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000196
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性分子全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-GITRL BsM_M和二聚体形式的CD3-GITRL BsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-GITRL BsM_M的核苷酸序列如SEQ ID NO.162所示，具体为：
Figure PCTCN2017096592-appb-000197
Figure PCTCN2017096592-appb-000198
具体地，二聚体形式的CD3-GITRL BsM_D的核苷酸序列如SEQ ID NO.164所示，具体为：
Figure PCTCN2017096592-appb-000199
表3-4.CD3-GITRL双特异性分子基因克隆中使用的引物
Figure PCTCN2017096592-appb-000200
实施例3-14：CD3-GITRL BsM_M和CD3-GITRL BsM_D的表达与纯化
一、CD3-GITRL BsM_M和CD3-GITRL BsM_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-GITRL BsM_M和CD3-GITRL BsM_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例3-13中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-GITRL BsM_M和CD3-GITRL BsM_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-GITRL BsM_M和CD3-GITRL BsM_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图3-11所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-GITRL BsM_M和CD3-GITRL BsM_D重组蛋白的纯度均＞95％；其中CD3-GITRL BsM_M重组蛋白的理论分子量为41.8kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，由于GITRL胞外区结构域存在翻译后N-糖基化修饰，因此实际分子量与理论值相比偏大，该双特异性分子为糖基化的单体形式(图3-11A)；CD3-GITRL BsM_D重组蛋白的理论分子量为49.7kDa，还原条件下该蛋白电泳条带所呈现分子量与糖基化的单体一致，非还原条件下电泳条带所呈现分子量与糖基化的二聚体一致(图3-11B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性分子为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-GITRL BsM_M为单体形式，CD3-GITRL BsM_D为二聚体形式。
因此，可得知，单体形式的CD3-GITRL BsM_M的氨基酸序列如SEQ ID NO.161所示，具体为：
Figure PCTCN2017096592-appb-000201
Figure PCTCN2017096592-appb-000202
二聚体形式的CD3-GITRL BsM_D的氨基酸序列如SEQ ID NO.163所示，具体为：
Figure PCTCN2017096592-appb-000203
抗CD3 scFv的氨基酸序列如SEQ ID NO.169所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.170所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.171所示。
GITRL胞外区结构域的氨基酸序列如SEQ ID NO.175所示，具体为：
Figure PCTCN2017096592-appb-000204
单体形式的CD3-4-1BBL BsM_M中连接片段的氨基酸序列如SEQ ID NO.135所示。
二聚体形式的CD3-4-1BBL BsM_D中连接片段的氨基酸序列如SEQ ID NO.137所示。
实施例3-15：ELISA检测CD3-GITRL BsM_M和CD3-GITRL BsM_D的CD3抗原及正共刺激分子GITR结合活性
ELISA操作步骤：
1.重组蛋白包被：人类CD3-hFc与人类GITR-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，蛋白浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-GITRL BsM_M或CD3-GITRL BsM_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3-12A和图3-12B所示：图3-12A说明CD3-GITRL BsM_M与抗原CD3-hFc和T细胞正共刺激分子GITR-hFc均具有体外结合活性，其中GITR结合活性较CD3结合活性更高；图3-12B说明CD3-GITRL BsM_D与抗原CD3-hFc和T细胞正共刺激分子GITR-hFc同样具有体外结合活性，其中GITR结合活性更高。
实施例3-16：CD3-GITRL双特异性分子介导的CIK(Cytokine induced killer)细胞增 殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异性分子CD3-GITRL BsM_M、二聚体形式的双特异性分子CD3-GITRL BsM_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性分子CD3-GITRL BsM_M 10ng/ml)；实验组2(溶液状态下添加双特异性分子CD3-GITRL BsM_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图3-13所示，单体和二聚体形式的CD3-GITRL双特异性分子单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-GITRL BsM_M或二聚体形式的CD3-GITRL BsM_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-GITRL双特异性分子均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例3-17：CD3-CD70 BsM_M和CD3-CD70 BsM_D真核表达载体的构建
在本发明中，融合抗T细胞表面人类CD3蛋白scFv结构域和T细胞正共刺激分子配体CD70胞外区结构域的双特异性分子被命名为CD3-CD70BsM。
一、CD3-CD70 BsM_M和CD3-CD70 BsM_D构建方案设计
单体形式的CD3-CD70 BsM_M具体构建方案为：抗CD3 scFv和CD70胞外区序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-CD70 BsM_D具体构建方案为：抗CD3 scFv和CD70胞外区序列之间通过IgD铰链区作为Linker相连。
为使双特异性分子在哺乳细胞中进行表达，针对抗CD3 scFv，CD70胞外区及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.178所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.179所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.177所示。
具体地，CD70胞外区的核苷酸序列如SEQ ID NO.184所示，具体为：
Figure PCTCN2017096592-appb-000205
Figure PCTCN2017096592-appb-000206
单体形式的CD3-CD70 BsM_M连接片段的核苷酸序列如SEQ ID NO.136所示。
二聚体形式的CD3-CD70 BsM_D连接片段的核苷酸序列如SEQ ID NO.138所示。
为使双特异性分子在CHO-S细胞中表达并成功分泌到培养基中，选择了分泌型表达信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.185所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.186所示。
二、CD3-CD70 BsM_M和CD3-CD70 BsM_D真核表达载体构建
本发明双特异性分子的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性分子，分别设计了如表3-5所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD70 BsM_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD70-F和pcDNA3.1-CD70-R扩增出抗CD3 scFv、(GGGGS)3 Linker、CD70胞外区的基因序列；针对CD3-CD70 BsM_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD70-F和pcDNA3.1-CD70-R扩增出抗CD3 scFv、IgD铰链区、CD70胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000207
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性分子全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD70 BsM_M和二聚体形式的CD3-CD70 BsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD70 BsM_M的核苷酸序列如SEQ ID NO.166所示，具体为：
Figure PCTCN2017096592-appb-000208
Figure PCTCN2017096592-appb-000209
具体地，二聚体形式的CD3-CD70 BsM_D的核苷酸序列如SEQ ID NO.168所示，具体为：
Figure PCTCN2017096592-appb-000210
表3-5.CD3-CD70双特异性分子基因克隆中使用的引物
Figure PCTCN2017096592-appb-000211
Figure PCTCN2017096592-appb-000212
实施例3-18：CD3-CD70 BsM_M和CD3-CD70 BsM_D的表达与纯化
