WO2018120843A1 - 一种三功能分子及其应用 - Google Patents

一种三功能分子及其应用 Download PDF

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WO2018120843A1
WO2018120843A1 PCT/CN2017/096594 CN2017096594W WO2018120843A1 WO 2018120843 A1 WO2018120843 A1 WO 2018120843A1 CN 2017096594 W CN2017096594 W CN 2017096594W WO 2018120843 A1 WO2018120843 A1 WO 2018120843A1
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seq
amino acid
acid sequence
set forth
chain antibody
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PCT/CN2017/096594
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WO2018120843A9 (zh
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陈帅
朱化星
廖远平
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上海近岸生物科技有限公司
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Priority claimed from CN201611260817.8A external-priority patent/CN108264561B/zh
Priority claimed from CN201611258691.0A external-priority patent/CN108264559B/zh
Priority claimed from CN201611258643.1A external-priority patent/CN106589129B/zh
Priority claimed from CN201611256659.9A external-priority patent/CN108264558B/zh
Application filed by 上海近岸生物科技有限公司 filed Critical 上海近岸生物科技有限公司
Priority to US16/474,555 priority Critical patent/US11535666B2/en
Priority to EP17888652.9A priority patent/EP3564265A4/en
Publication of WO2018120843A1 publication Critical patent/WO2018120843A1/zh
Publication of WO2018120843A9 publication Critical patent/WO2018120843A9/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/2818—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a trifunctional molecule and an application thereof.
  • the human CD19 antigen is a transmembrane glycoprotein of 95kDa, which belongs to the immunoglobulin superfamily. In addition to its expression on the surface of normal B lymphocytes, CD19 is also highly expressed in B cell malignancies. Therefore, the anti-CD19 monoclonal full-length antibody has been Developed for the treatment of acute/chronic lymphocytic leukemia and B cell lymphoma (Wang K et al, Experimental Hematology & Oncology, 1:36-42, 2012).
  • CD3/CD8 double positive T cells can specifically recognize antigenic peptide/MHC class I complexes on the surface of target cells, self-activation After the release of perforin (Peforin), the target cells rupture and death, can also secrete cytotoxin and granzyme (Granzyme) and other DNA damage caused by the target cell nucleus, causing apoptosis of target cells.
  • CTLs Cytotoxic T lymphocytes
  • BsAb Bi-specific antibody
  • CAR-T Chimeric antigen receptor T-cell immunotherapy
  • bispecific antibody targeting CD19 is an anti-CD19/anti-CD3 bispecific T cell engager (BiTE), and its structure is two single-chain antibodies (Single The -chain variable fragment (scFv) domain is covalently linked by a flexible linker fragment (Linker) (Goebeler ME et al, Leukemia & Lymphoma, 57: 1021-1032, 2016).
  • Linker flexible linker fragment
  • the TCR/CD3 complex on the surface of CD8-positive T cells and the endogenous antigen peptide/MHC class I complex on the surface of antigen-presenting cells (APC) are specifically recognized.
  • CD3 This leads to the interaction of CD3 with the cytoplasmic segment of the co-receptor CD8, activating the protein tyrosine kinase linked to the tail of the cytoplasmic segment, and making the immunoreceptor tyrosine-based activation motif of the CD3 cytoplasmic region.
  • Tyrosine phosphorylation in ITAM initiates a signaling cascade that activates transcription factors that allow T cells to be initially activated.
  • Anti-CD19/anti-CD3 BiTE bispecific antibody has the binding activity of human CD3 and CD19 antigens, can form a cell junction between T cells and tumor B cells, and simultaneously gives T cell initial activation signal, which enhances its tumor cells. Killing targeting.
  • the BiTE bispecific antibody does not have the Fc fragment of the full-length antibody, and the protein has a small molecular weight ( ⁇ 54 kDa), so it can cross the hematuria barrier and the cerebral blood barrier during tumor treatment, and has low bioavailability, requiring continuous intravenous injection. It is administered with certain neurotoxicity.
  • T cells In humans requires a dual signaling pathway (Baxter AG et al, Nature Reviews Immunology, 2: 439-446, 2002).
  • the antigenic peptide-MHC molecular complex on the surface of APC cells interacts with the TCR/CD3 complex on the surface of T cells to generate a first signal, allowing T cells to be initially activated, followed by costimulatory ligands on the surface of APC cells (eg CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1, PD-L2, Galectin-9, and HVEM, etc.)
  • Co-stimulatory molecules such as CD28 corresponding to the surface of T cells.
  • CD28 4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, BTLA, etc. interact to produce a second signal (co-stimulatory signal): where CD28 4-1BB, ICOS, OX40, GITR, CD40L, and CD27 are positive co-stimulatory molecules, which are produced by interaction with the corresponding ligands (CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD70, etc.).
  • the second signal (positive costimulatory signal) can lead to complete activation of T cells; while CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, and BTLA are negative co-stimulatory molecules, and the corresponding ligands (CD80)
  • the second signal (negative co-stimulatory signal) generated by the interaction of CD86, PD-L1, PD-L2, Galectin-9, HVEM, etc. is mainly down-regulated and terminated. The activation. Studies have shown that only the first signaling pathway can not fully activate T cells, which in turn leads to its loss. It can even produce activation induced cell death (AICD).
  • a bispecific antibody against a tumor antigen/anti-T cell positive (negative) costimulatory molecule can be used in combination with an anti-tumor antigen/anti-CD3 bispecific antibody to enhance T cell activation and tumors.
  • Cell killing efficiency Jung G et al, Int J Cancer, 91: 225-230, 2001; Kodama H et al, Immunol Lett, 81: 99-106, 2002.
  • this method has many inconveniences in the actual operation process, for example, it increases the workload and production cost of recombinant bispecific antibody expression and purification, and the relative proportion of the two bispecific antibodies needs to be optimized when activating the expanded T cells.
  • CAR-T technology can better solve the problem of T cell activation.
  • the construction of CAR typically includes a tumor-associated antigen binding region (eg, a CD19 antigen binding region, typically derived from an anti-CD19 monoclonal full-length antibody scFv fragment), an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
  • the intracellular signal region is responsible for mediating the activation of T cells.
  • the first stimulation signal is completed by the tyrosine activation motif on the CD3 ⁇ chain.
  • the expansion of the first stimulation signal is achieved by the CD28 costimulatory signal, and the T is promoted.
  • CAR-T technology itself also has some shortcomings: First, the technology relies on virus transfection to genetically engineer T cells, which is cumbersome and requires high experimental conditions. Secondly, it needs to be activated in vitro after specific use. When CAR-T cells are returned to the patient, the dose is more difficult to control the antibody. In addition, the rapid increase in the number of CAR-T cells entering the patient can lead to Cytokine storm, which is excessive in a short period of time. Cytokines that cause side effects such as high fever, low pressure, shock, and even death.
  • a trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding and activating a T cell surface CD3 molecule, and capable of binding and activating a T cell surface The third domain of the CD28 molecule.
  • the trifunctional molecule is capable of binding to and activating the T cell surface CD3 molecule and the CD28 molecule while recognizing CD19, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD19 antibody
  • the second functional domain is an anti-CD3 antibody
  • the third 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 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third Hinge between functional domains by immunoglobulin IgD
  • the strand fragments are joined, and the hinge region fragment of the immunoglobulin IgD is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is shown in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain is linked to the N-terminus of the third functional domain.
  • the first functional domain is a single chain antibody against CD19
  • the second domain is a single chain antibody against CD3
  • the third domain is a single chain antibody against CD28, the single chain antibody A heavy chain variable region and a light chain variable region are included.
  • the amino acid sequence of the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • 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 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-CD19 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 amino acid sequence of the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the trifunctional molecule in monomeric form is also set forth in SEQ ID NO.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is set forth in SEQ ID NO.
  • a trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and capable of binding and activating T cells The third functional domain of the co-stimulatory molecule.
  • the trifunctional molecule is capable of binding to and activating T cell surface CD3 molecules and T cell positive costimulatory molecules while recognizing CD19, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an antibody against CD19
  • the second functional domain is an anti-CD3 antibody
  • the third 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 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is shown in SEQ ID NO.
  • the connecting fragment 2 can be connected to each other by a disulfide bond Form a dimer.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain is linked to the N-terminus of the third functional domain.
  • the first functional domain is a single chain antibody against CD19
  • the second domain is a single chain antibody against CD3
  • the third domain is a single chain antibody against a T cell positive costimulatory molecule
  • the single chain antibody includes a heavy chain variable region and a light chain variable region.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • 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 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-CD19 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 trifunctional molecule in monomeric form is SEQ ID NO. 59, SEQ ID NO. 63, SEQ ID NO. 67, SEQ ID NO. 71, SEQ ID NO. 75 or SEQ. Shown as any of ID NO.79.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is SEQ ID NO. 61, SEQ ID NO. 65, SEQ ID NO. 69, SEQ ID NO. 73, SEQ ID NO. Shown as any of SEQ ID NO. 81.
  • a trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding and activating a T cell surface CD3 molecule, and capable of binding and activating T cells The third functional domain of the co-stimulatory molecule.
  • the trifunctional molecule is capable of binding to and activating T cell surface CD3 molecules and T cell positive costimulatory molecules while binding to CD19, thereby generating a first signal and a second signal required for T cell activation.
  • the first functional domain is an anti-CD19 antibody
  • the second functional domain is an anti-CD3 antibody
  • the third 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 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is set forth in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain is linked to the N-terminus of the third functional domain.
  • the first functional domain is a single chain antibody against CD19
  • the single chain antibody against CD19 comprises a heavy chain variable region and a light chain variable region.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the second 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-CD19 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 third functional domain is a ligand extracellular domain domain of a T cell positive costimulatory molecule.
  • 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 monomeric form of the trifunctional molecule is as defined in SEQ ID NO. 177, SEQ ID NO. 181, SEQ ID NO. 185, SEQ ID NO. 189 or SEQ ID NO. One is shown.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is as set forth in any one of SEQ ID NO. 179, SEQ ID NO. 183, SEQ ID NO. 187, SEQ ID NO. 191 or SEQ ID NO.
  • a trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding and activating a T cell surface CD3 molecule, and capable of binding and blocking T Cell negative spur The third functional domain of the molecule.
  • the trifunctional molecule is capable of binding to and activating a T cell surface CD3 molecule, binding and blocking a T cell negative costimulatory molecule while recognizing CD19, thereby generating a first signal and a second signal required for T cell activation. .
  • the first functional domain is an antibody against CD19
  • the second functional domain is an anti-CD3 antibody
  • the third 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 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is set forth in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the C-terminus of the first functional domain is linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain is linked to the N-terminus of the third functional domain.
  • the first functional domain is a single chain antibody against CD19
  • the second domain is a single chain antibody against CD3
  • the third domain is a single chain antibody against a T cell negative costimulatory molecule
  • the single chain antibody includes a heavy chain variable region and a light chain variable region.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • 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 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-CD19 single chain antibody is set forth in SEQ ID NO. 282.
  • 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-PD-1 single chain antibody 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 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 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 monomeric form of the trifunctional molecule is SEQ ID NO. 258, SEQ ID NO. 262, SEQ ID NO. 266, SEQ ID NO. 270, SEQ ID NO. 274 or SEQ. Shown as any of ID NO.278.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is any one of SEQ ID NO. 260, SEQ ID NO. 264, SEQ ID NO. 268, SEQ ID NO. 272, SEQ ID NO. 276 or SEQ ID NO. Shown.
  • a polynucleotide encoding the aforementioned trifunctional 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 trifunctional molecule comprises: constructing an expression vector containing a gene sequence of a trifunctional molecule, and then transforming an expression vector containing a gene sequence of the trifunctional molecule into a host cell to induce expression, The trifunctional 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 trifunctional molecule for the preparation of a medicament for the treatment of a tumor.
  • a therapeutic composition for tumor therapy comprising the aforementioned trifunctional molecule and at least one pharmaceutically acceptable carrier or excipient.
  • the tumor is a tumor whose cell surface is CD19 positive.
  • a method of treating a tumor in vitro comprising administering the aforementioned trifunctional molecule or tumor therapeutic pharmaceutical composition to a tumor patient.
  • the method can be for non-therapeutic purposes.
  • the tumor is a tumor whose cell surface is CD19 positive.
  • the present invention has the following beneficial effects:
  • the present invention is capable of binding to a first domain of CD19, a second domain capable of binding and activating a T cell surface CD3 molecule, and a third domain capable of binding and activating a T cell surface CD28 molecule fused to the same protein peptide
  • the chain is produced by eukaryotic cell expression system, 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 use is convenient; and the anti-CD19/anti-CD3 bispecific antibody and the anti-CD19/anti-CD28 bispecific If the antibodies are used in combination, the two bispecific antibodies need to be separately expressed and purified, the preparation process is more complicated, the workload and production cost are significantly increased, and the relative proportions of the two are optimized.
  • the trifunctional molecule of the invention is capable of generating a second stimulation signal of T cell activation, and further enhances the activation effect on T cells while imparting T cell targeting, thereby increasing secretion of cytokines and anti-apoptotic proteins, thereby effectively avoiding
  • the phenomenon of T cell inability and death, the mediated T cell binding and killing of CD19-positive target cells can achieve or even better than the anti-CD19/anti-CD3 BiTE bispecific antibody, and the amount of protein is less.
  • the trifunctional molecule of the present invention does not involve the steps of virus-mediated transgene, in vitro T cell culture and reinfusion, and is more convenient to use and dose controllable, after entering the patient's body. The risk of causing excessive release of cytokines is small, avoiding the side effects of using CAR-T.
  • the present invention also binds to the first functional domain of CD19, the second functional domain capable of binding and activating T cell surface CD3 molecules, and the third functional domain capable of binding and activating T cell positive costimulatory molecules.
  • Protein peptide chain Produced by eukaryotic cell expression system 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 convenient to use; and the anti-CD19/anti-CD3 bispecific antibody is positively stimulated with anti-CD19/anti-T cells. If the molecular bispecific antibody is used in combination, the two bispecific antibodies need to be separately expressed and purified, the preparation process is more complicated, the workload and production cost are significantly increased, and the relative proportion of the two needs to be optimized.
  • the trifunctional molecule of the present invention is capable of generating a second (positive) stimulation signal for T cell activation, further enhancing the activation effect on T cells, conferring cytokines and anti-apoptosis while imparting T cell targeting.
  • the increase of protein secretion effectively avoids the phenomenon of T cell insufficiency and death.
  • the mediated T cell killing of CD19 positive target cells can achieve or even better than the anti-CD19/anti-CD3 BiTE bispecific antibody, and the protein dosage less.
  • the trifunctional molecule of the present invention does not involve the steps of virus-mediated transgene, in vitro T cell culture and reinfusion, and is more convenient to use and dose controllable, after entering the patient's body. The risk of causing excessive release of cytokines is small, avoiding the side effects of using CAR-T.
  • the present invention also binds to the first functional domain of CD19, the second functional domain capable of binding and activating T cell surface CD3 molecules, and the third functional domain capable of binding and activating T cell positive costimulatory molecules.
  • the protein peptide chain forms a trifunctional molecule and is produced by a eukaryotic cell expression system.
  • the expression product has a single structure, a simple purification process, high protein yield, stable preparation process and product, and convenient use; and anti-CD19/anti-CD3 bispecific antibody and anti-CD CD19/anti-T cell positive costimulatory molecule bispecific antibody If used in combination, the two bispecific antibodies need to be separately expressed and purified, the preparation process is more complicated, the workload and production cost are significantly increased, and the relative need of the two should be optimized.
  • the trifunctional molecule of the present invention is capable of generating a second (positive) stimulation signal for T cell activation, further enhancing the activation effect on T cells, conferring cytokines and anti-apoptosis while imparting T cell targeting.
  • the increase of protein secretion effectively avoids the phenomenon of T cell insufficiency and death.
  • the mediated T cell killing of CD19 positive target cells can achieve or even better than the anti-CD19/anti-CD3 BiTE bispecific antibody, and the protein dosage less.
  • the trifunctional molecule of the present invention does not involve the steps of virus-mediated transgene, in vitro T cell culture and reinfusion, and is more convenient to use, and the dosage is controllable, and enters the patient's body.
  • the risk of causing excessive release of cytokines is small, avoiding the side effects of using CAR-T.
  • the present invention is also capable of binding to a first domain of CD19, a second domain capable of binding and activating a T cell surface CD3 molecule, and a third domain capable of binding and blocking a T cell negative costimulatory molecule.
  • the same protein peptide chain is produced by eukaryotic cell expression system, and the expression product has a single structure, simple purification process, high protein yield, stable preparation process and product, and convenient use; and anti-CD19/anti-CD3 bispecific antibody and anti-CD19/anti-antibody If the T cell positive (negative) costimulatory molecule bispecific antibody is used in combination, the two bispecific antibodies need to be separately expressed and purified, the preparation process is more complicated, the workload and production cost are significantly increased, and the relative ratio of the two needs to be optimized.
  • the trifunctional molecule of the present invention can block or down-regulate the second (negative) stimulation signal of T cell activation, further enhances the activation effect on T cells, and makes cytokines and antibiotics while imparting T cell targeting. Increased secretion of apoptotic proteins effectively prevents T cell inability and death, and the mediated T cell killing of CD19-positive target cells can achieve or even better than anti-CD19/anti-CD3 BiTE bispecific antibodies. And the amount of protein is less.