一、CD3-CD70 BsM_M和CD3-CD70 BsM_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD70 BsM_M和CD3-CD70 BsM_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例3-17中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD70 BsM_M和CD3-CD70 BsM_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD70 BsM_M和CD3-CD70 BsM_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图3-14所示。从图中可以看出，经ProteinL亲和层析柱纯化后，CD3-CD70 BsM_M和CD3-CD70 BsM_D重组蛋白的纯度均＞95％；其中CD3-CD70 BsM_M重组蛋白的理论分子量为44.4kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，由于CD70胞外区结构域存在翻译后N-糖基化修饰，因此实际分子量与理论值相比偏大，该双特异性分子为糖基化的单体形式(图3-14A)；CD3-CD70 BsM_D重组蛋白的理论分子量为52.3kDa，还原条件下该蛋白电泳条带所呈现分子量与糖基化的单体一致，非还原条件下电泳条带所呈现分子量与糖基化的二聚体一致(图3-14B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性分子为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD70 BsM_M为单体形式，CD3-CD70 BsM_D为二聚体形式。
因此，可得知，单体形式的CD3-CD70 BsM_M的氨基酸序列如SEQ ID NO.165所示，具体为：
Figure PCTCN2017096592-appb-000213
Figure PCTCN2017096592-appb-000214
二聚体形式的CD3-CD70 BsM_D的氨基酸序列如SEQ ID NO.167所示，具体为：
Figure PCTCN2017096592-appb-000215
抗CD3 scFv的氨基酸序列如SEQ ID NO.169所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.170所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.171所示。
CD70胞外区结构域的氨基酸序列如SEQ ID NO.176所示，具体为：
Figure PCTCN2017096592-appb-000216
单体形式的CD3-4-1BBL BsM_M中连接片段的氨基酸序列如SEQ ID NO.135所示。
二聚体形式的CD3-4-1BBL BsM_D中连接片段的氨基酸序列如SEQ ID NO.137所示。
实施例3-19：ELISA检测CD3-CD70 BsM_M和CD3-CD70 BsM_D的CD3抗原及正共刺激分子CD27结合活性
ELISA操作步骤：
1.重组蛋白包被：人类CD3-hFc与人类CD27-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，蛋白浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD70 BsM_M或CD3-CD70 BsM_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3-15A和图3-15B所示：图3-15A说明CD3-CD70 BsM_M与抗原CD3-hFc和T细胞正共刺激分子CD27-hFc均具有体外结合活性，其中CD27结合活性较 CD3结合活性更高；图3-15B说明CD3-CD70 BsM_D与抗原CD3-hFc和T细胞正共刺激分子CD27-hFc同样具有体外结合活性，其中CD27结合活性更高。
实施例3-20：CD3-CD70双特异性分子介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异性分子CD3-CD70 BsM_M、二聚体形式的双特异性分子CD3-CD70 BsM_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD285ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性分子CD3-CD70 BsM_M10ng/ml)；实验组2(溶液状态下添加双特异性分子CD3-CD70 BsM_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图3-16所示，单体和二聚体形式的CD3-CD70双特异性分子单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-CD70 BsM_M或二聚体形式的CD3-CD70 BsM_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-CD70双特异性分子均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-1：CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子PD-1蛋白为靶点的双特异性抗体被命名为CD3-PD-1 BsAb。
一、CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D构建方案设计
单体形式的CD3-PD-1 BsAb_M具体构建方案为：抗CD3 scFv和抗PD-1 scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-PD-1 BsAb_D具体构建方案为：抗CD3 scFv和抗PD-1 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗PD-1 scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示，具体为：
Figure PCTCN2017096592-appb-000217
Figure PCTCN2017096592-appb-000218
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示，具体为：
Figure PCTCN2017096592-appb-000219
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示，具体为：
Figure PCTCN2017096592-appb-000220
具体地，抗PD-1 scFv的重链可变区的核苷酸序列如SEQ ID NO.267所示，具体为：
Figure PCTCN2017096592-appb-000221
具体地，抗PD-1 scFv的轻链可变区的核苷酸序列如SEQ ID NO.268所示，具体为：
Figure PCTCN2017096592-appb-000222
具体地，抗PD-1 scFv的核苷酸序列如SEQ ID NO.266所示，具体为：
Figure PCTCN2017096592-appb-000223
Figure PCTCN2017096592-appb-000224
单体形式的CD3-PD-1 BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示，具体为：
Figure PCTCN2017096592-appb-000225
二聚体形式的CD3-PD-1 BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示，具体为：
Figure PCTCN2017096592-appb-000226
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示，具体为：
Figure PCTCN2017096592-appb-000227
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示，具体为：
Figure PCTCN2017096592-appb-000228
二、CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-1所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-PD-1 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-PD-1-F和pcDNA3.1-PD-1-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗PD-1 scFv的基因序列；针对CD3-PD-1 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-PD-1-F和pcDNA3.1-PD-1-R扩增出抗CD3 scFv、IgD铰链区、抗PD-1 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000229
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-PD-1 BsAb_M和二聚体形式的CD3-PD-1 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-PD-1 BsAb_M的核苷酸序列如SEQ ID NO.219所示，具体为：
Figure PCTCN2017096592-appb-000230
具体地，二聚体形式的CD3-PD-1 BsAb_D的核苷酸序列如SEQ ID NO.221所示，具体为：
Figure PCTCN2017096592-appb-000231
Figure PCTCN2017096592-appb-000232
表4-1.CD3-PD-1双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000233
实施例4-2：CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D的表达与纯化
一、CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-1中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-2所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D重组蛋白的纯度均＞95％；其中CD3-PD-1 BsAb_M重组蛋白的理论分子量为52.5kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-2A)；CD3-PD-1 BsAb_D重组蛋白的理论分子量为60.4kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-2B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-PD-1 BsAb_M为单体形式，CD3-PD-1 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-PD-1 BsAb_M的氨基酸序列如SEQ ID NO.218所示，具体为：