  • the trifunctional molecule of the present invention does not involve the steps of virus-mediated transgene, in vitro T cell culture and reinfusion, and is more convenient to use and dose controllable, after entering the patient's body. The risk of causing excessive release of cytokines is small, avoiding the side effects of using CAR-T.
  • Figure 1-1 A. Structural map of the monomeric form of anti-CD19/CD3/anti-CD28 trispecific antibody (CD19-CD3-CD28 TsAb_M); B. Dimeric form of anti-CD19/anti-CD3/anti-CD28 trispecific A structural diagram of the antibody (CD19-CD3-CD28 TsAb_D).
  • Figure 1-2 A. Purified CD19-CD3-CD28 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD19-CD3-CD28 TsAb_M; lane 3: non-reducing CD19-CD3- CD28 TsAb_M; B. Purified CD19-CD3-CD28 TsAb_D SDS-PAGE analysis map; Lane 1: molecular weight protein Marker; Lane 2: reducing CD19-CD3-CD28 TsAb_D; Lane 3: non-reducing CD19-CD3-CD28 TsAb_D.
  • Figure 1-3A ELISA identification of CD19-CD3-CD28 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 results of CD19-CD3-CD28 TsAb_D; the curves in the figure represent 4 test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 Trispecific antibody and bispecific antibody-mediated cell-binding experiments. Raji lymphoma cells were used as CD19-positive target cells, and Jurkat cells were used as CD3 and CD28-positive effector cells to detect different concentrations of CD19-CD3.
  • Figure 1-5A Trispecific antibody and bispecific antibody-mediated cell killing experiments, using Raji lymphoma cells as CD19-positive target cells, and CIK (Cytokine induced killer) cells as CD3 and CD28-positive killer effect cells, respectively
  • Figure 1-5B Trispecific antibody and bispecific antibody-mediated cell killing experiments.
  • Figure 2-1 A Structural map of the monomeric form of anti-CD19/anti-CD3/anti-T cell positive costimulatory molecule trispecific antibody; B. Dimeric form of anti-CD19/anti-CD3/anti-T cell positive costimulatory molecule A structural map of a trispecific antibody.
  • Figure 2-2 A. Purified CD19-CD3-4-1BB TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BB TsAb_M; lane 3: non-reducing CD19-CD3-4-1BBTsAb_M; B. Purified CD19-CD3-4-1BB TsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BB TsAb_D; lane 3: Non-reducing CD19-CD3-4-1BB TsAb_D.
  • Figure 2-3A ELISA identification of CD19-CD3-4-1BB TsAb_M, the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g / ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 2-3B ELISA identification of CD19-CD3-4-1BB TsAb_D, the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g / ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 2-5 A. Purified CD19-CD3-ICOS TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-ICOS TsAb_M; lane 3: non-reducing CD19-CD3- ICOS TsAb_M; B. Purified CD19-CD3-ICOS TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD19-CD3-ICOS TsAb_D; lane 3: non-reducing CD19-CD3-ICOS TsAb_D .
  • Figure 2-6A ELISA identification of CD19-CD3-ICOS TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-6B ELISA identification of CD19-CD3-ICOS TsAb_D, the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-7 CD19-CD3-ICOS trispecific antibody-mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-.
  • Figure 2-8 A. Purified CD19-CD3-OX40 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-OX40 TsAb_M; lane 3: non-reducing CD19-CD3- OX40 TsAb_M; B. Purified CD19-CD3-OX40 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: non-reducing CD19-CD3-OX40 TsAb_D; lane 3: reducing CD19-CD3-OX40 TsAb_D .
  • Figure 2-9A ELISA identification of CD19-CD3-OX40 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-9B ELISA identification of CD19-CD3-OX40 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-10 CD19-CD3-OX40 trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-OX40 TsAb_M, CD19-CD3-OX40 TsAb_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • Figure 2-11 A. Purified CD19-CD3-GITR TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-GITR TsAb_M; lane 3: non-reducing CD19-CD3- GITR TsAb_M; B. Purified CD19-CD3-GITR TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-GITR TsAb_D; lane 3: non-reducing CD19-CD3-GITR TsAb_D .
  • Figure 2-12A ELISA identification of CD19-CD3-GITR TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-12B ELISA identification of CD19-CD3-GITR TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-13 CD19-CD3-GITR trispecific antibody-mediated cell killing assay using Raji lymphoma cells as CD19-positive target cells, CIK cells as CD3-positive killer effector cells, were tested for the killing efficiency of CIK cells against Raji cells mediated by different concentrations of CD19-CD3-GITR TsAb_M, CD19-CD3-GITR TsAb_D and CD19-CD3 BsAb, respectively.
  • Figure 2-14 A. Purified CD19-CD3-CD40L TsAb_M SDS-PAGE analysis, Lane 1: molecular weight protein Marker; Lane 2: reducing CD19-CD3-CD40L TsAb_M; Lane 3: non-reducing CD19-CD3- CD40L TsAb_M; B. Purified CD19-CD3-CD40L TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CD40L TsAb_D; lane 3: non-reducing CD19-CD3-CD40L TsAb_D .
  • Figure 2-15A ELISA identification of CD19-CD3-CD40L TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-15B ELISA identification of CD19-CD3-CD40L TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-17 A. Purified CD19-CD3-CD27 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CD27 TsAb_M; lane 3: non-reducing CD19-CD3- CD27 TsAb_M; B. Purified CD19-CD3-CD27 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CD27 TsAb_D; lane 3: non-reducing CD19-CD3-CD27 TsAb_D .
  • Figure 2-18A ELISA identification of CD19-CD3-CD27 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 2-18B ELISA identification of CD19-CD3-CD27 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 3-1 A. Structure of the monomeric form of anti-CD19/anti-CD3/T cell positive costimulatory molecule ligand TsM; B. Dimeric form of anti-CD19/anti-CD3/T cell positive costimulatory molecule ligand TsM Structure diagram.
  • Figure 3-2 A. Purified CD19-CD3-4-1BBL TsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BBL TsM_M; lane 3: non-reducing CD19-CD3-4-1BBL TsM_M; B. Purified CD19-CD3-4-1BBL TsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BBL TsM_D; lane 3 : Non-reducing CD19-CD3-4-1BBL TsM_D.
  • Figure 3-3A ELISA identification of CD19-CD3-4-1BBL TsM_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen 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 CD19-CD3-4-1BBL TsM_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3 -hFc; ⁇ coated with 1 ⁇ g / ml recombinant protein 4-1BB-hFc; Does not coat the results of any protein assay.
  • Figure 3-4 CD19-CD3-4-1BBL trispecific molecular mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK (Cytokine induced killer) cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-4-1BBL TsM_M, CD19-CD3-4-1BBL TsM_D and CD19.
  • CIK Cytokine induced killer
  • Figure 3-5 A. Purified CD19-CD3-B7RP-1 TsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-B7RP-1 TsM_M; lane 3: non-reducing CD19-CD3-B7RP-1 TsM_M; B. Purified CD19-CD3-B7RP-1 TsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-B7RP-1 TsM_D; lane 3 : Non-reducing CD19-CD3-B7RP-1 TsM_D.
  • Figure 3-6A ELISA identification of CD19-CD3-B7RP-1 TsM_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3 -hFc; ⁇ coated with 1 ⁇ g / ml recombinant protein ICOS-hFc; Does not coat the results of any protein assay.
  • Figure 3-6B ELISA identification of CD19-CD3-B7RP-1 TsM_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3 -hFc; ⁇ coated with 1 ⁇ g / ml recombinant protein ICOS-hFc; Does not coat the results of any protein assay.
  • Figure 3-7 CD19-CD3-B7RP-1 trispecific molecular mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-B7RP-1 TsM_M, CD19-CD3-B7RP-1 TsM_D and CD19-CD3 BsAb.
  • the killing efficiency of the guided CIK cells against Raji cells; effector cells: target cells (E:T ratio) 1:1, killing time: 3 h.
  • Figure 3-8 A. Purified CD19-CD3-OX40L TsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-OX40L TsM_M; lane 3: non-reducing CD19-CD3- OX40L TsM_M; B. Purified CD19-CD3-OX40L TsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-OX40L TsM_D; lane 3: non-reducing CD19-CD3-OX40L TsM_D .
  • Figure 3-9A ELISA identification results of CD19-CD3-OX40L TsM_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen 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 CD19-CD3-OX40L TsM_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc ; ⁇ coated with 1 ⁇ g / ml recombinant protein OX40-hFc; Does not coat the results of any protein assay.
  • Figure 3-10 CD19-CD3-OX40L trispecific molecular mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-OX40L TsM_M, CD19-CD3-OX40L TsM_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • Figure 3-11 A. Purified CD19-CD3-GITRL TsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-GITRL TsM_M; lane 3: non-reducing CD19-CD3- GITRL TsM_M; B. Purified CD19-CD3-GITRL TsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-GITRL TsM_D; lane 3: non-reducing CD19-CD3-GITRL TsM_D .
  • Figure 3-12A ELISA identification of CD19-CD3-GITRL TsM_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen 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 CD19-CD3-GITRL TsM_D, the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc ; ⁇ coated with 1 ⁇ g / ml recombinant protein GITR-hFc; Does not coat the results of any protein assay.
  • Figure 3-13 CD19-CD3-GITRL trispecific molecular mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-GITRL TsM_M, CD19-CD3-GITRL TsM_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • Figure 3-14 A. Purified CD19-CD3-CD70 TsM_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD19-CD3-CD70 TsM_M; lane 3: non-reducing CD19-CD3- CD70 TsM_M; B. Purified CD19-CD3-CD70 TsM_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CD70 TsM_D; lane 3: non-reducing CD19-CD3-CD70 TsM_D .
  • Figure 3-15A ELISA identification of CD19-CD3-CD70 TsM_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen 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 CD19-CD3-CD70 TsM_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ coated with 1 ⁇ g/ml recombinant antigen CD3-hFc ; ⁇ coated with 1 ⁇ g / ml recombinant protein CD27-hFc; Does not coat the results of any protein assay.
  • Figure 3-16 CD19-CD3-CD70 trispecific molecular mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-CD70 TsM_M, CD19-CD3-CD70 TsM_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • Figure 4-1 A. Structural map of the monomeric form of anti-CD19/anti-CD3/anti-T cell negative costimulatory molecule trispecific antibody; B. Dimeric form of anti-CD19/anti-CD3/anti-T cell negative costimulatory molecule A structural map of a trispecific antibody.
  • Figure 4-2 A. Purified CD19-CD3-PD-1 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-PD-1 TsAb_M; lane 3: non-reducing CD19-CD3-PD-1 TsAb_M; B. Purified CD19-CD3-PD-1 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-PD-1 TsAb_D; lane 3 : Non-reducing CD19-CD3-PD-1 TsAb_D.
  • Figure 4-3A ELISA identification of CD19-CD3-PD-1 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-3B ELISA identification of CD19-CD3-PD-1 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-4 CD19-CD3-PD-1 trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK (Cytokine induced killer) cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-PD-1 TsAb_M, CD19-CD3-PD-1 TsAb_D and CD19.
  • CIK Cytokine induced killer
  • Figure 4-5 A. Purified CD19-CD3-CTLA-4 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CTLA-4 TsAb_M; lane 3: non-reducing CD19-CD3-CTLA-4 TsAb_M; B. Purified CD19-CD3-CTLA-4 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-CTLA-4 TsAb_D; lane 3 : Non-reducing CD19-CD3-CTLA-4 TsAb_D.
  • Figure 4-6A ELISA identification of CD19-CD3-CTLA-4 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-6B ELISA identification of CD19-CD3-CTLA-4 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-7 CD19-CD3-CTLA-4 trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-CTLA-4 TsAb_M, CD19-CD3-CTLA-4 TsAb_D and CD19-CD3 BsAb.
  • the killing efficiency of the guided CIK cells against Raji cells; effector cells: target cells (E:T ratio) 1:1, killing time: 3 h.
  • Figure 4-8 A. Purified CD19-CD3-LAG-3 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-LAG-3 TsAb_M; lane 3: non-reducing CD19-CD3-LAG-3 TsAb_M; B. Purified CD19-CD3-LAG-3 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-LAG-3 TsAb_D; lane 3 : Non-reducing CD19-CD3-LAG-3 TsAb_D.
  • Figure 4-9A ELISA identification of CD19-CD3-LAG-3 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-9B ELISA identification of CD19-CD3-LAG-3 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-10 CD19-CD3-LAG-3 trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-LAG-3 TsAb_M, CD19-CD3-LAG-3 TsAb_D and CD19-CD3 BsAb.
  • the killing efficiency of the guided CIK cells against Raji cells; effector cells: target cells (E:T ratio) 1:1, killing time: 3 h.
  • Figure 4-11 A. Purified CD19-CD3-TIM-3 TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-TIM-3 TsAb_M; lane 3: non-reducing CD19-CD3-TIM-3 TsAb_M; B. Purified CD19-CD3-TIM-3 TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-TIM-3 TsAb_D; lane 3 : Non-reducing CD19-CD3-TIM-3 TsAb_D.
  • Figure 4-12A ELISA identification of CD19-CD3-TIM-3 TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-12B ELISA identification of CD19-CD3-TIM-3 TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 determination.
  • Figure 4-13 CD19-CD3-TIM-3 trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect different concentrations of CD19-CD3-TIM-3 TsAb_M, CD19-CD3-TIM-3 TsAb_D and CD19-CD3 BsAb.
  • the killing efficiency of the guided CIK cells against Raji cells; effector cells: target cells (E:T ratio) 1:1, killing time: 3 h.
  • Figure 4-14 A. Purified CD19-CD3-TIGIT TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-TIGIT TsAb_M; lane 3: non-reducing CD19-CD3- TIGIT TsAb_M; B. Purified CD19-CD3-TIGIT TsAb_D SDS-PAGE analysis map, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-TIGIT TsAb_D; lane 3: non-reducing CD19-CD3-TIGIT TsAb_D.
  • Figure 4-15A ELISA identification of CD19-CD3-TIGIT TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 4-15B ELISA identification of CD19-CD3-TIGIT TsAb_D, the curves in the figure represent 4 test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 4-16 CD19-CD3-TIGIT trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-TIGIT TsAb_M, CD19-CD3-TIGIT TsAb_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • Figure 4-17 A. Purified CD19-CD3-BTLA TsAb_M SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reduced CD19-CD3-BTLA TsAb_M; lane 3: non-reducing CD19-CD3- BTLA TsAb_M; B. Purified CD19-CD3-BTLA TsAb_D SDS-PAGE analysis, lane 1: molecular weight protein Marker; lane 2: reducing CD19-CD3-BTLA TsAb_D; lane 3: non-reducing CD19-CD3-BTLA TsAb_D .
  • Figure 4-18A ELISA identification of CD19-CD3-BTLA TsAb_M.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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 4-18B ELISA identification of CD19-CD3-BTLA TsAb_D.
  • the curves in the figure represent four test results: ⁇ coated with 1 ⁇ g/ml recombinant antigen CD19-hFc; ⁇ 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.
  • FIG. 4-19 CD19-CD3-BTLA trispecific antibody mediated cell killing assay.
  • Raji lymphoma cells were used as CD19-positive target cells, and CIK cells were used as CD3-positive killer effector cells to detect CIK cells mediated by different concentrations of CD19-CD3-BTLA TsAb_M, CD19-CD3-BTLA TsAb_D and CD19-CD3 BsAb.
  • Killing efficiency against Raji cells; effector cells: target cells (E:T ratio) 1:1, kill time: 3 h.