Figure PCTCN2017096592-appb-000234
二聚体形式的CD3-PD-1 BsAb_D的氨基酸序列如SEQ ID NO.220所示，具体为：
Figure PCTCN2017096592-appb-000235
Figure PCTCN2017096592-appb-000236
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示，具体为：
Figure PCTCN2017096592-appb-000237
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示，具体为：
Figure PCTCN2017096592-appb-000238
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示，具体为：
Figure PCTCN2017096592-appb-000239
抗PD-1 scFv的氨基酸序列如SEQ ID NO.245所示，具体为：
Figure PCTCN2017096592-appb-000240
抗PD-1 scFv的重链可变区的氨基酸序列如SEQ ID NO.246所示，具体为：
Figure PCTCN2017096592-appb-000241
抗PD-1 scFv的轻链可变区的氨基酸序列如SEQ ID NO.247所示，具体为：
Figure PCTCN2017096592-appb-000242
单体形式的CD3-PD-1 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD3-PD-1 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示，具体为：
Figure PCTCN2017096592-appb-000243
实施例4-3：ELISA检测CD3-PD-1 BsAb_M和CD3-PD-1 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类PD-1-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl， 8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-PD-1 BsAb_M或CD3-PD-1 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-3A和图4-3B所示：图4-3A说明CD3-PD-1 BsAb_M与重组抗原CD3-hFc和PD-1-hFc均具有体外结合活性，其中PD-1结合活性较CD3结合活性更高；图4-3B说明CD3-PD-1 BsAb_D与重组抗原CD3-hFc和PD-1-hFc同样具有体外结合活性，其中PD-1结合活性更高。
实施例4-4：CD3-PD-1双特异抗体介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-PD-1 BsAb_M、二聚体形式的双特异抗体CD3-PD-1 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD285ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-PD-1 BsAb_M10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-PD-1 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4-4所示，单体和二聚体形式的CD3-PD-1双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-PD-1 BsAb_M或二聚体形式的CD3-PD-1 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-PD-1双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-5：CD3-PD-1双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-4中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测， 三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-5所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-PD-1 BsAb_M培养的CIK细胞IFN-γ相对分泌量为2.45，溶液状态下添加二聚体形式的CD3-PD-1 BsAb_D培养的CIK细胞IFN-γ相对分泌量为4.12，因此本发明制备的两种形式的CD3-PD-1双特异抗体均更有利于活化CIK细胞并诱导IFN-γ的分泌，其中二聚体形式效果更佳。
实施例4-6：CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子CTLA-4蛋白为靶点的双特异性抗体被命名为CD3-CTLA-4 BsAb。
一、CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D构建方案设计
单体形式的CD3-CTLA-4 BsAb_M具体构建方案为：抗CD3 scFv和抗CTLA-4 scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-CTLA-4 BsAb_D具体构建方案为：抗CD3 scFv和抗CTLA-4 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗CTLA-4 scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示。
具体地，抗CTLA-4 scFv的重链可变区的核苷酸序列如SEQ ID NO.270所示，具体为：
Figure PCTCN2017096592-appb-000244
具体地，抗CTLA-4 scFv的轻链可变区的核苷酸序列如SEQ ID NO.271所示，具体为：
Figure PCTCN2017096592-appb-000245
具体地，抗CTLA-4 scFv的核苷酸序列如SEQ ID NO.269所示，具体为：
Figure PCTCN2017096592-appb-000246
Figure PCTCN2017096592-appb-000247
单体形式的CD3-CTLA-4 BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示。
二聚体形式的CD3-CTLA-4 BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示。
二、CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-2所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CTLA-4 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CTLA-4-F和pcDNA3.1-CTLA-4-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗CTLA-4 scFv的基因序列；针对CD3-CTLA-4 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CTLA-4-F和pcDNA3.1-CTLA-4-R扩增出抗CD3 scFv、IgD铰链区、抗CTLA-4 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000248
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CTLA-4 BsAb_M和二聚体形式的CD3-CTLA-4 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CTLA-4 BsAb_M的核苷酸序列如SEQ ID NO.223所示，具体为：
Figure PCTCN2017096592-appb-000249
Figure PCTCN2017096592-appb-000250
具体地，二聚体形式的CD3-CTLA-4 BsAb_D的核苷酸序列如SEQ ID NO.225所示，具体为：
Figure PCTCN2017096592-appb-000251
Figure PCTCN2017096592-appb-000252
表4-2.CD3-CTLA-4双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000253
实施例4-7：CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D的表达与纯化
一、CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-6中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(BufferA)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-6所示。从图中可以看出，经Protein L亲和层析柱 纯化后，CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D重组蛋白的纯度均＞95％；其中CD3-CTLA-4 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-6A)；CD3-CTLA-4 BsAb_D重组蛋白的理论分子量为61.2kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-6B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CTLA-4 BsAb_M为单体形式，CD3-CTLA-4 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CTLA-4 BsAb_M的氨基酸序列如SEQ ID NO.222所示，具体为：
Figure PCTCN2017096592-appb-000254
二聚体形式的CD3-CTLA-4 BsAb_D的氨基酸序列如SEQ ID NO.224所示，具体为：
Figure PCTCN2017096592-appb-000255
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