  • BsAb Bi-specific Antibody
  • TsAb Tri-specific Antibody
  • TsM Tri-specific Molecule
  • BiTE Bi-specific T cell engager
  • 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
  • Co-stimulatory molecule co-stimulatory molecule
  • CD19-CD3-CD28 TsAb_M monomeric form of anti-CD19/anti-CD3/anti-CD28 trispecific antibody
  • CD19-CD3-CD28 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-CD28 trispecific antibody
  • CD19-CD3-4-1BB TsAb_M Monoclonal form of anti-CD19/anti-CD3/anti-4-1BB trispecific antibody
  • CD19-CD3-4-1BB TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-4-1BB trispecific antibody
  • CD19-CD3-ICOS TsAb_M anti-CD19/anti-CD3/anti-ICOS trispecific antibody in monomeric form
  • CD19-CD3-ICOS TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-ICOS trispecific antibody
  • CD19-CD3-OX40 TsAb_M anti-CD19/anti-CD3/anti-OX40 trispecific antibody in monomeric form
  • CD19-CD3-OX40 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-OX40 trispecific antibody
  • CD19-CD3-GITR TsAb_M monomeric form of anti-CD19/anti-CD3/anti-GITR trispecific antibody
  • CD19-CD3-GITR TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-GITR trispecific antibody
  • CD19-CD3-CD40L TsAb_M monomeric form of anti-CD19/anti-CD3/anti-CD40L trispecific antibody
  • CD19-CD3-CD40L TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-CD40L trispecific antibody
  • CD19-CD3-CD27 TsAb_M anti-CD19/anti-CD3/anti-CD27 trispecific antibody in monomeric form
  • CD19-CD3-CD27 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-CD27 trispecific antibody
  • 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
  • CD19-CD3-4-1BBL TsM_M Monomeric form of anti-CD19/anti-CD3/4-1BBL trispecific molecule
  • CD19-CD3-4-1BBL TsM_D a dimeric form of anti-CD19/anti-CD3/4-1BBL trispecific molecule
  • CD19-CD3-B7RP-1 TsM_M Monomeric form of anti-CD19/anti-CD3/B7RP-1 trispecific molecule
  • CD19-CD3-B7RP-1 TsM_D a dimeric form of anti-CD19/anti-CD3/B7RP-1 trispecific molecule
  • CD19-CD3-OX40L TsM_M Monomeric form of anti-CD19/anti-CD3/OX40L trispecific molecule
  • CD19-CD3-OX40L TsM_D a dimeric form of anti-CD19/anti-CD3/OX40L trispecific molecule
  • CD19-CD3-GITRL TsM_M Monomeric form of anti-CD19/anti-CD3/GITRL trispecific molecule
  • CD19-CD3-GITRL TsM_D a dimeric form of anti-CD19/anti-CD3/GITRL trispecific molecule
  • CD19-CD3-CD70 TsM_M Monomeric form of anti-CD19/anti-CD3/CD70 trispecific molecule
  • CD19-CD3-CD70 TsM_D a dimeric form of anti-CD19/anti-CD3/CD70 trispecific molecule
  • CD19-CD3-PD-1 TsAb_M anti-CD19/anti-CD3/anti-PD-1 trispecific antibody in monomeric form
  • CD19-CD3-PD-1 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-PD-1 trispecific antibody
  • CD19-CD3-CTLA-4 TsAb_M monomeric form of anti-CD19/anti-CD3/anti-CTLA-4 trispecific antibody
  • CD19-CD3-CTLA-4 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-CTLA-4 trispecific antibody
  • CD19-CD3-LAG-3 TsAb_M monomeric form of anti-CD19/anti-CD3/anti-LAG-3 trispecific antibody
  • CD19-CD3-LAG-3 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-LAG-3 trispecific antibody
  • CD19-CD3-TIM-3 TsAb_M Monoclonal form of anti-CD19/anti-CD3/anti-TIM-3 trispecific antibody
  • CD19-CD3-TIM-3 TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-TIM-3 trispecific antibody
  • CD19-CD3-TIGIT TsAb_M monomeric form of anti-CD19/anti-CD3/anti-TIGIT trispecific antibody
  • CD19-CD3-TIGIT TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-TIGIT trispecific antibody
  • CD19-CD3-BTLA TsAb_M monomeric form of anti-CD19/anti-CD3/anti-BTLA trispecific antibody
  • CD19-CD3-BTLA TsAb_D a dimeric form of anti-CD19/anti-CD3/anti-BTLA trispecific antibody.
  • a trifunctional molecule of the invention comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a third capable of binding and activating a T cell surface CD28 molecule Functional domain.
  • the trifunctional molecule is capable of binding to and activating the T cell surface CD3 molecule and the CD28 molecule while recognizing CD19, 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, the second functional domain, and the third functional domain, as long as it can bind and activate the T cell surface CD3 molecule and the CD28 molecule while recognizing CD19, thereby generating T cell activation.
  • the first signal and the second signal are sufficient.
  • the first functional domain can be an anti-CD19 antibody
  • the second functional domain can be an anti-CD3 antibody
  • the third 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 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 segment 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • 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 linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain may be linked to the N-terminus of the third functional domain.
  • the present invention is also not particularly limited to the ligation fragment 1 and the ligation fragment 2 as long as it does not limit the object of the present invention.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is shown in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the schematic structure of the trifunctional molecule is shown in Figure 1-1.
  • the trifunctional molecule may be in a monomeric form or a dimeric form.
  • a schematic diagram of the structure of the monomeric form of the trifunctional molecule of the present invention is shown in Figure 1-1A.
  • the structure of the trifunctional molecule contains a first functional domain that binds to the CD19 antigen and a second function that binds to the CD3 antigen. Domain, a third domain that binds to a CD28 antigen, the first domain is a single chain antibody (scFv) that binds to a CD19 antigen, and the second domain is a single chain antibody (scFv) that binds to a CD3 antigen.
  • the third domain is a single chain antibody (scFv) that binds to the CD28 antigen.
  • scFv single chain antibody
  • the dimeric form of the trifunctional molecule of the invention has an antigen binding potency that is twice that of the monomeric form.
  • T cell activation Due to the doubling of the first signal (CD3) and the second signal (CD28) of T cell activation, T cell activation is more sufficient, and the killing effect on target cells is stronger; the CD19 single-chain antibody domain is doubled to target cells.
  • the identification is also more precise, so the dimer has a better use effect than the monomer.
  • the first functional domain is a single chain antibody against CD19.
  • the anti-CD19 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-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD19 single-chain antibody is shown in SEQ ID NO.
  • the second 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 third 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 monomeric form of the trifunctional molecule is set forth in SEQ ID NO.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is set forth in SEQ ID NO.
  • Another trifunctional molecule of the invention comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a T cell positive costimulatory molecule capable of binding and activating T cells.
  • the third functional domain comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a T cell positive costimulatory molecule capable of binding and activating T cells.
  • the trifunctional molecule is capable of binding to and activating a T cell surface CD3 molecule and a T cell positive costimulatory molecule while recognizing CD19, 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, the second functional domain, and the third functional domain, as long as it can bind and activate T cell surface CD3 molecules and T cell positive costimulatory molecules while recognizing CD19, thereby producing T cells.
  • the first signal and the second signal required for activation may be used.
  • the first functional domain can be an antibody against CD19
  • the second functional domain can be an anti-CD3 antibody
  • the third 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 segment 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited. For example, it may be that the C-terminus of the first functional domain is connected to the N-terminus of the second functional domain; The C-terminus of the second functional domain is linked to the N-terminus of the third functional domain.
  • the present invention is also not particularly limited to the ligation fragment 1 and the ligation fragment 2 as long as it does not limit the object of the present invention.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is shown in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the schematic structure of the trifunctional molecule is shown in Figure 2-1.
  • the trifunctional molecule may be in a monomeric form or a dimeric form.
  • a schematic diagram of the structure of the monomeric form of the trifunctional molecule of the present invention is shown in Figure 2-1A.
  • the structure of the trifunctional molecule contains a first functional domain that binds to the CD19 antigen and a second function that binds to the CD3 antigen.
  • scFv single chain antibody
  • scFv single chain antibody
  • scFv single-chain antibody
  • a second domain two third domains that bind to a T cell positive co-stimulatory molecule antigen
  • the first domain being a single chain antibody (scFv) that binds to a CD19 antigen
  • the second domain is a CD3
  • An antigen-binding single-chain antibody (scFv) which is a single-chain antibody (scFv) that binds to a T cell positive costimulatory molecule antigen.
  • the dimeric form of the trifunctional molecule of the invention has an antigen binding potency that is twice that of the monomeric form.
  • T cell activation first signal CD3
  • T cell positive costimulatory molecule T cell activation second signal
  • CD19 single chain antibody domain doubles It also makes the recognition of target cells more precise, so the dimer has better use effect than the monomer.
  • 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. 52, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the human 4-1BB extracellular domain is shown in SEQ ID NO. 53, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human ICOS is shown in SEQ ID NO. 54, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the human OX40 extracellular domain is set forth in SEQ ID NO. 55, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human GITR is shown in SEQ ID NO. 56, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human CD40L is shown in SEQ ID NO. 57, specifically:
  • T cell positive costimulatory molecule The amino acid sequence of the extracellular domain of human CD27 is shown in SEQ ID NO. 58, specifically:
  • the first functional domain is a single chain antibody against CD19.
  • the anti-CD19 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-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD19 single-chain antibody is shown in SEQ ID NO.
  • the second 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 third 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 anti-anti The amino acid sequence of the light chain variable region of the single chain antibody of CD27 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 trifunctional molecule in monomeric form is SEQ ID NO. 59, SEQ ID NO. 63, SEQ ID NO. 67, SEQ ID NO. 71, SEQ ID NO. 75 or SEQ. Shown as any of ID NO.79.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is any one of SEQ ID NO. 61, SEQ ID NO. 65, SEQ ID NO. 69, SEQ ID NO. 73, SEQ ID NO. 77 or SEQ ID NO. Shown. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • Another trifunctional molecule of the invention comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a T cell positive costimulatory molecule capable of binding and activating T cells.
  • the third functional domain comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a T cell positive costimulatory molecule capable of binding and activating T cells.
  • the trifunctional molecule is capable of binding to and activating a T cell surface CD3 molecule and a T cell positive costimulatory molecule while binding to CD19, 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 4-1BB, ICOS, OX40, GITR, CD40L or CD27.
  • the present invention has no particular limitation on the first functional domain, the second functional domain, and the third functional domain, as long as it can bind and activate T cell surface CD3 molecules and T cell positive costimulatory molecules while recognizing CD19, thereby producing T cells.
  • the first signal and the second signal required for activation may be used.
  • the first functional domain may be an antibody against CD19
  • the second functional domain may be an anti-CD3 antibody
  • the third functional domain may be a ligand extracellular 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 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 segment 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • 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 linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain may be linked to the N-terminus of the third functional domain.
  • the present invention is also not particularly limited to the ligation fragment 1 and the ligation fragment 2 as long as it does not limit the object of the present invention.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is set forth in SEQ ID NO.
  • the connecting fragment 2 can be linked to each other by a disulfide bond Join to form a dimer.
  • the schematic structure of the trifunctional molecule is shown in Figure 3-1.
  • the trifunctional molecule may be in a monomeric form or a dimeric form.
  • the schematic diagram of the structure of the monomeric trifunctional molecule of the present invention is shown in A of Figure 3-1, and the structure of the trifunctional molecule contains a first functional domain that binds to the CD19 antigen, and a binding to the CD3 antigen.
  • a second domain, a third domain that binds to a T cell positive costimulatory molecule the first domain is a single chain antibody (scFv) that binds to a CD19 antigen, and the second domain is a CD3 antigen.
  • scFv single chain antibody
  • a single-chain antibody which is a ligand extracellular domain of a T cell positive costimulatory molecule.
  • a schematic diagram of the structure of the dimeric form of the trifunctional molecule of the present invention is shown in B of Figure 3-1, the structure of the trifunctional molecule contains two first functional domains that bind to the CD19 antigen, and two with the CD3 antigen.
  • a second domain of binding a third domain that binds to a T cell positive costimulatory molecule
  • the first domain is a single chain antibody (scFv) that binds to a CD19 antigen
  • the second domain is A CD3 antigen-binding single-chain antibody (scFv), which is a ligand extracellular domain domain of a T cell positive costimulatory molecule.
  • the dimeric form of the trifunctional molecule of the invention has an antigen binding potency that is twice that of the monomeric form.
  • T cell activation Due to the doubling of the first signal (CD3) and the second signal (positive costimulatory signal) of T cell activation, T cell activation is more sufficient, and the killing effect on target cells is stronger; doubling of CD19 scFv domain makes it target Cell identification is also more precise, so dimers have better use than monomers.
  • the T cell positive costimulatory molecule may be human 4-1BB (UniProt ID: Q07011), the amino acid sequence is shown in SEQ ID NO. 167, 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 as SEQ ID NO. 169, the ligand is human B7RP-1 (UniProt ID: O75144), and the amino acid sequence is SEQ ID. Shown in NO.170.
  • the T cell positive costimulatory molecule may be human OX40 (UniProt ID: P43489), the amino acid sequence is shown as SEQ ID NO. 171, the ligand is human OX40L (UniProt ID: P23510), and the amino acid sequence is SEQ ID NO. 172 Shown.
  • the T cell positive costimulatory molecule may be human GITR (UniProt ID: Q9Y5U5), the amino acid sequence is shown as SEQ ID NO. 173, the ligand is human GITRL (UniProt ID: Q9UNG2), and the amino acid sequence is SEQ ID NO. 174 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. 175, the ligand is human CD70 (UniProt ID: P32970), and the amino acid sequence is SEQ ID NO. 176 shows.
  • the first functional domain is a single chain antibody against CD19.
  • the anti-CD19 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-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD19 single-chain antibody is shown in SEQ ID NO.
  • the second 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 third 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 Any of the CD70 extracellular domain domains.
  • 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 trifunctional molecule is as defined in SEQ ID NO. 177, SEQ ID NO. 181, SEQ ID NO. 185, SEQ ID NO. 189 or SEQ ID NO.
  • One is shown.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is as set forth in any one of SEQ ID NO. 179, SEQ ID NO. 183, SEQ ID NO. 187, SEQ ID NO. 191 or SEQ ID NO. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • Another trifunctional molecule of the invention comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a T cell negative costimulatory molecule capable of binding and blocking The third functional domain.
  • the trifunctional molecule is capable of binding to and activating a T cell surface CD3 molecule, binding and blocking a T cell negative costimulatory molecule while recognizing CD19, 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, the second functional domain, and the third functional domain, as long as it can bind and activate the T cell surface CD3 molecule, bind and block the T cell negative costimulatory molecule while recognizing CD19.
  • the first signal and the second signal required for T cell activation can be produced.
  • the first functional domain can be an antibody against CD19
  • the second functional domain can be an anti-CD3 antibody
  • the third 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 segment 1, and the second functional domain and the third functional domain are connected by a connection segment 2.
  • 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 linked to the N-terminus of the second functional domain; the C-terminus of the second functional domain may be linked to the N-terminus of the third functional domain.
  • the present invention is also not particularly limited to the ligation fragment 1 and the ligation fragment 2 as long as it does not limit the object of the present invention.
  • the ligation fragment 1 and the ligation fragment 2 are 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.
  • a bifunctional molecule in a monomeric form is exemplified, wherein the first functional domain and the second functional domain are linked by a linker 1 in a G4S unit, the second functional domain and the third functional function.
  • the domains are connected by a connected segment 2 in units of G4S.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the ligated fragment 2 contains three G4S units, and 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 linker 1 in units of G4S, a second functional domain and a third
  • the functional domains are joined by a hinge region fragment of immunoglobulin IgD, which is the hinge Ala90-Val170 of immunoglobulin IgD.
  • the ligation fragment 1 contains a G4S unit, and the amino acid sequence of the ligation fragment is shown in SEQ ID NO.
  • the amino acid sequence of the ligated fragment 2 is set forth in SEQ ID NO.
  • the linking fragment 2 can be linked to each other by a disulfide bond to form a dimer.
  • the schematic structure of the trifunctional molecule is shown in Figure 4-1.
  • the trifunctional molecule may be in a monomeric form or a dimeric form.
  • the schematic diagram of the structure of the monomeric trifunctional molecule of the present invention is shown in A of Figure 4-1.
  • the structure of the trifunctional molecule contains a first functional domain that binds to the CD19 antigen, and a binding to the CD3 antigen.
  • a single-chain antibody (scFv) which is a single-chain antibody (scFv) that binds to a T cell negative costimulatory molecule antigen.
  • a schematic diagram of the structure of the dimeric form of the trifunctional molecule of the present invention is shown in B of Figure 4-1. The structure of the trifunctional molecule contains two first functional domains that bind to the CD19 antigen, and two with the CD3 antigen.
  • the first domain is a single chain antibody (scFv) that binds to a CD19 antigen
  • the second domain is A single-chain antibody (scFv) that binds to a CD3 antigen, which is a single-chain antibody (scFv) that binds to a T cell negative costimulatory molecule antigen.
  • the dimeric form of the trifunctional molecule of the invention has an antigen binding potency that is twice that of the monomeric form.
  • T cell activation Due to the doubling of the T-cell activation first signal (CD3) and the second signal (negative co-stimulatory signal blocked), T cell activation is more sufficient, and the killing effect on target cells is stronger; CD19 single-chain antibody domain Doubling makes the recognition of target cells more precise, so dimers have better use than monomers.
  • the T cell negative costimulatory molecules may be PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, 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. 252, 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. 253, 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. 254, 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. 255, 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. 256, 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. 257, specifically:
  • the first functional domain is a single chain antibody against CD19.
  • the anti-CD19 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-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-CD19 single-chain antibody is shown in SEQ ID NO.
  • the second 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 third 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 , a single-chain antibody against TIGIT or a single-chain antibody against BTLA.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the light chain variable region of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the anti-PD-1 single chain antibody is set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 single chain antibody 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 monomeric form of the trifunctional molecule is SEQ ID NO. 258, SEQ ID NO. 262, SEQ ID NO. 266, SEQ ID NO. 270, SEQ ID NO. 274 or SEQ. Shown as any of ID NO.278.
  • the amino acid sequence of the dimeric form of the trifunctional molecule is any one of SEQ ID NO. 260, SEQ ID NO. 264, SEQ ID NO. 268, SEQ ID NO. 272, SEQ ID NO. 276 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 trifunctional 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 trifunctional 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-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD19 is shown in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region encoding 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 of the single-chain antibody encoding the anti-CD3 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 encoding the anti-CD28 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD28 is shown in SEQ ID NO.
  • the nucleotide sequence of the ligated fragment 1 having the amino acid sequence shown in SEQ ID NO. 23 is shown in SEQ ID NO.
  • nucleotide sequence of the ligated fragment 2 having the amino acid sequence shown in SEQ ID NO. 25 is shown in SEQ ID NO.
  • the nucleotide sequence of the ligated fragment 1 encoding the amino acid sequence shown in SEQ ID NO. 27 is shown in SEQ ID NO.
  • the nucleotide sequence of the ligated fragment 2 encoding the amino acid sequence shown in SEQ ID NO. 29 is shown in SEQ ID NO.
  • nucleotide sequence encoding the trifunctional molecule in monomeric form is set forth in SEQ ID NO.
  • nucleotide sequence encoding the trifunctional molecule in the dimeric form is set forth in SEQ ID NO.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD19 is shown in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the 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 of the single-chain antibody encoding the anti-CD3 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. Coding the anti-antibody
  • the nucleotide sequence of the 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.