抗CTLA-4 scFv的氨基酸序列如SEQ ID NO.248所示，具体为：
Figure PCTCN2017096592-appb-000256
抗CTLA-4 scFv的重链可变区的氨基酸序列如SEQ ID NO.249所示，具体为：
Figure PCTCN2017096592-appb-000257
抗CTLA-4 scFv的轻链可变区的氨基酸序列如SEQ ID NO.250所示，具体为：
Figure PCTCN2017096592-appb-000258
单体形式的CD3-CTLA-4 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示。
二聚体形式的CD3-CTLA-4 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示。
实施例4-8：ELISA检测CD3-CTLA-4 BsAb_M和CD3-CTLA-4 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CTLA-4-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CTLA-4 BsAb_M或CD3-CTLA-4 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-7A和4-7B所示：图4-7A说明CD3-CTLA-4 BsAb_M与重组抗原CD3-hFc和CTLA-4-hFc均具有体外结合活性，其中CTLA-4结合活性较CD3结合活性更高；图4-7B说明CD3-CTLA-4 BsAb_D与重组抗原CD3-hFc和CTLA-4-hFc同样具有体外结合活性，其中CTLA-4结合活性更高。
实施例4-9：CD3-CTLA-4双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CTLA-4 BsAb_M、二聚体形式的双特异抗体CD3-CTLA-4 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-CTLA-4 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CTLA-4 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的 密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4-8所示，单体和二聚体形式的CD3-CTLA-4双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-CTLA-4 BsAb_M或二聚体形式的CD3-CTLA-4 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-CTLA-4双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-10：CD3-CTLA-4双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-9中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测，三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-9所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-CTLA-4 BsAb_M培养的CIK细胞IFN-γ相对分泌量为1.94，溶液状态下添加二聚体形式的CD3-CTLA-4 BsAb_D培养的CIK细胞IFN-γ相对分泌量为2.85，因此本发明制备的两种形式的CD3-CTLA-4双特异抗体均更有利于活化CIK细胞，诱导IFN-γ的分泌，其中二聚体形式效果更佳。
实施例4-11：CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子LAG-3蛋白为靶点的双特异性抗体被命名为CD3-LAG-3 BsAb。
一、CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D构建方案设计
单体形式的CD3-LAG-3 BsAb_M具体构建方案为：抗CD3scFv和抗LAG-3 scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-LAG-3 BsAb_D具体构建方案为：抗CD3 scFv和抗LAG-3 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗LAG-3 scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示。
具体地，抗LAG-3 scFv的重链可变区的核苷酸序列如SEQ ID NO.273所示，具体为：
Figure PCTCN2017096592-appb-000259
具体地，抗LAG-3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.274所示，具体为：
Figure PCTCN2017096592-appb-000260
Figure PCTCN2017096592-appb-000261
具体地，抗LAG-3 scFv的核苷酸序列如SEQ ID NO.272所示，具体为：
Figure PCTCN2017096592-appb-000262
单体形式的CD3-LAG-3 BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示。
二聚体形式的CD3-LAG-3 BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示。
二、CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-3所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-LAG-3 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-LAG-3-F和pcDNA3.1-LAG-3-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗LAG-3 scFv的基因序列；针对CD3-LAG-3 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-LAG-3-F和pcDNA3.1-LAG-3-R扩增出抗CD3 scFv、IgD铰链区、抗LAG-3 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000263
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-LAG-3 BsAb_M和二聚体形式的CD3-LAG-3 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-LAG-3 BsAb_M的核苷酸序列如SEQ ID NO.227所示，具体为：
Figure PCTCN2017096592-appb-000264
具体地，二聚体形式的CD3-LAG-3 BsAb_D的核苷酸序列如SEQ ID NO.229所示，具体为：
Figure PCTCN2017096592-appb-000265
Figure PCTCN2017096592-appb-000266
表4-3.CD3-LAG-3双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000267
实施例4-12：CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D的表达与纯化
一、CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-11中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-10所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D重组蛋白的纯度均＞95％；其中CD3-LAG-3 BsAb_M重组蛋白的理论分子量为53.5kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-10A)；CD3-LAG-3 BsAb_D重组蛋白的理论分子量为61.4kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-10B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-LAG-3 BsAb_M为单体形式，CD3-LAG-3 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-LAG-3 BsAb_M的氨基酸序列如SEQ ID NO.226所示，具体为：
Figure PCTCN2017096592-appb-000268
二聚体形式的CD3-LAG-3 BsAb_D的氨基酸序列如SEQ ID NO.228所示，具体为：
Figure PCTCN2017096592-appb-000269
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
抗LAG-3 scFv的氨基酸序列如SEQ ID NO.251所示，具体为：
Figure PCTCN2017096592-appb-000270
抗LAG-3 scFv的重链可变区的氨基酸序列如SEQ ID NO.252所示，具体为：
Figure PCTCN2017096592-appb-000271
抗LAG-3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.253所示，具体为：
Figure PCTCN2017096592-appb-000272
单体形式的CD3-LAG-3 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示。
二聚体形式的CD3-LAG-3 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示。
实施例4-13：ELISA检测CD3-LAG-3 BsAb_M和CD3-LAG-3 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类LAG-3-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-LAG-3 BsAb_M或CD3-LAG-3 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-11A和图4-11B所示：图4-11A说明CD3-LAG-3 BsAb_M与重组抗原CD3-hFc和LAG-3-hFc均具有体外结合活性，其中LAG-3结合活性较CD3结合活性更高；图4-11B说明CD3-LAG-3 BsAb_D与重组抗原CD3-hFc和LAG-3-hFc同样具有体外结合活性，其中LAG-3结合活性更高。