  • the nucleotide sequence of the ligated fragment 1 encoding the amino acid sequence shown in SEQ ID NO. 44 is shown in SEQ ID NO.
  • the nucleotide sequence of the ligated fragment 2 encoding the amino acid sequence shown in SEQ ID NO. 46 is shown in SEQ ID NO.
  • nucleotide sequence of the ligated fragment 1 having the amino acid sequence shown in SEQ ID NO. 48 is shown in SEQ ID NO.
  • the nucleotide sequence of the ligated fragment 2 encoding the amino acid sequence shown in SEQ ID NO. 50 is shown in SEQ ID NO.
  • nucleotide sequence encoding the trifunctional molecule in monomeric form is SEQ ID NO. 60, SEQ ID NO. 64, SEQ ID NO. 68, SEQ ID NO. 72, SEQ ID NO. 76 or SEQ ID NO. Any of the .80 is shown.
  • a nucleotide sequence encoding a trifunctional molecule in the form of a dimer is SEQ ID NO. 62, SEQ ID NO. 66, SEQ ID NO. 70, SEQ ID NO. 74, SEQ ID NO. 78 or SEQ ID NO. Either shown.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD19 is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the 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 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. 159 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 161 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 163 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the ligated fragment of the amino acid sequence set forth in SEQ ID NO. 165 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the trifunctional molecule in monomeric form is as set forth in any one of SEQ ID NO. 178, SEQ ID NO. 182, SEQ ID NO. 186, SEQ ID NO. 190 or SEQ ID NO. Show.
  • the nucleotide sequence encoding the trifunctional molecule in the dimeric form is SEQ ID NO. 180, SEQ ID NO. 184, SEQ ID NO. 188, SEQ ID Shown as any of NO. 192 or SEQ ID NO.
  • nucleotide sequence encoding the heavy chain variable region of the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region encoding the anti-CD19 single chain antibody is set forth in SEQ ID NO.
  • nucleotide sequence of the single-chain antibody encoding the anti-CD19 is set forth in SEQ ID NO.
  • the nucleotide sequence of the heavy chain variable region encoding the anti-CD3 single chain antibody is set forth in SEQ ID NO.
  • the 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 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.
  • nucleotide sequence of ligated fragment 1 encoding the amino acid sequence set forth in SEQ ID NO. 244 is set forth in SEQ ID NO.
  • nucleotide sequence of the ligated fragment 2 encoding the amino acid sequence shown in SEQ ID NO. 246 is set forth in SEQ ID NO.
  • nucleotide sequence of ligated fragment 1 encoding the amino acid sequence set forth in SEQ ID NO. 248 is set forth in SEQ ID NO.
  • nucleotide sequence of the ligated fragment 2 encoding the amino acid sequence shown in SEQ ID NO. 250 is set forth in SEQ ID NO.
  • nucleotide sequence encoding the trifunctional molecule in monomeric form is SEQ ID NO. 259, SEQ ID NO. 263, SEQ ID NO. 267, SEQ ID NO. 271, SEQ ID NO. 275 or SEQ ID NO. Any of the .279 is shown.
  • a nucleotide sequence encoding a trifunctional molecule in the form of a dimer is as set forth in SEQ ID NO. 261, SEQ ID NO. 265, SEQ ID NO. 269, SEQ ID NO. 273, SEQ ID NO. 277 or SEQ ID NO. Any of the 281 is shown.
  • the expression vector of the present invention contains a polynucleotide encoding the trifunctional 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 trifunctional molecule of the present invention comprises: constructing an expression vector containing a gene sequence of a trifunctional molecule, and then transforming an expression vector containing a gene sequence of the trifunctional molecule 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 trifunctional molecule of the present invention can be used for a tumor therapeutic drug.
  • the tumor is a tumor whose cell surface is CD19 positive.
  • human peripheral blood mononuclear cells are used as experimental materials, and the above-mentioned monomer-form structure prepared by the present invention includes the first function capable of binding to CD19. a domain, a second functional domain capable of binding and activating a T cell surface CD3 molecule, and a trifunctional molecule in a dimeric form capable of binding and activating a third functional domain of a T cell surface CD28 molecule, including a structure capable of binding to CD19 a functional domain, a second domain capable of binding and activating a T cell surface CD3 molecule, and a trifunctional molecule capable of binding and activating a third domain of a T cell surface CD28 molecule, and a purchased anti-CD19/anti-CD3 BiTE bispecific antibody ( CD19-CD3 BsAb) was applied to CIK cells (CD3 + CD56 + ) and CCL-86 Raji lymphoma cells (CD19 + ) prepared from human blood PBMC of the same donor source
  • the killing efficiency of the CIK cells to the Raji cells was significantly improved, and the targeted killing activity against the CD19 positive tumor cells was superior to the anti-CD19/anti-CD3 BiTE bispecific antibody ( CD19-CD3 BsAb).
  • the structure prepared by the present invention includes a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that activate the third domain of the T cell positive costimulatory molecule have in vitro binding activity to CD19, CD3 and the corresponding T cell positive costimulatory molecule recombinant antigen, which can promote the targeted killing of CD19 positive target cells by T cells.
  • the dimer has a better effect than the monomer.
  • the structure prepared by the present invention comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that activate the third domain of the T cell positive costimulatory molecule have in vitro binding activity with CD19 recombinant antigen, CD3 recombinant antigen and corresponding T cell positive costimulatory molecule recombinant protein, which can promote T cell to CD19 positive target cells.
  • the targeted killing, and the dimer has a better effect than the monomer.
  • the structure prepared by the present invention comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that block the third domain of the T cell negative costimulatory molecule have in vitro binding activity to CD19, CD3 and the corresponding T cell negative costimulatory molecule recombinant antigen, which can promote the targeting of T cells to CD19 positive target cells. Killed, and the dimer has a better effect than the monomer.
  • the tumor therapeutic pharmaceutical composition of the present invention comprises the aforementioned trifunctional molecule and at least one pharmaceutically acceptable carrier or excipient.
  • the tumor is a tumor whose cell surface is CD19 positive.
  • the pharmaceutical composition provided by the present invention can be present in various dosage forms, such as an injection for intravenous injection or the like, a percutaneous absorption agent for subcutaneous injection, external application of the epidermis, etc., for nasal spray, throat, oral cavity, epidermis, mucous membrane, etc.
  • a spray for dripping nose, eyes, ears, etc., for suppositories such as anorectal, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc., and lungs
  • Pharmaceutical preparations and other compositions for parenteral administration can be prepared according to a conventional method in the pharmaceutical field.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the pharmaceutical composition may also be added with a flavoring agent and a sweetener Wait.
  • the pharmaceutical preparations as described above can be used clinically in mammals, including humans and animals, and can be administered by intravenous injection or by mouth, nose, skin, lung inhalation or the like.
  • a preferred weekly dose of the above medicament is 0.1-5 mg/kg body weight, and a preferred course of treatment is 10 to 30 days. Administration once or in divided doses. Regardless of the method of administration, the optimal dosage for an individual should be based on the particular treatment.
  • the method of treating a tumor in vitro of the present invention comprises administering the aforementioned trifunctional molecule or tumor therapeutic pharmaceutical composition to a tumor patient.
  • the tumor is a tumor whose cell surface is CD19 positive.
  • the method can be for non-therapeutic purposes.
  • human peripheral blood mononuclear cells (PBMC) are used as experimental materials, and the above-mentioned monomer-form structure prepared by the present invention includes the first function capable of binding to CD19.
  • a domain, a second functional domain capable of binding and activating a T cell surface CD3 molecule, and a trifunctional molecule in a dimeric form capable of binding and activating a third functional domain of a T cell surface CD28 molecule including a structure capable of binding to CD19 a functional domain, a second domain capable of binding and activating a T cell surface CD3 molecule, and a trifunctional molecule capable of binding and activating a third domain of a T cell surface CD28 molecule, and a purchased anti-CD19/anti-CD3 BiTE bispecific antibody ( CD19-CD3 BsAb) was applied to CIK cells (CD3 + CD56 + ) and CCL-86 Raji lymphoma cells (CD19 + ) prepared from human blood PBMC of the same donor source, respectively.
  • CD19-CD3 BsAb purchased anti-CD19/anti-CD3 BiTE bispecific antibody
  • the killing efficiency of the CIK cells to the Raji cells was significantly improved, and the targeted killing activity against the CD19 positive tumor cells was superior to the anti-CD19/anti-CD3 BiTE bispecific antibody ( CD19-CD3 BsAb).
  • the present invention aims at the deficiency of anti-CD19/anti-CD3 BiTE bispecific antibody and CAR-T technology targeting CD19, and constructs a trifunctional molecule capable of simultaneously recognizing CD19, CD3 and CD28 by genetic engineering and antibody engineering.
  • the molecule has obvious advantages in the preparation process and practical application: it further enhances the efficacy of activated T cells while conferring T cells on the targeting of CD19-positive cells, and the T cells to CD19-positive target cells mediated by the addition alone.
  • the combination and killing effect are superior to the anti-CD19/anti-CD3 BiTE bispecific antibody, and it is superior to the CAR-T technology targeting CD19 in the convenience of use.
  • the structure prepared by the present invention comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that activate the third domain of the T cell positive costimulatory molecule have in vitro binding activity to CD19, CD3 and the corresponding T cell positive costimulatory molecule recombinant antigen, which can promote the targeted killing of CD19 positive target cells by T cells.
  • the dimer has a better effect than the monomer.
  • the present invention is directed to the deficiency of anti-CD19/anti-CD3 BiTE bispecific antibody and CAR-T technology targeting CD19, and is capable of simultaneously recognizing CD19, CD3 and any T cell positive costimulatory molecule by genetic engineering and antibody engineering methods.
  • the trifunctional molecule The molecule has obvious advantages in the preparation process and practical application: it further enhances the efficacy of activated T cells while conferring T cells on the targeting of CD19-positive cells, and the T cells to CD19-positive target cells mediated by the addition alone.
  • the killing effect is superior to the anti-CD19/anti-CD3 BiTE bispecific antibody, and is superior to the CAR-T technology targeting CD19 in convenience of use.
  • the structure prepared by the present invention comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that activate the third domain of the T cell positive costimulatory molecule have in vitro binding activity with CD19 recombinant antigen, CD3 recombinant antigen and corresponding T cell positive costimulatory molecule recombinant protein, which can promote T cell to CD19 positive target cells.
  • the targeted killing, and the dimer has a better effect than the monomer.
  • the present invention is directed to the deficiency of anti-CD19/anti-CD3 BiTE bispecific antibody and CAR-T technology targeting CD19, and is capable of simultaneously recognizing CD19, CD3 and any T cell positive costimulatory molecule by genetic engineering and antibody engineering methods.
  • Tri-specific Molecule TsM
  • the molecule has obvious advantages in the preparation process and practical application: it further enhances the efficacy of activated T cells while imparting T cells to the targeting of CD19 positive cells.
  • the T cells mediated by the single addition have better killing effect on CD19-positive target cells than the anti-CD19/anti-CD3 BiTE bispecific antibody, and are superior to the CAR-T technology targeting CD19 in convenience of use.
  • the structure prepared by the present invention comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding and activating T cell surface CD3 molecules, and capable of binding and
  • the trifunctional molecules that block the third domain of the T cell negative costimulatory molecule have in vitro binding activity to CD19, CD3 and the corresponding T cell negative costimulatory molecule recombinant antigen, which can promote the targeting of T cells to CD19 positive target cells. Killed, and the dimer has a better effect than the monomer.
  • the present invention aims to simultaneously recognize CD19, CD3 and any T cell negative costimulatory molecule by genetic engineering and antibody engineering methods against the deficiency of anti-CD19/anti-CD3 BiTE bispecific antibody and CAR-T technology targeting CD19.
  • Tri-specific antibody TsAb
  • the molecule has obvious advantages in the preparation process and practical application: it further enhances the efficacy of activated T cells while conferring T cells on the targeting of CD19-positive cells, and the T cells to CD19-positive target cells mediated by the addition alone.
  • the killing effect is superior to the anti-CD19/anti-CD3 BiTE bispecific antibody, and is superior to the CAR-T technology targeting CD19 in 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 prior literature, 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. 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.
  • a TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells, human CD3 and CD28 proteins on the surface of T cells is designated as CD19-CD3-CD28 TsAb.
  • the specific construction of the monomeric form of CD19-CD3-CD28 TsAb_M is: anti-CD19 scFv, anti-CD3 scFv and anti-CD28 scFv sequences are linked by a linker (Linker), in particular, by anti-CD19 scFv and anti-CD3 scFv Fragment 1 (Linker 1) was ligated and the anti-CD3 scFv and anti-CD28 scFv sequences were ligated by Linker 2 (Linker 2).
  • Linker 1 linker 1
  • Linker 2 Linker 2
  • the specific construction of the dimeric form of CD19-CD3-CD28 TsAb_D is: anti-CD19 scFv, anti-CD3 scFv and anti-CD28 scFv sequences are linked by a linker (Linker), specifically, between anti-CD19 scFv and anti-CD3 scFv Linker 1 (Linker 1) is ligated, and the IgD hinge region is between the anti-CD3 scFv and anti-CD28 scFv sequences. (Ala90-Val170) is connected as Linker 2 (Linker 2).
  • codon optimization of expression of the mammalian system was performed for the anti-CD19 scFv, anti-CD3 scFv, and anti-CD28 scFv sequences.
  • nucleotide sequence of the heavy chain variable region of the anti-CD19 scFv is as shown in SEQ ID NO. 15, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD19 scFv is shown in SEQ ID NO. 16, specifically:
  • nucleotide sequence of the anti-CD19 scFv is shown in SEQ ID NO. 14, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 18, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 19, specifically:
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 17, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-CD28 scFv is shown in SEQ ID NO. 21, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD28 scFv is shown in SEQ ID NO. 22, specifically:
  • nucleotide sequence of the anti-CD28 scFv is shown in SEQ ID NO. 20, specifically:
  • Linker 1 The nucleotide sequence of the dimeric form of CD19-CD3-CD28 TsAb_D Linker 1 (Linker 1) is shown in SEQ ID NO. 28, specifically:
  • amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO. 31, specifically:
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO. 32, 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-CD19-F and CD19-R, CD19-G4S- were used, respectively.
  • CD3-F and CD3-R CD3-(GGGGS) 3 -CD28-F and pcDNA3.1-CD28-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) 3 Linker 2+ anti-CD28
  • Escherichia coli DH5 ⁇ was transformed, and positive clones were identified by colony PCR, and the recombinants (recombinant plasmids) identified as positive were identified by sequencing. The correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD19-CD3-CD28 TsAb_M is shown in SEQ ID NO. 2, specifically:
  • the nucleotide sequence of the dimeric form of CD19-CD3-CD28 TsAb_D is shown in SEQ ID NO. 4, specifically:
  • 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.
  • CD19-CD3-CD28 TsAb_M and CD19-CD3-CD28 TsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electrophoresis patterns under reducing and non-reducing conditions are shown in Figure 1-2.
  • the purity of CD19-CD3-CD28 TsAb_M and CD19-CD3-CD28 TsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column: CD19-CD3-CD28 TsAb_M recombinant protein
  • the theoretical molecular weight is 81.3kDa.
  • the protein Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer. Therefore, the trispecific antibody is in monomeric form (Fig. 1-2A); CD19-CD3-CD28 TsAb_D
  • the theoretical molecular weight of the recombinant protein is 89.1kDa.
  • the molecular weight of the protein Under the reducing condition, the molecular weight of the protein is consistent with the monomer.
  • the molecular weight of the electrophoresis band is consistent with the dimer ( ⁇ 180kDa) (Fig. 1-2B). It is indicated that the two protein molecules can be linked to each other through the IgD hinge region to form a disulfide bond, and thus the trispecific antibody is in the form of a dimer.
  • CD19-CD3-CD28 TsAb_M was a single In bulk form
  • CD19-CD3-CD28 TsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD19-CD3-CD28 TsAb_M is as shown in SEQ ID NO. 1, specifically:
  • the amino acid sequence of the dimeric form of CD19-CD3-CD28 TsAb_D is shown in SEQ ID NO. 3, specifically:
  • amino acid sequence of the anti-CD19 scFv is shown in SEQ ID NO. 5, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO. 6, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO. 7, specifically:
  • amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 8, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 9, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 10, specifically:
  • amino acid sequence of the anti-CD28 scFv is shown in SEQ ID NO. 11, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD28 scFv is set forth in SEQ ID NO. 12, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD28 scFv is set forth in SEQ ID NO. 13, specifically:
  • Linker 1 in the monomeric form of CD19-CD3-CD28 TsAb_M is shown in SEQ ID NO. 23, specifically: GGGGS.
  • Linker 2 in the monomeric form of CD19-CD3-CD28 TsAb_M is shown in SEQ ID NO. 25, specifically: GGGGSGGGGSGGGGS.
  • Linker 1 in the dimeric form of CD19-CD3-CD28 TsAb_D is shown in SEQ ID NO. 27, specifically: GGGGS.
  • Linker 2 in the dimeric form of CD19-CD3-CD28 TsAb_D is shown in SEQ ID NO. 29, specifically:
  • Example 1-3 ELISA assay for antigen binding activity of CD19-CD3-CD28 TsAb_M and CD19-CD3-CD28 TsAb_D
  • Recombinant antigen coating human CD19-hFc, 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. The volume was 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, 950ml H 2 O, adjusted to pH 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, 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 CD19-CD3-CD28 TsAb_M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc and CD28-hFc, wherein CD28 binding activity The activity is the highest, the CD19 binding activity is second, and the CD3 binding activity is weak.
  • Figure 1-3B shows that CD19-CD3-CD28 TsAb_D has the same in vitro binding activity as the recombinant antigens CD19-hFc, CD3-hFc and CD28-hFc, among which CD28 binding activity The highest, CD19 binding activity followed, CD3 binding activity was weak.
  • Example 1-4 Trispecific antibody and bispecific antibody mediated cell engagement assay
  • CD19-positive target cells Using CCL-86 Raji lymphoma cells (purchased from ATCC) as CD19-positive target cells, TIB-152 Jurkat cells (purchased from ATCC) as CD3 and CD28-positive effector cells, comparing the monomeric forms of TiTE III of the present invention Specific antibody (CD19-CD3-CD28 TsAb_M), dimeric form of TiTE trispecific antibody (CD19-CD3-CD28 TsAb_D) and anti-CD19/anti-CD3 BiTE bispecific antibody (CD19-CD3 BsAb, purchased from Wujiang near shore Protein Technology Co., Ltd. mediated differences in cell-binding activity.
  • CD19-CD3-CD28 TsAb_M with final concentration of 45, 0.45 and 0.0045 ng/ml respectively.
  • CD19-CD3-CD28 TsAb_D let stand for 5 min; use cells without any antibody as a blank control;
  • CD19-CD3-CD28 TsAb_M TiTE trispecific antibody
  • CD19-CD3-CD28 TsAb_D CD19-CD3-CD28 TsAb_D
  • anti-CD19/anti-CD3 BiTE bispecific antibody CD19-CD3 BsAb
  • CD19-CD3 BsAb shows little difference in cell binding activity at higher concentrations; under the condition of adding medium concentration antibody (0.45 ng/ml) , CD19-CD3-CD28 TsAb_M and CD19-CD3-CD28 TsAb_D can still cause Raji cells to form agglomerated with Jurkat cells, CD19-CD3 BsAb can cause a small amount of agglomeration of two cells (Fig.
  • TiTE trispecific antibody is superior to BiTE bispecific antibody at medium concentration; CD19-CD3-CD28 TsAb_D can still cause Raji cells and Jurkat with low concentration of antibody (0.0045 ng/ml) The cells are clearly clustered, CD19-CD3-CD28 TsAb_M can cause a small number of cells to form a cluster, while CD19-CD3 BsAb
  • the method of agglomerating cells indicates that the dimeric form of the TiTE trispecific antibody has a better cell binding activity at low concentrations than the monomeric form of TiTE trispecific antibody, whereas the BiTE bispecific antibody Cell-free binding activity at low concentrations.
  • Example 1-5 Trispecific antibody and bispecific antibody mediated cell killing assay
  • the above-mentioned monomeric form of the TiTE trispecific antibody CD19-CD3-CD28 TsAb_M and the dimeric form of the TiTE trispecific antibody were prepared by using human peripheral blood mononuclear cell (PBMC) as experimental material.
  • PBMC peripheral blood mononuclear cell
  • CD19-CD3-CD28 TsAb_D and purchased anti-CD19/anti-CD3 BiTE bispecific antibody (CD19-CD3 BsAb) were applied to human donor PBMC-derived CIK cells (CD3 + CD56 + ) and CCL-86 Raji, respectively, from the same donor source. Lymphoma cells (CD19 + ) were tested for cell death, and the difference in killing efficiency of CCL-86 Raji target cells by three antibody-mediated CIK effector cells was compared.
  • PBMC was resuspended in CIK basal medium (90% X-vivo 15 + 10% FBS) (purchased from Gbico), adjusted to a cell density of 1 ⁇ 10 6 /ml, and added to the whole Long antibody Anti-CD3 (5ug/ml), full-length antibody Anti-CD28 (5ug/ml) and NovoNectin (25ug/ml) coated T25 flask (full-length antibody and NovoNectin were purchased from Wujiang nearshore protein technology) Ltd.), adding cytokine IFN- ⁇ (200ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.) and IL-1 ⁇ (2ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.), placed in the incubator The culture was carried out under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 was added to continue the culture, and every 2-3 days was counted and 1 ⁇ 10 6 was added with CIK basal medium supplemented with 500 U/ml IL-2.
  • CD19-CD3-CD28 TsAb_M and CD19-CD3-CD28 TsAb_D antibody samples mix at room temperature for 3-5 min, co-culture for 3 h at 37 °C, add 10 uL of CCK8 per well, continue reaction for 2-3 h at 37 ° C.
  • the OD 450 value was measured by a microplate reader, and the cell killing efficiency was calculated according to the following formula. Each group of experiments was repeatedly tested 3 times; and the cell killing efficiency without adding any antibody was used as a blank control.
  • CD19-CD3-CD28 TsAb_M The effect of CD19-CD3-CD28 TsAb_M was second. When the E:T ratio was 1:5, the killing efficiency was about 30% and 23% respectively. And 26%, when the E:T ratio is 1:1, the killing efficiency is about 86%, 82% and 81%. The effect of CD19-CD3 BsAb is the weakest, and the killing efficiency when the E:T ratio is 1:5.
  • Example 2-1 Construction of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D eukaryotic expression vector
  • a TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells, T cell surface human CD3 and T cell positive costimulatory molecule 4-1BB protein is named CD19-CD3-4- 1BB TsAb.
  • the specific construction of the monomeric form of CD19-CD3-4-1BB TsAb_M is: anti-CD19 scFv, anti-CD3 scFv and anti-4-1BB scFv sequences are linked by a linker (Linker), specifically, anti-CD19 scFv and anti-CD3 scFv Linked to each other by Linker 1 (Linker 1), the anti-CD3 scFv and anti-4-1BB scFv sequences were ligated by Linker 2 (Linker 2).
  • Linker 1 Linker 1
  • Linker 2 Linker 2
  • the specific construction of the dimeric form of CD19-CD3-4-1BB TsAb_D is: anti-CD19 scFv, anti-CD3 scFv and anti-4-1BB scFv sequences are linked by a linker (Linker), specifically, anti-CD19 scFv and anti-CD3
  • Linker 1 specifically, anti-CD19 scFv and anti-CD3
  • the scFvs were ligated by Linker 1 and the anti-CD3 scFv and anti-4-1BB scFv sequences were ligated with the IgD hinge region (Ala 90-Val 170) as Linker 2 (Linker 2).
  • nucleotide sequence of the heavy chain variable region of the anti-CD19 scFv is shown in SEQ ID NO. 108, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO. 109, specifically:
  • nucleotide sequence of the anti-CD19 scFv is shown in SEQ ID NO. 107, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 111, specifically:
  • nucleotide sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 112, specifically:
  • nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 110, specifically:
  • nucleotide sequence of the heavy chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 114, specifically:
  • nucleotide sequence of the light chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 115, specifically:
  • nucleotide sequence of the anti-4-1BB scFv is shown in SEQ ID NO. 113, specifically:
  • the nucleotide sequence of the monomeric form of CD19-CD3-4-1BB TsAb_M Linker 1 is set forth in SEQ ID NO. 45, specifically: GGTGGCGGAGGGTCC.
  • the nucleotide sequence of the dimeric form of CD19-CD3-4-1BB TsAb_D Linker 1 is set forth in SEQ ID NO. 49, specifically: GGTGGCGGAGGGTCC.
  • amino acid sequence of the secretory expression signal peptide is shown in SEQ ID NO. 131, specifically:
  • the nucleotide sequence of the secretory expression signal peptide is shown in SEQ ID NO. 132, 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 required 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-CD19-F and CD19-R, CD19- were used, respectively.
  • G4S-CD3-F and CD3-R CD3-(GGGGS) 3 -4-1BB-F and pcDNA3.1-4-1BB-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) 3 Linker 2+ anti-4-1BB scFv gene sequence; for CD19-CD3-4-1BB TsAb_D cloning construction, the first use of primers pcDNA3.1-Sig-F and Sig-R to amplify the signal peptide fragment, and then separately Using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-4-1BB-F and pcDNA3.1-4-1BB- R amplified the gene sequences of anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomeric and dimeric forms of the trispecific antibody and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII.
  • Escherichia coli DH5 ⁇ was transformed, and positive clones were identified by colony PCR, and the recombinants (recombinant plasmids) identified as positive were identified by sequencing.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD19-CD3-4-1BB TsAb_M is shown in SEQ ID NO. 60, specifically:
  • nucleotide sequence of the dimeric form of CD19-CD3-4-1BB TsAb_D is shown in SEQ ID NO. 62, specifically:
  • Example 2-2 Expression and purification of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_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.
  • CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electropherograms under reducing and non-reducing conditions are shown in Figure 2-2.
  • the purified protein of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D was purified by Protein L affinity chromatography column.
  • the average molecular weight is >95%: the theoretical molecular weight of CD19-CD3-4-1BB TsAb_M recombinant protein is 80.6kDa.
  • the protein Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer, so the trispecificity
  • the antibody is in monomeric form (Fig. 2-2A); the theoretical molecular weight of the recombinant protein of CD19-CD3-4-1BB TsAb_D is 88.4 kDa.
  • the molecular weight of the protein Under the reducing condition, the molecular weight of the protein is consistent with the monomer, and the electrophoresis is carried out under non-reducing conditions.
  • the band exhibits a molecular weight consistent with the dimer (about 180 kDa) (Fig. 2-2B), indicating that the two protein molecules can be linked to each other via the IgD hinge region to form a disulfide bond, and thus the trispecific antibody is in the form of a dimer.
  • CD19-CD3-4-1BB TsAb_M In monomeric form, CD19-CD3-4-1BB TsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD19-CD3-4-1BB TsAb_M is as shown in SEQ ID NO. 59, specifically:
  • amino acid sequence of the dimeric form of CD19-CD3-4-1BB TsAb_D is shown in SEQ ID NO. 61, specifically:
  • amino acid sequence of the anti-CD19 scFv is set forth in SEQ ID NO. 83, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO. 84, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO. 85, specifically:
  • amino acid sequence of the anti-CD3 scFv is set forth in SEQ ID NO. 86, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 87, specifically:
  • amino acid sequence of the light chain variable region of the anti-CD3 scFv is set forth in SEQ ID NO. 88, specifically:
  • amino acid sequence of the anti-4-1BB scFv is shown in SEQ ID NO. 89, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 90, specifically:
  • amino acid sequence of the light chain variable region of the anti-4-1BB scFv is shown in SEQ ID NO. 91, specifically:
  • the amino acid sequence of the monomeric form of CD19-CD3-4-1BB TsAb_M Linker 1 is set forth in SEQ ID NO. 44, specifically: GGGGS.
  • the amino acid sequence of the monomeric form of CD19-CD3-4-1BB TsAb_M Linker 2 is set forth in SEQ ID NO. 46, specifically: GGGGSGGGGSGGGGS.
  • Linker 1 The amino acid sequence of the dimeric form of CD19-CD3-4-1BB TsAb_D Linker 1 (Linker 1) is set forth in SEQ ID NO. 48, specifically: GGGGS.
  • Linker 2 The amino acid sequence of the dimeric form of CD19-CD3-4-1BB TsAb_D Linker 2 (Linker 2) is set forth in SEQ ID NO. 50, specifically:
  • Example 2-3 ELISA assay for antigen binding activity of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D
  • Recombinant antigen coating human CD19-hFc, 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.
  • the coating volume was 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, 950ml H 2 O, adjusted to pH 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, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Stop solution (1 M HCl) was added, 100 ⁇ l/well, and the absorbance (OD 450 ) was read at a wavelength of 450 nm on a microplate reader.
  • Figure 2-3A illustrates that CD19-CD3-4-1BB TsAb_M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc and 4-1BB-hFc, Among them, 4-1BB has the highest binding activity, CD19 binding activity is second, and CD3 binding activity is weak;
  • Figure 2-3B shows that CD19-CD3-4-1BB TsAb_D is identical to recombinant antigens CD19-hFc, CD3-hFc and 4-1BB-hFc. It has in vitro binding activity, in which 4-1BB has the highest binding activity, CD19 binding activity is second, and CD3 binding activity is weak.
  • PBMC was resuspended in CIK basal medium (90% X-vivo 15 + 10% FBS) (purchased from Gbico), adjusted to a cell density of 1 ⁇ 10 6 /ml, and added to the whole Long antibody Anti-CD3 (5ug/ml), full-length antibody Anti-CD28 (5ug/ml) and NovoNectin (25ug/ml) coated T25 flask (full-length antibody and NovoNectin were purchased from Wujiang nearshore protein technology) Ltd.), adding cytokine IFN- ⁇ (200ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.) and IL-1 ⁇ (2ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.), placed in the incubator The culture was carried out under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 was added to continue the culture, and every 2 to 3 days was counted and 1 ⁇ 10 6 was added with CIK basal medium supplemented with 500 U/ml IL-2.
  • CD3-4-1BB TsAb_D antibody sample mix at room temperature for 3 to 5 min, co-culture for 3 h at 37 ° C, add 10 ⁇ l of CCK-8 per well, continue reaction at 37 ° C for 2 to 3 h, then measure the OD 450 value with a microplate reader.
  • the cell killing efficiency was calculated according to the following formula, and each group of experiments was repeated 3 times; at the same time, the cell killing efficiency without adding any antibody was used as a blank control.
  • CD19-CD3 BsAb The effect is the weakest, the killing efficiency is about 80%, 54% and 54% respectively; under the condition of adding lower concentration antibody (3.125ng/ml), CD19-CD3-4-1BB TsAb_D and CD19-CD3-4-1BB
  • the killing efficiency of Tjab cells mediated by TsAb_M was still significantly improved, and the killing efficiency was about 87% and 80%, respectively, while CD19-CD3 BsAb had no effect compared with the blank control.
  • Example 2-5 Construction of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D eukaryotic expression vector
  • a TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells, T cell surface human CD3 and T cell positive costimulatory molecule ICOS protein is named CD19-CD3-ICOS TsAb.
  • the specific construction of the monomeric form of CD19-CD3-ICOS TsAb_M is: anti-CD19 scFv, anti-CD3 scFv and anti-ICOS scFv sequences are linked by a linker (Linker), in particular, by anti-CD19 scFv and anti-CD3 scFv Fragment 1 (Linker 1) was ligated, and the anti-CD3 scFv and anti-ICOS scFv sequences were ligated by Linker 2 (Linker 2).
  • Linker 1 linker 1
  • Linker 2 Linker 2
  • the specific construction of the dimeric form of CD19-CD3-ICOS TsAb_D is: anti-CD19 scFv, anti-CD3 scFv and anti-ICOS scFv sequences are linked by a linker (Linker), specifically, between anti-CD19 scFv and anti-CD3 scFv Linker 1 (Linker 1) was ligated, and the IgD hinge region (Ala 90-Val 170) was ligated as Linker 2 between the anti-CD3 scFv and the anti-ICOS scFv sequences.
  • Linker specifically, between anti-CD19 scFv and anti-CD3 scFv Linker 1 (Linker 1) was ligated, and the IgD hinge region (Ala 90-Val 170) was ligated as Linker 2 between the anti-CD3 scFv and the anti-ICOS scFv sequences.
  • codon optimization of expression of the mammalian system was performed against the anti-CD19 scFv, anti-CD3 scFv, and anti-ICOS scFv sequences.
  • nucleotide sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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 set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 117, specifically:
  • nucleotide sequence of the light chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 118, specifically:
  • nucleotide sequence of the anti-ICOS scFv is shown in SEQ ID NO. 116, specifically:
  • 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 set forth 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. All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the required 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-CD19-F and CD19-R, CD19-G4S- were used, respectively.
  • CD3-F and CD3-R CD3-(GGGGS) 3 -ICOS-F and pcDNA3.1-ICOS-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) 3 Linker 2+ anti-ICOS
  • Escherichia coli DH5 ⁇ was transformed, and positive clones were identified by colony PCR, and the recombinants (recombinant plasmids) identified as positive were identified by sequencing. The correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD19-CD3-ICOS TsAb_M is shown in SEQ ID NO. 64, specifically:
  • nucleotide sequence of the dimeric form of CD19-CD3-ICOS TsAb_D is shown in SEQ ID NO. 66, specifically:
  • Example 2-6 Expression and purification of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_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.
  • CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electrophoresis patterns under reducing and non-reducing conditions are shown in Figure 2-5.
  • the purity of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column: among the recombinant proteins of CD19-CD3-ICOS TsAb_M
  • the theoretical molecular weight is 80.7kDa.
  • the protein Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer. Therefore, the trispecific antibody is in monomeric form (Fig. 2-5A); CD19-CD3-ICOS TsAb_D
  • the theoretical molecular weight of the recombinant protein is 88.6kDa.
  • the molecular weight of the protein Under the reducing condition, the molecular weight of the protein is consistent with the monomer.
  • the molecular weight of the electrophoresis band is consistent with the dimer (about 180kDa) (Fig. 2-5B). It is indicated that the two protein molecules can be linked to each other through the IgD hinge region to form a disulfide bond, and thus the trispecific antibody is in the form of a dimer.
  • CD19-CD3-ICOS TsAb_M was single.