实施例4-14：CD3-LAG-3双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-LAG-3 BsAb_M、二聚体形式的双特异抗体CD3-LAG-3 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心 20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-LAG-3 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-LAG-3 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线。
检测结果如图4-12所示，单体和二聚体形式的CD3-LAG-3双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-LAG-3 BsAb_M或二聚体形式的CD3-LAG-3 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-LAG-3双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-15：CD3-LAG-3双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-14中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测，三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-13所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-LAG-3 BsAb_M培养的CIK细胞IFN-γ相对分泌量为2.25，溶液状态下添加二聚体形式的CD3-LAG-3 BsAb_D培养的CIK细胞IFN-γ相对分泌量为3.37，因此本发明制备的两种形式的CD3-LAG-3双特异抗体均更有利于活化CIK细胞，诱导IFN-γ的分泌，其中二聚体形式效果更佳。
实施例4-16：CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子TIM-3蛋白为靶点的双特异性抗体被命名为CD3-TIM-3BsAb。
一、CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D构建方案设计
单体形式的CD3-TIM-3 BsAb_M具体构建方案为：抗CD3 scFv和抗TIM-3 scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-TIM-3 BsAb_D具体构建方案为：抗CD3 scFv和抗TIM-3 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗TIM-3 scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示。
具体地，抗TIM-3 scFv的重链可变区的核苷酸序列如SEQ ID NO.276所示，具体为：
Figure PCTCN2017096592-appb-000273
具体地，抗TIM-3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.277所示，具体为：
Figure PCTCN2017096592-appb-000274
具体地，抗TIM-3 scFv的核苷酸序列如SEQ ID NO.275所示，具体为：
Figure PCTCN2017096592-appb-000275
单体形式的CD3-TIM-3 BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示。
二聚体形式的CD3-TIM-3 BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示。
二、CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-4所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-TIM-3 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-TIM-3-F和pcDNA3.1-TIM-3-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗TIM-3 scFv的基因序列；针 对CD3-TIM-3 BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-TIM-3-F和pcDNA3.1-TIM-3-R扩增出抗CD3 scFv、IgD铰链区、抗TIM-3 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000276
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-TIM-3 BsAb_M和二聚体形式的CD3-TIM-3 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-TIM-3 BsAb_M的核苷酸序列如SEQ ID NO.231所示，具体为：
Figure PCTCN2017096592-appb-000277
具体地，二聚体形式的CD3-TIM-3 BsAb_D的核苷酸序列如SEQ ID NO.233所示。具体为：
Figure PCTCN2017096592-appb-000278
Figure PCTCN2017096592-appb-000279
表4-4.CD3-TIM-3双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000280
实施例4-17：CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D的表达与纯化
一、CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-16中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-14所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D重组蛋白的纯度均＞95％；其中CD3-TIM-3 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-14A)；CD3-TIM-3 BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-14B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-TIM-3 BsAb_M为单体形式，CD3-TIM-3 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-TIM-3 BsAb_M的氨基酸序列如SEQ ID NO.230所示，具体为：
Figure PCTCN2017096592-appb-000281
二聚体形式的CD3-TIM-3 BsAb_D的氨基酸序列如SEQ ID NO.232所示，具体为：
Figure PCTCN2017096592-appb-000282
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
抗TIM-3 scFv的氨基酸序列如SEQ ID NO.254所示，具体为：
Figure PCTCN2017096592-appb-000283
抗TIM-3 scFv的重链可变区的氨基酸序列如SEQ ID NO.255所示，具体为：
Figure PCTCN2017096592-appb-000284
抗TIM-3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.256所示，具体为：
Figure PCTCN2017096592-appb-000285
单体形式的CD3-TIM-3 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示。
二聚体形式的CD3-TIM-3 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示。
实施例4-18：ELISA检测CD3-TIM-3 BsAb_M和CD3-TIM-3 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类TIM-3-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-TIM-3 BsAb_M或CD3-TIM-3 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-15A和图4-15B所示：图4-15A说明CD3-TIM-3 BsAb_M与重组抗原CD3-hFc和TIM-3-hFc均具有体外结合活性，其中TIM-3结合活性较CD3结合活性更高；图4-15B说明CD3-TIM-3 BsAb_D与重组抗原CD3-hFc和TIM-3-hFc同样具有体外结合活性，其中TIM-3结合活性更高。
实施例4-19：CD3-TIM-3双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-TIM-3 BsAb_M、二聚体形式的双特异抗体CD3-TIM-3 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-TIM-3 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-TIM-3 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4-16所示，单体和二聚体形式的CD3-TIM-3双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-TIM-3 BsAb_M或二聚体形式的CD3-TIM-3 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-TIM-3双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-20：CD3-TIM-3双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-19中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测，三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-17所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-TIM-3 BsAb_M培养的CIK细胞IFN-γ相对分泌量为2.07，溶液状态下添加二聚体形式的CD3-TIM-3 BsAb_D培养的CIK细胞IFN-γ相对分泌量为3.04，因此本发明制备的两种形式的CD3-TIM-3双特异抗体均更有利 于活化CIK细胞，诱导IFN-γ的分泌，其中二聚体形式效果更佳。