  • CD19-CD3-ICOS TsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD19-CD3-ICOS TsAb_M is as shown in SEQ ID NO. 63, specifically:
  • amino acid sequence of the dimeric form of CD19-CD3-ICOS TsAb_D is shown in SEQ ID NO. 65, specifically:
  • amino acid sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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. 92, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 93, specifically:
  • amino acid sequence of the light chain variable region of the anti-ICOS scFv is set forth in SEQ ID NO. 94, specifically:
  • Example 2-7 ELISA for detection of antigen binding activity of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D
  • Recombinant antigen coating human CD19-hFc, human CD3-hFc and human ICOS-hFc fusion protein (purchased from Wujiang Coastal Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml. The volume was 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, 950ml H 2 O, adjusted to pH 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, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Stop solution (1 M HCl) was added, 100 ⁇ l/well, and the absorbance (OD 450 ) was read at a wavelength of 450 nm on a microplate reader.
  • Figure 2-6A illustrates that CD19-CD3-ICOS TsAb_M has in vitro binding activity to recombinant antigens CD19-hFc, CD3-hFc and ICOS-hFc, of which ICOS binding activity The highest, CD19 binding activity is second, CD3 binding activity is weak;
  • Figure 2-6B shows that CD19-CD3-ICOS TsAb_D has the same in vitro binding activity as recombinant antigens CD19-hFc, CD3-hFc and ICOS-hFc, of which ICOS has the highest binding activity.
  • CD19 binding activity is second, CD3 binding activity is weak.
  • Example 2-8 CD19-CD3-ICOS trispecific antibody-mediated cell killing assay
  • TiTE trispecific antibody CD19-CD3-ICOS TsAb_M
  • dimeric form of TiTE III were prepared by using human peripheral blood mononuclear cell (PBMC) as experimental material.
  • PBMC peripheral blood mononuclear cell
  • Specific antibody CD19-CD3-ICOS TsAb_D
  • anti-CD19/anti-CD3 BiTE bispecific antibody CD19-CD3 BsAb, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • CD3 + CD56 + CD56 +
  • CD19 + purchased from ATCC
  • PBMC was resuspended in CIK basal medium (90% X-vivo 15 + 10% FBS) (purchased from Gbico), adjusted to a cell density of 1 ⁇ 10 6 /ml, and added to the whole Long antibody Anti-CD3 (5ug/ml), full-length antibody Anti-CD28 (5ug/ml) and NovoNectin (25ug/ml) coated T25 flask (full-length antibody and NovoNectin were purchased from Wujiang nearshore protein technology) Ltd.), adding cytokine IFN- ⁇ (200ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.) and IL-1 ⁇ (2ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.), placed in the incubator The culture was carried out under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 was added to continue the culture, and every 2 to 3 days was counted and 1 ⁇ 10 6 was added with CIK basal medium supplemented with 500 U/ml IL-2.
  • the results are shown in Figure 2-7.
  • Raji target cells E:T ratio
  • the killing efficiency of CIK cells against Raji cells for 3 h was about 23 without any antibody added.
  • the killing efficiency of CIK cells against Raji cells was significantly increased under the condition of adding higher concentration of antibody (25, 12.5, 6.25 ng/ml), and the cell killing effect mediated by CD19-CD3-ICOS TsAb_D
  • the killing efficiencies were about 94%, 94%, and 82%, respectively.
  • the effect of CD19-CD3-ICOS TsAb_M was second, the killing efficiency was about 92%, 86%, and 84%, and the effect of CD19-CD3 BsAb was the weakest.
  • the killing efficiencies were approximately 80%, 54%, and 54%, respectively.
  • CHO cell pairs mediated by CD19-CD3-ICOS TsAb_D and CD19-CD3-ICOS TsAb_M were added at lower concentrations of antibody (3.125 ng/ml).
  • the killing efficiency of Raji cells was still significantly improved, and the killing efficiency was about 76% and 71%, respectively, while CD19-CD3 BsAb had no effect compared with the blank control.
  • Example 2-9 Construction of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D eukaryotic expression vector
  • a TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells, T cell surface human CD3 and T cell positive costimulatory molecule OX40 protein is named CD19-CD3-OX40 TsAb.
  • the specific construction of the monomeric form of CD19-CD3-OX40 TsAb_M is: anti-CD19 scFv, anti-CD3 scFv and anti-OX40 scFv sequences are linked by a linker (Linker), in particular, by anti-CD19 scFv and anti-CD3 scFv Fragment 1 (Linker 1) was ligated and the anti-CD3 scFv and anti-OX40 scFv sequences were ligated by Linker 2 (Linker 2).
  • Linker 1 linker 1
  • Linker 2 Linker 2
  • the specific construction of the dimeric form of CD19-CD3-OX40 TsAb_D is: anti-CD19 scFv, anti-CD3 scFv and anti-OX40 scFv sequences are linked by a linker (Linker), in particular, between anti-CD19 scFv and anti-CD3 scFv Linker 1 (Linker 1) was ligated, and the IgD hinge region (Ala 90-Val 170) was ligated as Linker 2 (Linker 2) between the anti-CD3 scFv and the anti-OX40 scFv sequences.
  • Linker linker
  • codon optimization of expression of the mammalian system was performed for the anti-CD19 scFv, anti-CD3 scFv, and anti-OX40 scFv sequences.
  • nucleotide sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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 set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO. 120, specifically:
  • nucleotide sequence of the light chain variable region of the anti-OX40 scFv is shown in SEQ ID NO. 121, specifically:
  • nucleotide sequence of the anti-OX40 scFv is shown in SEQ ID NO. 119, specifically:
  • 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 set forth 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 2-3 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-CD19-F and CD19-R, CD19-G4S- were used, respectively.
  • CD3-F and CD3-R CD3-(GGGGS) 3 -OX40-F and pcDNA3.1-OX40-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) 3 Linker 2+ anti-OX40
  • CD19-G4S-CD3-F and CD3-R CD3-IgD-F and IgD-R, IgD-OX40-F and pcDNA3.1-OX40-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker 2, anti-OX40 scFv gene sequence.
  • the PCR one-step cloning kit (purchased from Wujiang Nearshore Protein Technology Co., Ltd.) spliced the full-length gene sequences of the monomeric and dimeric forms of the trispecific antibody and cloned them seamlessly into the pcDNA3.1 linearized by EcoRI and HindIII.
  • Escherichia coli DH5 ⁇ was transformed, and positive clones were identified by colony PCR, and the recombinants (recombinant plasmids) identified as positive were identified by sequencing.
  • the correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD19-CD3-OX40 TsAb_M is shown in SEQ ID NO. 68, specifically:
  • the nucleotide sequence of the dimeric form of CD19-CD3-OX40 TsAb_D is shown in SEQ ID NO. 70, specifically:
  • Example 2-10 Expression and purification of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_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.
  • CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electropherograms under reducing and non-reducing conditions are shown in Figure 2-8.
  • the purity of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column: CD19-CD3-OX40 TsAb_M recombinant protein
  • the theoretical molecular weight is 80.1kDa.
  • the protein Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer. Therefore, the trispecific antibody is in monomeric form (Fig. 2-8A); CD19-CD3-OX40 TsAb_D
  • the theoretical molecular weight of the recombinant protein is 88.0kDa. 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 (about 180kDa) (Fig. 2-8B). It is indicated that the two protein molecules can be linked to each other through the IgD hinge region to form a disulfide bond, and thus the trispecific antibody is in the form of a dimer.
  • CD19-CD3-OX40 TsAb_M was a single In bulk form
  • CD19-CD3-OX40 TsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD19-CD3-OX40 TsAb_M is as shown in SEQ ID NO. 67, specifically:
  • amino acid sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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. 95, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO. 96, specifically:
  • amino acid sequence of the light chain variable region of the anti-OX40 scFv is set forth in SEQ ID NO. 97, specifically:
  • Example 2-11 ELISA assay for antigen binding activity of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D
  • Recombinant antigen coating human CD19-hFc, 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. The volume was 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, 950ml H 2 O, adjusted to pH 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, a blocking solution of PBSA (PBS + 2% BSA (V/W)), 200 ⁇ l/well was added. Blocked at 37 ° C for 1 hour;
  • Stop solution (1 M HCl) was added, 100 ⁇ l/well, and the absorbance (OD 450 ) was read at a wavelength of 450 nm on a microplate reader.
  • Figure 2-9A illustrates that CD19-CD3-OX40 TsAb_M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc and OX40-hFc, wherein CD19 binding activity The highest, OX40 binding activity followed, CD3 binding activity was weak;
  • Figure 2-9B shows that CD19-CD3-OX40 TsAb_D has the same in vitro binding activity as recombinant antigens CD19-hFc, CD3-hFc and OX40-hFc, with the highest CD19 binding activity.
  • the OX40 binding activity is second, and the CD3 binding activity is weak.
  • Example 2-12 CD19-CD3-OX40 trispecific antibody-mediated cell killing assay
  • TiTE trispecific antibody CD19-CD3-OX40 TsAb_M
  • dimer form of TiTE III were prepared by using human peripheral blood mononuclear cell (PBMC) as experimental material.
  • PBMC peripheral blood mononuclear cell
  • Specific antibody CD19-CD3-OX40 TsAb_D
  • anti-CD19/anti-CD3 BiTE bispecific antibody CD19-CD3 BsAb, purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • CD3 + CD56 + CD56 +
  • CD19 + purchased from ATCC
  • PBMC was resuspended in CIK basal medium (90% X-vivo 15 + 10% FBS) (purchased from Gbico), adjusted to a cell density of 1 ⁇ 10 6 /ml, and added to the whole Long antibody Anti-CD3 (5ug/ml), full-length antibody Anti-CD28 (5ug/ml) and NovoNectin (25ug/ml) coated T25 flask (full-length antibody and NovoNectin were purchased from Wujiang nearshore protein technology) Ltd.), adding cytokine IFN- ⁇ (200ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.) and IL-1 ⁇ (2ng/ml, purchased from Wujiang Nearshore Protein Technology Co., Ltd.), placed in the incubator The culture was carried out under the conditions of saturated humidity, 37 ° C, and 5.0% CO 2 .
  • IL-2 purchased from Wujiang Nearshore Protein Technology Co., Ltd.
  • 500 U/ml of IL-2 was added to continue the culture, and every 2 to 3 days was counted and 1 ⁇ 10 6 was added with CIK basal medium supplemented with 500 U/ml IL-2.
  • the results are shown in Figure 2-10.
  • Raji target cells E:T ratio
  • the killing efficiency of CIK cells against Raji cells for 3 h was about 23 without any antibody added. %;
  • the killing efficiency of CIK cells on Raji cells was significantly increased under the condition of adding higher concentration of antibody (25, 12.5, 6.25 ng/ml), and the cell killing effect mediated by CD19-CD3-OX40 TsAb_D
  • the killing efficiencies are about 96%, 92%, and 86%, respectively.
  • the effect of CD19-CD3-OX40 TsAb_M is second, the killing efficiency is about 89%, 82%, and 80%, and the effect of CD19-CD3 BsAb is the weakest.
  • the killing efficiencies were approximately 80%, 54%, and 54%, respectively.
  • CHO cell pairs mediated by CD19-CD3-OX40 TsAb_D and CD19-CD3-OX40 TsAb_M were added at lower concentrations of antibody (3.125 ng/ml).
  • the killing efficiency of Raji cells was still significantly improved, and the killing efficiency was about 72% and 68%, respectively, while CD19-CD3 BsAb had little effect compared with the blank control.
  • Example 2-13 Construction of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D eukaryotic expression vector
  • a TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells, T cell surface human CD3 and T cell positive costimulatory molecule GITR protein is named CD19-CD3-GITR TsAb.
  • the specific construction of the monomeric form of CD19-CD3-GITR TsAb_M is: anti-CD19 scFv, anti-CD3 scFv and anti-GITR scFv sequences are linked by a linker (Linker), in particular, by anti-CD19 scFv and anti-CD3 scFv Fragment 1 (Linker 1) was ligated and the anti-CD3 scFv and anti-GITR scFv sequences were ligated by Linker 2 (Linker 2).
  • Linker 1 linker 1
  • Linker 2 Linker 2
  • the specific construction scheme of the dimeric form of CD19-CD3-GITR TsAb_D is: anti-CD19 scFv, anti-CD3 scFv
  • the sequence of the anti-GITR scFv is linked by a linker (Linker), specifically, the anti-CD19 scFv and the anti-CD3 scFv are linked by Linker 1 (Linker 1), and the IgD hinge region is interposed between the anti-CD3 scFv and the anti-GITR scFv sequence. (Ala 90-Val 170) is connected as Linker 2 (Linker 2).
  • codon optimization of expression of the lactation system was performed against the anti-CD19 scFv, anti-CD3 scFv, and anti-GITR scFv sequences.
  • nucleotide sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • nucleotide sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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 set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 123, specifically:
  • nucleotide sequence of the light chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 124, specifically:
  • nucleotide sequence of the anti-GITR scFv is shown in SEQ ID NO. 122, specifically:
  • 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 set forth 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, all primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the required 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-CD19-F and CD19-R, CD19-G4S- were used, respectively.
  • CD3-F and CD3-R CD3-(GGGGS) 3 -GITR-F and pcDNA3.1-GITR-R amplify anti-CD19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) 3 Linker 2+ anti-GITR
  • Escherichia coli DH5 ⁇ was transformed, and positive clones were identified by colony PCR, and the recombinants (recombinant plasmids) identified as positive were identified by sequencing. The correct recombinant (recombinant plasmid) was then sequenced and plasmid extracted for transfection of CHO-S cells.
  • nucleotide sequence of the monomeric form of CD19-CD3-GITR TsAb_M is shown in SEQ ID NO. 72, specifically:
  • nucleotide sequence of the dimeric form of CD19-CD3-GITR TsAb_D is shown in SEQ ID NO. 74, specifically:
  • Example 2-14 Expression and purification of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_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.
  • CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D recombinant proteins were analyzed by SDS-PAGE, and the electropherograms under reducing and non-reducing conditions are shown in Figure 2-11.
  • the purity of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D recombinant protein was >95% after purification by Protein L affinity chromatography column: among the recombinant proteins of CD19-CD3-GITR TsAb_M
  • the theoretical molecular weight is 80.1kDa.
  • the protein Under the reducing and non-reducing conditions, the protein exhibits a single electrophoresis band, and the molecular weight is consistent with the monomer, so the trispecific antibody is in monomeric form (Fig. 2-11A); CD19-CD3-GITR TsAb_D
  • the theoretical molecular weight of the recombinant protein is 88.0kDa.
  • the molecular weight of the protein Under the reducing condition, the molecular weight of the protein is consistent with the monomer.
  • the molecular weight of the electrophoresis band is consistent with the dimer (about 180kDa) (Fig. 2-11B). It is indicated that the two protein molecules can be linked to each other through the IgD hinge region to form a disulfide bond, and thus the trispecific antibody is in the form of a dimer.
  • CD19-CD3-GITR TsAb_M was a single In bulk form
  • CD19-CD3-GITR TsAb_D is in the form of a dimer.
  • amino acid sequence of the monomeric form of CD19-CD3-GITR TsAb_M is as shown in SEQ ID NO. 71, specifically:
  • amino acid sequence of the dimeric form of CD19-CD3-GITR TsAb_D is shown in SEQ ID NO. 73, specifically:
  • amino acid sequence of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the heavy chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • amino acid sequence of the light chain variable region of the anti-CD19 scFv is set forth in SEQ ID NO.