实施例4-21：CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子TIGIT蛋白为靶点的双特异性抗体被命名为CD3-TIGIT BsAb。
一、CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D构建方案设计
单体形式的CD3-TIGIT BsAb_M具体构建方案为：抗CD3 scFv和抗TIGIT scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-TIGIT BsAb_D具体构建方案为：抗CD3 scFv和抗TIGIT scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗TIGIT scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示。
具体地，抗TIGIT scFv的重链可变区的核苷酸序列如SEQ ID NO.279所示，具体为：
Figure PCTCN2017096592-appb-000286
具体地，抗TIGIT scFv的轻链可变区的核苷酸序列如SEQ ID NO.280所示，具体为：
Figure PCTCN2017096592-appb-000287
具体地，抗TIGIT scFv的核苷酸序列如SEQ ID NO.278所示，具体为：
Figure PCTCN2017096592-appb-000288
单体形式的CD3-TIGIT BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示。
二聚体形式的CD3-TIGIT BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示。
二、CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-5所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-TIGIT BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-TIGIT-F和pcDNA3.1-TIGIT-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗TIGIT scFv的基因序列；针对CD3-TIGIT BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-TIGIT-F和pcDNA3.1-TIGIT-R扩增出抗CD3 scFv、IgD铰链区、抗TIGIT scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000289
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-TIGIT BsAb_M和二聚体形式的CD3-TIGIT BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-TIGIT BsAb_M的核苷酸序列如SEQ ID NO.235所示，具体为：
Figure PCTCN2017096592-appb-000290
Figure PCTCN2017096592-appb-000291
具体地，二聚体形式的CD3-TIGIT BsAb_D的核苷酸序列如SEQ ID NO.237所示，具体为：
Figure PCTCN2017096592-appb-000292
表4-5.CD3-TIGIT双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000293
实施例4-22：CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D的表达与纯化
一、CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-21中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-18所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D重组蛋白的纯度均＞95％；其中CD3-TIGIT BsAb_M重组蛋白的理论分子量为54.0kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-18A)；CD3-TIGIT BsAb_D 重组蛋白的理论分子量为61.9kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-18B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-TIGIT BsAb_M为单体形式，CD3-TIGIT BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-TIGIT BsAb_M的氨基酸序列如SEQ ID NO.234所示，具体为：
Figure PCTCN2017096592-appb-000294
二聚体形式的CD3-TIGIT BsAb_D的氨基酸序列如SEQ ID NO.236所示，具体为：
Figure PCTCN2017096592-appb-000295
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
抗TIGIT scFv的氨基酸序列如SEQ ID NO.257所示，具体为：
Figure PCTCN2017096592-appb-000296
抗TIGIT scFv的重链可变区的氨基酸序列如SEQ ID NO.258所示，具体为：
Figure PCTCN2017096592-appb-000297
抗TIGIT scFv的轻链可变区的氨基酸序列如SEQ ID NO.259所示，具体为：
Figure PCTCN2017096592-appb-000298
单体形式的CD3-TIGIT BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示。
二聚体形式的CD3-TIGIT BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示。
实施例4-23：ELISA检测CD3-TIGIT BsAb_M和CD3-TIGIT BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类TIGIT-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/LNaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-TIGIT BsAb_M或CD3-TIGIT BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-19A和图4-19B所示：图4-19A说明CD3-TIGIT BsAb_M与重组抗原CD3-hFc和TIGIT-hFc均具有体外结合活性，其中TIGIT结合活性较CD3结合活性更高；图4-19B说明CD3-TIGIT BsAb_D与重组抗原CD3-hFc和TIGIT-hFc同样具有体外结合活性，其中TIGIT结合活性更高。
实施例4-24：CD3-TIGIT双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-TIGIT BsAb_M、二聚体形式的双特异抗体CD3-TIGIT BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD285ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-TIGIT BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-TIGIT BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4-20所示，单体和二聚体形式的CD3-TIGIT双特异性抗体单一使用对 CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-TIGIT BsAb_M或二聚体形式的CD3-TIGIT BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-TIGIT双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-25：CD3-TIGIT双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-24中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测，三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-21所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-TIGIT BsAb_M培养的CIK细胞IFN-γ相对分泌量为1.66，溶液状态下添加二聚体形式的CD3-TIGIT BsAb_D培养的CIK细胞IFN-γ相对分泌量为2.30，因此本发明制备的两种形式的CD3-TIGIT双特异抗体均更有利于活化CIK细胞，诱导IFN-γ的分泌，其中二聚体形式效果更佳。
实施例4-26：CD3-BTLA BsAb_M和CD3-BTLA BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞负共刺激分子BTLA蛋白为靶点的双特异性抗体被命名为CD3-BTLA BsAb。
一、CD3-BTLA BsAb_M和CD3-BTLA BsAb_D构建方案设计
单体形式的CD3-BTLA BsAb_M具体构建方案为：抗CD3 scFv和抗BTLA scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-BTLA BsAb_D具体构建方案为：抗CD3 scFv和抗BTLA scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗BTLA scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.264所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.265所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.263所示。