  • 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. 98, specifically:
  • amino acid sequence of the heavy chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 99, specifically:
  • amino acid sequence of the light chain variable region of the anti-GITR scFv is set forth in SEQ ID NO. 100, specifically:
  • Example 2-15 ELISA assay for antigen binding activity of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D
  • Recombinant antigen coating human CD19-hFc, human CD3-hFc and human GITR-hFc fusion protein (purchased from Wujiang Coastal Protein Technology Co., Ltd.) were coated with 96-well plates at an antigen concentration of 1 ⁇ g/ml. The volume was 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, 950ml H 2 O, adjusted to pH 7.4 with 1mol / L HCl or 1mol / L NaOH, hydration to 1L;
  • PBS coating buffer

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Abstract

提供一种三功能分子及其应用。所述三功能分子的结构中包括第一功能域、第二功能域和第三功能域，能够同时结合CD19、CD3和T细胞正（负）共刺激因子，从而产生T细胞活化所需的第一信号和第二信号，在赋予T细胞对CD19阳性细胞靶向性的同时进一步提高了活化T细胞的功效。所述三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

一种三功能分子及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种三功能分子及其应用。
背景技术
人类CD19抗原是大小为95kDa的跨膜糖蛋白，从属于免疫球蛋白超家族，除表达于正常B淋巴细胞表面，CD19也高表达于B细胞恶性肿瘤，因此抗CD19单克隆全长抗体已被开发应用于治疗急/慢性淋巴细胞性白血病以及B细胞淋巴瘤(Wang K等人，Experimental Hematology & Oncology，1：36-42，2012)。鉴于抗CD19单克隆抗体无法有效募集细胞毒性T淋巴细胞(Cytotoxic T lymphocyte，CTL，该类CD3/CD8双阳性T细胞可特异性识别靶细胞表面的抗原肽/MHC I类分子复合物，自身活化后释放穿孔素(Peforin)，导致靶细胞裂解死亡，也可分泌细胞毒素和颗粒酶(Granzyme)等致使靶细胞核的DNA损伤，引起靶细胞凋亡)，人们进一步设计和开发了可衔接T细胞和淋巴瘤B细胞的双特异性抗体(Bi-specific antibody，BsAb)以及基因工程嵌合抗原受体T细胞免疫疗法(Chimeric antigen receptor T-cell immunotherapy，CAR-T)(Zhukovsky EA等人，Current Opinion in Immunology，40：24-35，2016)。
目前一种比较成熟的靶向CD19的双特异性抗体类型是抗CD19/抗CD3的双特异性T细胞衔接器(Bi-specific T cell engager，BiTE)，其结构是两个单链抗体(Single-chain variable fragment，scFv)功能域通过具有柔性的连接肽片段(Linker)共价串联(Goebeler ME等人，Leukemia & Lymphoma，57：1021-1032，2016)。在机体的细胞免疫过程中，CD8阳性T细胞表面的TCR/CD3复合物与抗原递呈细胞(Antigen presenting cell，APC)表面的内源性抗原肽/MHC I类分子复合物发生特异性识别，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。抗CD19/抗CD3 BiTE双特异性抗体由于具有人类CD3和CD19两种抗原的结合活性，能够在T细胞与肿瘤B细胞间形成细胞衔接，同时给予T细胞初步活化信号，提高了其对肿瘤细胞的杀伤靶向性。但是，BiTE双特异性抗体不具有全长抗体的Fc片段，蛋白分子量较小(～54kDa)，因此在肿瘤治疗的过程中可穿越尿血屏障和脑血屏障，生物利用度低，需持续静脉注射给药，同时具有一定的神经毒性。
此外，人体内T细胞的活化需要依赖双信号传递途径(Baxter AG等人，Nature Reviews Immunology，2：439-446，2002)。首先，APC细胞表面的抗原肽-MHC分子复合物与T细胞表面的TCR/CD3复合物相互作用产生第一信号，使得T细胞初步活化，之后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、Galectin-9和HVEM等)与T细胞表面相应的共刺激分子(Co-stimulatory molecule，例如CD28、4-1BB、ICOS、OX40、GITR、CD40L、CD27、CTLA-4、PD-1、LAG-3、TIM-3、TIGIT和BTLA等)相互作用产生第二信号(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、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。已有研究表明仅有第一信号传递途径无法充分活化T细胞，相反会导致其失 能甚至产生激活诱导的T细胞死亡(Activation induced cell death，AICD)。为解决这一问题，可将抗肿瘤抗原/抗T细胞正(负)共刺激分子的双特异性抗体与抗肿瘤抗原/抗CD3的双特异性抗体联合使用，以提高T细胞的活化及肿瘤细胞杀伤效率(Jung G等人，Int J Cancer，91：225-230，2001；Kodama H等人，Immunol Lett，81：99-106，2002)。但该方法在实际操作过程中存在诸多不便，例如会增加重组双特异性抗体表达与纯化的工作量及生产成本，活化扩增T细胞时还需优化两种双特异性抗体的相对比例。相比之下，CAR-T技术能更好地解决T细胞的活化问题。CAR的构建通常包括：肿瘤相关抗原结合区(例如CD19抗原结合区，通常来源于抗CD19单克隆全长抗体的scFv片段)、胞外铰链区、跨膜区以及胞内信号区。其中胞内信号区负责介导T细胞的激活，一方面通过CD3ζ链上的酪氨酸激活基序来完成第一刺激信号，一方面通过CD28共刺激信号实现第一刺激信号的扩大，促进T细胞增殖与活化，并导致细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟等。但CAR-T技术本身也存在着一些不足：首先，该技术依赖病毒转染对T细胞进行基因改造，步骤繁琐，对实验条件要求较高；其次，具体使用时需将体外扩增活化后的CAR-T细胞回输至病人体内，剂量把控相对抗体药物具有更大难度；此外，CAR-T细胞进入患者体内后数量急剧增加可导致细胞因子风暴(Cytokine storm)，短时期内产生超量的细胞因子，从而引起高烧、低压、休克甚至死亡等副反应。
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三功能分子及其应用。
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，提供一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域。
优选地，所述三功能分子能够在识别CD19的同时，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CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2连接。
优选地，所述连接片段1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.23所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.25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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.27所示。所述连接片段2的氨基酸序列如SEQ ID NO.29所示。所述连接片段2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末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。
优选地，所述第一功能域为抗CD19的单链抗体，所述第二功能域为抗CD3的单链抗体，所述第三功能域为抗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CD19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CD19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CD3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CD3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的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.12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.13所示。
本发明一些实施例中，列举了所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.5所示。所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.8所示。所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.11所示。
本发明一些实施例中，还列举了单体形式的三功能分子的氨基酸序列如SEQ ID NO.1所示。二聚体形式的三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，提供另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域。
优选地，所述三功能分子能够在识别CD19的同时，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CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2连接。
优选地，所述连接片段1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.44所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.46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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.48所示。所述连接片段2的氨基酸序列如SEQ ID NO.50所示。所述连接片段2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末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。
优选地，所述第一功能域为抗CD19的单链抗体，所述第二功能域为抗CD3的单链抗体，所述第三功能域为抗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CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.84所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.85所示。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.87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.88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.90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.91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.93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.94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.96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.97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.99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.100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.102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.103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.105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.106所示。
本发明一些实施例中，列举了所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.83所示。所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.86所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.89所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.92所示。所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.95所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.98所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.101所示。所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.104所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.59、SEQ ID NO.63、SEQ ID NO.67、SEQ ID NO.71、SEQ ID NO.75或SEQ ID NO.79之任一所示。
在本案的较佳案例中，二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.61、SEQ ID NO.65、SEQ ID NO.69、SEQ ID NO.73、SEQ ID NO.77或SEQ ID NO.81之任一所示。
本发明的第三方面，提供另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域。
优选地，所述三功能分子，能够在结合于CD19的同时，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CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2连接。
优选地，所述连接片段1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.159所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.161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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.163所示。所述连接片段2的氨基酸序列如SEQ ID NO.165所示。所述连接片段2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末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。
优选地，所述第一功能域为抗CD19的单链抗体，所述抗CD19的单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.198所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.199所示。
优选地，所述第二功能域为抗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.201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.202所示。
本发明一些实施例中，列举了所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.197所示。所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.200所示。
优选地，所述第三功能域为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.203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.204所示。
优选地，所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.205所示。
优选地，所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.206所示。
优选地，所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.207所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.177、SEQ ID NO.181、SEQ ID NO.185、SEQ ID NO.189或SEQ ID NO.193之任一所示。二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.179、SEQ ID NO.183、SEQ ID NO.187、SEQ ID NO.191或SEQ ID NO.195之任一所示。
本发明的第四方面，提供另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺 激分子的第三功能域。
优选地，所述三功能分子能够在识别CD19的同时，结合并激活T细胞表面CD3分子、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CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2连接。
优选地，所述连接片段1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.244所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.246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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.248所示。所述连接片段2的氨基酸序列如SEQ ID NO.250所示。所述连接片段2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末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。
优选地，所述第一功能域为抗CD19的单链抗体，所述第二功能域为抗CD3的单链抗体，所述第三功能域为抗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CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.283所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.284所示。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.286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.287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.289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.290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.292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.293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.295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.296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.298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.299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.301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.302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.304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.305所示。
本发明一些实施例中，列举了所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.282所示。所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.285所示。所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.288所示。所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.291所示。所述抗LAG-3的单链抗体的氨基酸序列如SEQ ID NO.294所示。所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.297所示。所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.300所示。所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.303所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.258、SEQ ID NO.262、SEQ ID NO.266、SEQ ID NO.270、SEQ ID NO.274或SEQ ID NO.278之任一所示。二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.260、SEQ ID NO.264、SEQ ID NO.268、SEQ ID NO.272、SEQ ID NO.276或SEQ ID NO.280之任一所示。
本发明的第五方面，提供一种多核苷酸，其编码前述三功能分子。
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，提供一种制备前述三功能分子的方法，包括：构建含有三功能分子基因序列的表达载体，然后将含三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三功能分子。
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，提供前述三功能分子用于制备肿瘤治疗药物的用途。
本发明的第十方面，提供一种肿瘤治疗药物组合物，含有前述三功能分子及至少一种药学可接受的载体或赋形剂。所述肿瘤为细胞表面为CD19阳性的肿瘤。
本发明的第十一方面，公开了一种体外治疗肿瘤的方法，包括将前述三功能分子或肿瘤治疗药物组合物施用于肿瘤患者。所述方法可以是非治疗目的的。所述肿瘤为细胞表面为CD19阳性的肿瘤。
与现有技术相比，本发明具有如下有益效果：
(1)本发明将能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域融合于同一蛋白肽链，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，使用方便；而抗CD19/抗CD3双特异性抗体与抗CD19/抗CD28双特异性抗体如果联合使用，两个双特异性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，且使用时需优化两者的相对比例。本发明的三功能分子能够产生T细胞活化的第二刺激信号，在赋予T细胞靶向性的同时进一步提高了对T细胞的激活功效，使得细胞因子和抗细胞凋亡蛋白分泌增加，有效避免了T细胞失能及死亡的现象，所介导的T细胞对CD19阳性靶细胞的结合与杀伤能够达到甚至优于抗CD19/抗CD3 BiTE双特异性抗体的效果，且蛋白用量更少。本发明所述的三功能分子与靶向CD19的CAR-T技术相比，不涉及病毒介导转基因、体外T细胞培养及回输等操作步骤，使用更方便，剂量可控，进入病人机体后引起细胞因子过量释放的风险小，避免了使用CAR-T时的毒副作用。
(2)本发明还将能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域融合于同一蛋白肽链，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，使用方便；而抗CD19/抗CD3双特异性抗体与抗CD19/抗T细胞正共刺激分子双特异性抗体如果联合使用，两个双特异性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，且使用时需优化两者的相对比例。本发明所述的三功能分子，能够产生T细胞活化的第二(正)刺激信号，在赋予T细胞靶向性的同时进一步提高了对T细胞的激活功效，使得细胞因子和抗细胞凋亡蛋白分泌增加，有效避免了T细胞失能及死亡的现象，所介导的T细胞对CD19阳性靶细胞的杀伤能够达到甚至优于抗CD19/抗CD3 BiTE双特异性抗体的效果，且蛋白用量更少。本发明所述的三功能分子与靶向CD19的CAR-T技术相比，不涉及病毒介导转基因、体外T细胞培养及回输等操作步骤，使用更方便，剂量可控，进入病人机体后引起细胞因子过量释放的风险小，避免了使用CAR-T时的毒副作用。
(3)本发明还将能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域融合于同一蛋白肽链形成三功能分子，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，使用方便；而抗CD19/抗CD3双特异性抗体与抗CD19/抗T细胞正共刺激分子双特异性抗体如果联合使用，两个双特异性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，且使用时需优化两者的相对比例。本发明所述的三功能分子，能够产生T细胞活化的第二(正)刺激信号，在赋予T细胞靶向性的同时进一步提高了对T细胞的激活功效，使得细胞因子和抗细胞凋亡蛋白分泌增加，有效避免了T细胞失能及死亡的现象，所介导的T细胞对CD19阳性靶细胞的杀伤能够达到甚至优于抗CD19/抗CD3 BiTE双特异性抗体的效果，且蛋白用量更少。本发明所述的三功能分子，与靶向CD19的CAR-T技术相比，不涉及病毒介导转基因、体外T细胞培养及回输等操作步骤，使用更方便，剂量可控，进入病人机体后引起细胞因子过量释放的风险小，避免了使用CAR-T时的毒副作用。
(4)本发明还将能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺激分子的第三功能域融合于同一蛋白肽链，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，使用方便；而抗CD19/抗CD3双特异性抗体与抗CD19/抗T细胞正(负)共刺激分子双特异性抗体如果联合使用，两个双特异性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，且使用时需优化两者的相对比例。本发明所述的三功能分子，能够阻断或下调T细胞活化的第二(负)刺激信号，在赋予T细胞靶向性的同时进一步提高了对T细胞的激活功效，使得细胞因子和抗细胞凋亡蛋白分泌增加，有效避免了T细胞失能及死亡的现象，所介导的T细胞对CD19阳性靶细胞的杀伤能够达到甚至优于抗CD19/抗CD3 BiTE双特异性抗体的效果，且蛋白用量更少。本发明所述的三功能分子与靶向CD19的CAR-T技术相比，不涉及病毒介导转基因、体外T细胞培养及回输等操作步骤，使用更方便，剂量可控，进入病人机体后引起细胞因子过量释放的风险小，避免了使用CAR-T时的毒副作用。
附图说明
图1-1：A.单体形式抗CD19/CD3/抗CD28三特异性抗体(CD19-CD3-CD28 TsAb_M)的结构图；B.二聚体形式抗CD19/抗CD3/抗CD28三特异性抗体(CD19-CD3-CD28 TsAb_D)的结构图。
图1-2：A.纯化的CD19-CD3-CD28 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD28 TsAb_M；泳道3：非还原性CD19-CD3-CD28  TsAb_M；B.纯化的CD19-CD3-CD28 TsAb_D SDS-PAGE分析图；泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD28 TsAb_D；泳道3：非还原性CD19-CD3-CD28 TsAb_D。
图1-3A：CD19-CD3-CD28 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD28-hFc；
Figure PCTCN2017096594-appb-000001
不包被任何抗原的测定结果。
图1-3B：CD19-CD3-CD28 TsAb_D的ELISA鉴定结果；图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD28-hFc；
Figure PCTCN2017096594-appb-000002
不包被任何抗原的测定结果。
图1-4：三特异抗体及双特异性抗体介导的细胞衔接实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，Jurkat细胞作为CD3和CD28阳性的效应细胞，分别检测不同浓度CD19-CD3-CD28 TsAb_M、CD19-CD3-CD28 TsAb_D以及CD19-CD3 BsAb的细胞衔接活性；A：未添加抗体的空白对照；B：添加高浓度CD19-CD3 BsAb(45ng/ml)的实验组；C：添加高浓度CD19-CD3-CD28 TsAb_M(45ng/ml)的实验组；D：添加高浓度CD19-CD3-CD28TsAb_D(45ng/ml)的实验组；E：添加中浓度CD19-CD3 BsAb(0.45ng/ml)的实验组；F：添加中浓度CD19-CD3-CD28 TsAb_M(0.45ng/ml)的实验组；G：添加中浓度CD19-CD3-CD28 TsAb_D(0.45ng/ml)的实验组；H：添加低浓度CD19-CD3 BsAb(0.0045ng/ml)的实验组；I：添加低浓度CD19-CD3-CD28 TsAb_M(0.0045ng/ml)的实验组；J：添加低浓度CD19-CD3-CD28 TsAb_D(0.0045ng/ml)的实验组。