具体地，抗BTLA scFv的重链可变区的核苷酸序列如SEQ ID NO.282所示，具体为：
Figure PCTCN2017096592-appb-000299
具体地，抗BTLA scFv的轻链可变区的核苷酸序列如SEQ ID NO.283所示，具体为：
Figure PCTCN2017096592-appb-000300
Figure PCTCN2017096592-appb-000301
具体地，抗BTLA scFv的核苷酸序列如SEQ ID NO.281所示，具体为：
Figure PCTCN2017096592-appb-000302
单体形式的CD3-BTLA BsAb_M连接片段的核苷酸序列如SEQ ID NO.209所示。
二聚体形式的CD3-BTLA BsAb_D连接片段的核苷酸序列如SEQ ID NO.211所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.284所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.285所示。
二、CD3-BTLA BsAb_M和CD3-BTLA BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4-6所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-BTLA BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-BTLA-F和pcDNA3.1-BTLA-R扩增出抗CD3 scFv、(GGGGS)3Linker、抗BTLA scFv的基因序列；针对CD3-BTLA BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-BTLA-F和pcDNA3.1-BTLA-R扩增出抗CD3 scFv、IgD铰链区、抗BTLA scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096592-appb-000303
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-BTLA BsAb_M和二聚体形式的CD3-BTLA BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-BTLA BsAb_M的核苷酸序列如SEQ ID NO.239所示，具体为：
Figure PCTCN2017096592-appb-000304
Figure PCTCN2017096592-appb-000305
具体地，二聚体形式的CD3-BTLA BsAb_D的核苷酸序列如SEQ ID NO.241所示，具体为：
Figure PCTCN2017096592-appb-000306
Figure PCTCN2017096592-appb-000307
表4-6.CD3-BTLA双特异性抗体基因克隆中使用的引物
Figure PCTCN2017096592-appb-000308
实施例4-27：CD3-BTLA BsAb_M和CD3-BTLA BsAb_D的表达与纯化
一、CD3-BTLA BsAb_M和CD3-BTLA BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力＞90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-BTLA BsAb_M和CD3-BTLA BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-26中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-BTLA BsAb_M和CD3-BTLA BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine，pH3.0
缓冲液C(Buffer C)：0.1M Glycine，pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris，pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-BTLA BsAb_M和CD3-BTLA BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-22所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-BTLA BsAb_M和CD3-BTLA BsAb_D重组蛋白的纯度均＞95％；其中CD3-BTLA BsAb_M重组蛋白的理论分子量为53.1kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图4-22A)；CD3-BTLA BsAb_D重组蛋白的理论分子量为61.0kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图4-22B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-BTLA BsAb_M为单体形式，CD3-BTLA BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-BTLA BsAb_M的氨基酸序列如SEQ ID NO.238所示，具体为：
Figure PCTCN2017096592-appb-000309
二聚体形式的CD3-BTLA BsAb_D的氨基酸序列如SEQ ID NO.240所示，具体为：
Figure PCTCN2017096592-appb-000310
抗CD3 scFv的氨基酸序列如SEQ ID NO.242所示。
抗CD3scFv的重链可变区的氨基酸序列如SEQ ID NO.243所示。
抗CD3scFv的轻链可变区的氨基酸序列如SEQ ID NO.244所示。
抗BTLA scFv的氨基酸序列如SEQ ID NO.260所示，具体为：
Figure PCTCN2017096592-appb-000311
抗BTLA scFv的重链可变区的氨基酸序列如SEQ ID NO.261所示，具体为：
Figure PCTCN2017096592-appb-000312
抗BTLA scFv的轻链可变区的氨基酸序列如SEQ ID NO.262所示，具体为：
Figure PCTCN2017096592-appb-000313
单体形式的CD3-BTLA BsAb_M中连接片段的氨基酸序列如SEQ ID NO.208所示。
二聚体形式的CD3-BTLA BsAb_D中连接片段的氨基酸序列如SEQ ID NO.210所示。
实施例4-28：ELISA检测CD3-BTLA BsAb_M和CD3-BTLA BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类BTLA-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃ 1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/LNaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-BTLA BsAb_M或CD3-BTLA BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图4-23A和图4-23B所示：图4-23A说明CD3-BTLA BsAb_M与重组抗原CD3-hFc和BTLA-hFc均具有体外结合活性，其中BTLA结合活性较CD3结合活性更高；图4-23B说明CD3-BTLA BsAb_D与重组抗原CD3-hFc和BTLA-hFc同样具有体外结合活性，其中BTLA结合活性更高。
实施例4-29：CD3-BTLA双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-BTLA BsAb_M、二聚体形式的双特异抗体CD3-BTLA BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心 20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-BTLA BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-BTLA BsAb_D 10ng/ml)。此外，三组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4-24所示，单体和二聚体形式的CD3-BTLA双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用，培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-BTLA BsAb_M或二聚体形式的CD3-BTLA BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-BTLA双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例4-30：CD3-BTLA双特异抗体诱导的CIK细胞IFN-γ分泌
操作步骤：
1、取实施例4-29中培养25天后的CIK细胞上清(调整到相同细胞密度，细胞数目为2×105个)100μl，37℃孵育45min，通过Human IFN-γELISA Kit(购自博士德生物)进行检测，三组实验每组取三个样品重复；
2、用PBS清洗三次，添加HRP标记的IFN-γ抗体，37℃孵育45min；
3、用PBS清洗三次，添加TMB 100μl显色，室温显色5-10min；
4、添加终止液HCl(1M)终止，450nm波长下读取吸光值。
结果如图4-25所示：其中Anti-CD3/Anti-CD28全长抗体联用培养的CIK细胞所分泌的IFN-γ数量定义为1，溶液状态下添加单体形式的CD3-BTLA BsAb_M培养的CIK细胞IFN-γ相对分泌量为1.54，溶液状态下添加二聚体形式的CD3-BTLA BsAb_D培养的CIK细胞IFN-γ相对分泌量为2.24，因此本发明制备的两种形式的CD3-BTLA双特异抗体均更有利于活化CIK细胞，诱导IFN-γ的分泌，其中二聚体形式效果更佳。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (19)

  1. 一种双功能分子，所述双功能分子选自以下之任一：(1)所述双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域；(2)所述双功能分子的结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并阻断T细胞负共刺激分子的第二功能域。
  2. 根据权利要求1所述的双功能分子，其特征在于：所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号；或者
    所述双功能分子能够在结合并激活T细胞表面CD3分子的同时结合并阻断T细胞负共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
  3. 根据权利要求1所述的双功能分子，其特征在于：所述第一功能域为抗CD3的抗体，所述第二功能域为抗T细胞正共刺激分子的抗体或T细胞正共刺激分子的配体胞外区结构域；或者
    所述第一功能域为抗CD3的抗体，所述第二功能域为抗T细胞负共刺激分子的抗体。
  4. 根据权利要求3所述的双功能分子，其特征在于，所述抗体选自Fab抗体、Fv抗体或单链抗体。
  5. 根据权利要求1所述的双功能分子，其特征在于，所述第一功能域和所述第二功能域通过连接片段连接。
  6. 根据权利要求5所述的双功能分子，其特征在于，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
  7. 根据权利要求6所述的双功能分子，其特征在于，以G4S为单位的连接片段的氨基酸序列如SEQ ID NO.17所示；免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.19所示。
  8. 根据权利要求1所述的双功能分子，其特征在于：所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗T细胞正共刺激分子的单链抗体或T细胞正共刺激分子的配体胞外区结构域，所述单链抗体包括重链可变区和轻链可变区；或者
    所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗T细胞负共刺激分子的单链抗体，所述单链抗体包括重链可变区和轻链可变区。
  9. 根据权利要求8所述的双功能分子，其特征在于，所述抗T细胞正共刺激分子的单链抗体选自抗CD28的单链抗体、抗4-1BB的单链抗体、抗ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一；所述T细胞正共刺激分子的配体胞外区结构域选自4-1BBL胞外区结构域、B7RP-1胞外区结构域、OX40L胞外区结构域、GITRL胞外区结构域或CD70胞外区结构域之任一；所述抗T细胞负共刺激分子的单链抗体选自抗PD-1的单链抗体、抗CTLA-4的单链抗体、抗LAG-3的单链抗体、抗TIM-3的单链抗体、抗TIGIT的单链抗体或抗BTLA的单链抗体之任一。
  10. 根据权利要求9所述的双功能分子，其特征在于，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.6所示；所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.7所示；所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示；所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10所示；所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.71所示；所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.72所示；所述抗ICOS的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.74所示；所述抗ICOS的单链抗 体的轻链可变区的氨基酸序列如SEQ ID NO.75所示；所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.77所示；所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.78所示；所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.80所示；所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.81所示；所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.83所示；所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.84所示；所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.86所示；所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.87所示；所述抗PD-1的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.246所示；所述抗PD-1的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.247所示；所述抗CTLA-4的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.249所示；所述抗CTLA-4的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.250所示；所述抗LAG-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.252所示；所述抗LAG-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.253所示；所述抗TIM-3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.255所示；所述抗TIM-3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.256所示；所述抗TIGIT的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.258所示；所述抗TIGIT的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.259所示；所述抗BTLA的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.261所示；所述抗BTLA的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.262所示。
  11. 根据权利要求9所述的双功能分子，其特征在于，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.5所示；所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8所示；所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.70所示；所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.73所示；所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.76所示；所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.79所示；所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.82所示；所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.85所示；所述4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.172所示；所述B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.173所示；所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.174所示；所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.175所示；所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.176所示；所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.245所示；所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.248所示；所述抗LAG-3的单链抗体的氨基酸序列如SEQ ID NO.251所示；所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.254所示；所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.257所示；所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.260所示。
  12. 根据权利要求1所述的双功能分子，其特征在于，所述双功能分子的氨基酸序列如SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.43、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.55、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.61、SEQ ID NO.63、SEQ ID NO.65、SEQ ID NO.149、SEQ ID NO.151、SEQ ID NO.153、SEQ ID NO.155、SEQ ID NO.157、SEQ ID NO.159、SEQ ID NO.161、SEQ ID NO.163、SEQ ID NO.165、SEQ ID NO.167或SEQ ID NO.218、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.226、SEQ ID NO.228、SEQ ID NO.230、SEQ ID NO.232、SEQ ID NO.234、SEQ ID NO.236、SEQ ID NO.238、或SEQ ID NO.240之任一所示。
  13. 一种多核苷酸，其编码如权利要求1～12任一项所述双功能分子。
  14. 一种表达载体，其含有如权利要求13所述的多核苷酸。
  15. 一种宿主细胞，其被如权利要求14所述的表达载体所转化。
  16. 如权利要求1～12任一项所述双功能分子的制备方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。
  17. 如权利要求1～12任一项所述双功能分子用于制备T细胞体外扩增剂的用途。
  18. 一种T细胞体外扩增剂，含有如权利要求1～12任一项所述双功能分子。
  19. 一种体外扩增T细胞的方法，包括步骤：将如权利要求1～12任一项所述双功能分子作用于T细胞。
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