图1-5A：三特异抗体及双特异性抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK(Cytokine induced killer)细胞作为CD3和CD28阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CD28 TsAb_M、CD19-CD3-CD28 TsAb_D以及CD19-CD3 BsAb所介导的CIK细胞对Raji细胞的杀伤效率；效应细胞∶靶细胞(E∶T比)＝1∶5，杀伤时间：3h。
图1-5B：三特异抗体及双特异性抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3和CD28阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CD28 TsAb_M、CD19-CD3-CD28 TsAb_D以及CD19-CD3 BsAb所介导的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2-1：A.单体形式抗CD19/抗CD3/抗T细胞正共刺激分子三特异性抗体的结构图；B.二聚体形式抗CD19/抗CD3/抗T细胞正共刺激分子三特异性抗体的结构图。
图2-2：A.纯化的CD19-CD3-4-1BB TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-4-1BB TsAb_M；泳道3：非还原性CD19-CD3-4-1BBTsAb_M；B.纯化的CD19-CD3-4-1BB TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-4-1BB TsAb_D；泳道3：非还原性CD19-CD3-4-1BB TsAb_D。
图2-3A：CD19-CD3-4-1BB TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原4-1BB-hFc；
Figure PCTCN2017096594-appb-000003
不包被任何抗原的测定结果。
图2-3B：CD19-CD3-4-1BB TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原4-1BB-hFc；
Figure PCTCN2017096594-appb-000004
不包被任何抗原的测定结果。
图2-4：CD19-CD3-4-1BB三特异抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK(Cytokine induced killer)细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-4-1BB TsAb_M、CD19-CD3-4-1BB TsAb_D以及CD19-CD3  BsAb所介导的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2-5：A.纯化的CD19-CD3-ICOS TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-ICOS TsAb_M；泳道3：非还原性CD19-CD3-ICOS TsAb_M；B.纯化的CD19-CD3-ICOS TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-ICOS TsAb_D；泳道3：非还原性CD19-CD3-ICOS TsAb_D。
图2-6A：CD19-CD3-ICOS TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原ICOS-hFc；
Figure PCTCN2017096594-appb-000005
不包被任何抗原的测定结果。
图2-6B：CD19-CD3-ICOS TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原ICOS-hFc；
Figure PCTCN2017096594-appb-000006
不包被任何抗原的测定结果。
图2-7：CD19-CD3-ICOS三特异抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-ICOS TsAb_M、CD19-CD3-ICOS TsAb_D以及CD19-CD3 BsAb所介导的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2-8：A.纯化的CD19-CD3-OX40 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-OX40 TsAb_M；泳道3：非还原性CD19-CD3-OX40 TsAb_M；B.纯化的CD19-CD3-OX40 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：非还原性CD19-CD3-OX40 TsAb_D；泳道3：还原性CD19-CD3-OX40 TsAb_D。
图2-9A：CD19-CD3-OX40 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原OX40-hFc；
Figure PCTCN2017096594-appb-000007
不包被任何抗原的测定结果。
图2-9B：CD19-CD3-OX40 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原OX40-hFc；
Figure PCTCN2017096594-appb-000008
不包被任何抗原的测定结果。
图2-10：CD19-CD3-OX40三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-OX40 TsAb_M、CD19-CD3-OX40 TsAb_D以及CD19-CD3 BsAb所介导的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2-11：A.纯化的CD19-CD3-GITR TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-GITR TsAb_M；泳道3：非还原性CD19-CD3-GITR TsAb_M；B.纯化的CD19-CD3-GITR TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-GITR TsAb_D；泳道3：非还原性CD19-CD3-GITR TsAb_D。
图2-12A：CD19-CD3-GITR TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原GITR-hFc；
Figure PCTCN2017096594-appb-000009
不包被任何抗原的测定结果。
图2-12B：CD19-CD3-GITR TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原GITR-hFc；
Figure PCTCN2017096594-appb-000010
不包被任何抗原的测定结果。
图2-13：CD19-CD3-GITR三特异抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为 CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-GITR TsAb_M、CD19-CD3-GITR TsAb_D以及CD19-CD3 BsAb所介导的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CD19-CD3-CD40L TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD40L TsAb_M；泳道3：非还原性CD19-CD3-CD40L TsAb_M；B.纯化的CD19-CD3-CD40L TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD40L TsAb_D；泳道3：非还原性CD19-CD3-CD40L TsAb_D。
图2-15A：CD19-CD3-CD40L TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD40L-hFc；
Figure PCTCN2017096594-appb-000011
不包被任何抗原的测定结果。
图2-15B：CD19-CD3-CD40L TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD40L-hFc；
Figure PCTCN2017096594-appb-000012
不包被任何抗原的测定结果。
图2-16：CD19-CD3-CD40L三特异抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CD40L TsAb_M、CD19-CD3-CD40L TsAb_D以及CD19-CD3 BsAb所介导的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2-17：A.纯化的CD19-CD3-CD27 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD27 TsAb_M；泳道3：非还原性CD19-CD3-CD27 TsAb_M；B.纯化的CD19-CD3-CD27 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD27 TsAb_D；泳道3：非还原性CD19-CD3-CD27 TsAb_D。
图2-18A：CD19-CD3-CD27 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD27-hFc；
Figure PCTCN2017096594-appb-000013
不包被任何抗原的测定结果。
图2-18B：CD19-CD3-CD27 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CD27-hFc；
Figure PCTCN2017096594-appb-000014
不包被任何抗原的测定结果。
图2-19：CD19-CD3-CD27三特异抗体介导的细胞杀伤实验，以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CD27 TsAb_M、CD19-CD3-CD27 TsAb_D以及CD19-CD3 BsAb所介导的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3-1：A.单体形式抗CD19/抗CD3/T细胞正共刺激分子配体TsM的结构图；B.二聚体形式抗CD19/抗CD3/T细胞正共刺激分子配体TsM的结构图。
图3-2：A.纯化的CD19-CD3-4-1BBL TsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-4-1BBL TsM_M；泳道3：非还原性CD19-CD3-4-1BBL TsM_M；B.纯化的CD19-CD3-4-1BBL TsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-4-1BBL TsM_D；泳道3：非还原性CD19-CD3-4-1BBL TsM_D。
图3-3A：CD19-CD3-4-1BBL TsM_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白4-1BB-hFc；
Figure PCTCN2017096594-appb-000015
不包被任何蛋白的测定结果。
图3-3B：CD19-CD3-4-1BBL TsM_D的ELISA鉴定结果，图中的曲线分别代表4种检 测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白4-1BB-hFc；
Figure PCTCN2017096594-appb-000016
不包被任何蛋白的测定结果。
图3-4：CD19-CD3-4-1BBL三特异分子介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK(Cytokine induced killer)细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-4-1BBL TsM_M、CD19-CD3-4-1BBL TsM_D以及CD19-CD3 BsAb所介导的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3-5：A.纯化的CD19-CD3-B7RP-1 TsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-B7RP-1 TsM_M；泳道3：非还原性CD19-CD3-B7RP-1 TsM_M；B.纯化的CD19-CD3-B7RP-1 TsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-B7RP-1 TsM_D；泳道3：非还原性CD19-CD3-B7RP-1 TsM_D。
图3-6A：CD19-CD3-B7RP-1 TsM_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白ICOS-hFc；
Figure PCTCN2017096594-appb-000017
不包被任何蛋白的测定结果。
图3-6B：CD19-CD3-B7RP-1 TsM_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白ICOS-hFc；
Figure PCTCN2017096594-appb-000018
不包被任何蛋白的测定结果。
图3-7：CD19-CD3-B7RP-1三特异分子介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-B7RP-1 TsM_M、CD19-CD3-B7RP-1 TsM_D以及CD19-CD3 BsAb所介导的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3-8：A.纯化的CD19-CD3-OX40L TsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-OX40L TsM_M；泳道3：非还原性CD19-CD3-OX40L TsM_M；B.纯化的CD19-CD3-OX40L TsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-OX40L TsM_D；泳道3：非还原性CD19-CD3-OX40L TsM_D。
图3-9A：CD19-CD3-OX40L TsM_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白OX40-hFc；
Figure PCTCN2017096594-appb-000019
不包被任何蛋白的测定结果。
图3-9B：CD19-CD3-OX40L TsM_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白OX40-hFc；
Figure PCTCN2017096594-appb-000020
不包被任何蛋白的测定结果。
图3-10：CD19-CD3-OX40L三特异分子介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-OX40L TsM_M、CD19-CD3-OX40L TsM_D以及CD19-CD3 BsAb所介导的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3-11：A.纯化的CD19-CD3-GITRL TsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-GITRL TsM_M；泳道3：非还原性CD19-CD3-GITRL TsM_M；B.纯化的CD19-CD3-GITRL TsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-GITRL TsM_D；泳道3：非还原性CD19-CD3-GITRL TsM_D。
图3-12A：CD19-CD3-GITRL TsM_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包 被1μg/ml重组蛋白GITR-hFc；
Figure PCTCN2017096594-appb-000021
不包被任何蛋白的测定结果。
图3-12B：CD19-CD3-GITRL TsM_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白GITR-hFc；
Figure PCTCN2017096594-appb-000022
不包被任何蛋白的测定结果。
图3-13：CD19-CD3-GITRL三特异分子介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-GITRL TsM_M、CD19-CD3-GITRL TsM_D以及CD19-CD3 BsAb所介导的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3-14：A.纯化的CD19-CD3-CD70 TsM_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD70 TsM_M；泳道3：非还原性CD19-CD3-CD70 TsM_M；B.纯化的CD19-CD3-CD70 TsM_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CD70 TsM_D；泳道3：非还原性CD19-CD3-CD70 TsM_D。
图3-15A：CD19-CD3-CD70 TsM_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白CD27-hFc；
Figure PCTCN2017096594-appb-000023
不包被任何蛋白的测定结果。
图3-15B：CD19-CD3-CD70 TsM_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组蛋白CD27-hFc；
Figure PCTCN2017096594-appb-000024
不包被任何蛋白的测定结果。
图3-16：CD19-CD3-CD70三特异分子介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CD70 TsM_M、CD19-CD3-CD70 TsM_D以及CD19-CD3 BsAb所介导的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4-1：A.单体形式抗CD19/抗CD3/抗T细胞负共刺激分子三特异性抗体的结构图；B.二聚体形式抗CD19/抗CD3/抗T细胞负共刺激分子三特异性抗体的结构图。
图4-2：A.纯化的CD19-CD3-PD-1 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-PD-1 TsAb_M；泳道3：非还原性CD19-CD3-PD-1 TsAb_M；B.纯化的CD19-CD3-PD-1 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-PD-1 TsAb_D；泳道3：非还原性CD19-CD3-PD-1 TsAb_D。
图4-3A：CD19-CD3-PD-1 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原PD-1-hFc；
Figure PCTCN2017096594-appb-000025
不包被任何抗原的测定结果。
图4-3B：CD19-CD3-PD-1 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原PD-1-hFc；
Figure PCTCN2017096594-appb-000026
不包被任何抗原的测定结果。
图4-4：CD19-CD3-PD-1三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK(Cytokine induced killer)细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-PD-1 TsAb_M、CD19-CD3-PD-1 TsAb_D以及CD19-CD3 BsAb所介导的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4-5：A.纯化的CD19-CD3-CTLA-4 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CTLA-4 TsAb_M；泳道3：非还原性CD19-CD3-CTLA-4 TsAb_M；B.纯化的CD19-CD3-CTLA-4 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-CTLA-4 TsAb_D；泳道3：非还原性CD19-CD3-CTLA-4 TsAb_D。
图4-6A：CD19-CD3-CTLA-4 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CTLA-4-hFc；
Figure PCTCN2017096594-appb-000027
不包被任何抗原的测定结果。
图4-6B：CD19-CD3-CTLA-4 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原CTLA-4-hFc；
Figure PCTCN2017096594-appb-000028
不包被任何抗原的测定结果。
图4-7：CD19-CD3-CTLA-4三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-CTLA-4 TsAb_M、CD19-CD3-CTLA-4 TsAb_D以及CD19-CD3 BsAb所介导的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4-8：A.纯化的CD19-CD3-LAG-3 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-LAG-3 TsAb_M；泳道3：非还原性CD19-CD3-LAG-3 TsAb_M；B.纯化的CD19-CD3-LAG-3 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-LAG-3 TsAb_D；泳道3：非还原性CD19-CD3-LAG-3 TsAb_D。
图4-9A：CD19-CD3-LAG-3 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原LAG-3-hFc；
Figure PCTCN2017096594-appb-000029
不包被任何抗原的测定结果。
图4-9B：CD19-CD3-LAG-3 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原LAG-3-hFc；
Figure PCTCN2017096594-appb-000030
不包被任何抗原的测定结果。
图4-10：CD19-CD3-LAG-3三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-LAG-3 TsAb_M、CD19-CD3-LAG-3 TsAb_D以及CD19-CD3 BsAb所介导的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4-11：A.纯化的CD19-CD3-TIM-3 TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-TIM-3 TsAb_M；泳道3：非还原性CD19-CD3-TIM-3 TsAb_M；B.纯化的CD19-CD3-TIM-3 TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-TIM-3 TsAb_D；泳道3：非还原性CD19-CD3-TIM-3 TsAb_D。
图4-12A：CD19-CD3-TIM-3 TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原TIM-3-hFc；
Figure PCTCN2017096594-appb-000031
不包被任何抗原的测定结果。
图4-12B：CD19-CD3-TIM-3 TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原TIM-3-hFc；
Figure PCTCN2017096594-appb-000032
不包被任何抗原的测定结果。
图4-13：CD19-CD3-TIM-3三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-TIM-3 TsAb_M、CD19-CD3-TIM-3 TsAb_D以及CD19-CD3 BsAb所介导的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4-14：A.纯化的CD19-CD3-TIGIT TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-TIGIT TsAb_M；泳道3：非还原性CD19-CD3-TIGIT TsAb_M；B.纯化的CD19-CD3-TIGIT TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-TIGIT TsAb_D；泳道3：非还原性CD19-CD3-TIGIT  TsAb_D。
图4-15A：CD19-CD3-TIGIT TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原TIGIT-hFc；
Figure PCTCN2017096594-appb-000033
不包被任何抗原的测定结果。
图4-15B：CD19-CD3-TIGIT TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原TIGIT-hFc；
Figure PCTCN2017096594-appb-000034
不包被任何抗原的测定结果。
图4-16：CD19-CD3-TIGIT三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-TIGIT TsAb_M、CD19-CD3-TIGIT TsAb_D以及CD19-CD3 BsAb所介导的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4-17：A.纯化的CD19-CD3-BTLA TsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-BTLA TsAb_M；泳道3：非还原性CD19-CD3-BTLA TsAb_M；B.纯化的CD19-CD3-BTLA TsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD19-CD3-BTLA TsAb_D；泳道3：非还原性CD19-CD3-BTLA TsAb_D。
图4-18A：CD19-CD3-BTLA TsAb_M的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原BTLA-hFc；
Figure PCTCN2017096594-appb-000035
不包被任何抗原的测定结果。
图4-18B：CD19-CD3-BTLA TsAb_D的ELISA鉴定结果，图中的曲线分别代表4种检测结果：■包被1μg/ml重组抗原CD19-hFc；●包被1μg/ml重组抗原CD3-hFc；▲包被1μg/ml重组抗原BTLA-hFc；
Figure PCTCN2017096594-appb-000036
不包被任何抗原的测定结果。
图4-19：CD19-CD3-BTLA三特异抗体介导的细胞杀伤实验。以Raji淋巴瘤细胞作为CD19阳性的靶细胞，CIK细胞作为CD3阳性的杀伤效应细胞，分别检测不同浓度CD19-CD3-BTLA TsAb_M、CD19-CD3-BTLA TsAb_D以及CD19-CD3 BsAb所介导的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。
具体实施方式
一、术语和缩略语：
CTL：细胞毒性T淋巴细胞(Cytotoxic T lymphocyte)
BsAb：双特异性抗体(Bi-specific Antibody)
TsAb：三特异性抗体(Tri-specific Antibody)
TsM：三特异性分子(Tri-specific Molecule)
BiTE：双特异性T细胞衔接器(Bi-specific T cell engager)
TiTE：三特异性T细胞衔接器(Tri-specific T cell engager)
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
Linker1：连接片段1
Linker2：连接片段2
Extracellular domain：胞外区
Co-stimulatory molecule：共刺激分子
CD19-CD3-CD28 TsAb_M：单体形式的抗CD19/抗CD3/抗CD28三特异性抗体
CD19-CD3-CD28 TsAb_D：二聚体形式的抗CD19/抗CD3/抗CD28三特异性抗体
CD19-CD3-4-1BB TsAb_M：单体形式的抗CD19/抗CD3/抗4-1BB三特异性抗体
CD19-CD3-4-1BB TsAb_D：二聚体形式的抗CD19/抗CD3/抗4-1BB三特异性抗体
CD19-CD3-ICOS TsAb_M：单体形式的抗CD19/抗CD3/抗ICOS三特异性抗体
CD19-CD3-ICOS TsAb_D：二聚体形式的抗CD19/抗CD3/抗ICOS三特异性抗体
CD19-CD3-OX40 TsAb_M：单体形式的抗CD19/抗CD3/抗OX40三特异性抗体
CD19-CD3-OX40 TsAb_D：二聚体形式的抗CD19/抗CD3/抗OX40三特异性抗体
CD19-CD3-GITR TsAb_M：单体形式的抗CD19/抗CD3/抗GITR三特异性抗体
CD19-CD3-GITR TsAb_D：二聚体形式的抗CD19/抗CD3/抗GITR三特异性抗体
CD19-CD3-CD40L TsAb_M：单体形式的抗CD19/抗CD3/抗CD40L三特异性抗体
CD19-CD3-CD40L TsAb_D：二聚体形式的抗CD19/抗CD3/抗CD40L三特异性抗体
CD19-CD3-CD27 TsAb_M：单体形式的抗CD19/抗CD3/抗CD27三特异性抗体
CD19-CD3-CD27 TsAb_D：二聚体形式的抗CD19/抗CD3/抗CD27三特异性抗体
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
CD19-CD3-4-1BBL TsM_M：单体形式的抗CD19/抗CD3/4-1BBL三特异性分子
CD19-CD3-4-1BBL TsM_D：二聚体形式的抗CD19/抗CD3/4-1BBL三特异性分子
CD19-CD3-B7RP-1 TsM_M：单体形式的抗CD19/抗CD3/B7RP-1三特异性分子
CD19-CD3-B7RP-1 TsM_D：二聚体形式的抗CD19/抗CD3/B7RP-1三特异性分子
CD19-CD3-OX40L TsM_M：单体形式的抗CD19/抗CD3/OX40L三特异性分子
CD19-CD3-OX40L TsM_D：二聚体形式的抗CD19/抗CD3/OX40L三特异性分子
CD19-CD3-GITRL TsM_M：单体形式的抗CD19/抗CD3/GITRL三特异性分子
CD19-CD3-GITRL TsM_D：二聚体形式的抗CD19/抗CD3/GITRL三特异性分子
CD19-CD3-CD70 TsM_M：单体形式的抗CD19/抗CD3/CD70三特异性分子
CD19-CD3-CD70 TsM_D：二聚体形式的抗CD19/抗CD3/CD70三特异性分子
CD19-CD3-PD-1 TsAb_M：单体形式的抗CD19/抗CD3/抗PD-1三特异性抗体
CD19-CD3-PD-1 TsAb_D：二聚体形式的抗CD19/抗CD3/抗PD-1三特异性抗体
CD19-CD3-CTLA-4 TsAb_M：单体形式的抗CD19/抗CD3/抗CTLA-4三特异性抗体
CD19-CD3-CTLA-4 TsAb_D：二聚体形式的抗CD19/抗CD3/抗CTLA-4三特异性抗体
CD19-CD3-LAG-3 TsAb_M：单体形式的抗CD19/抗CD3/抗LAG-3三特异性抗体
CD19-CD3-LAG-3 TsAb_D：二聚体形式的抗CD19/抗CD3/抗LAG-3三特异性抗体
CD19-CD3-TIM-3 TsAb_M：单体形式的抗CD19/抗CD3/抗TIM-3三特异性抗体
CD19-CD3-TIM-3 TsAb_D：二聚体形式的抗CD19/抗CD3/抗TIM-3三特异性抗体
CD19-CD3-TIGIT TsAb_M：单体形式的抗CD19/抗CD3/抗TIGIT三特异性抗体
CD19-CD3-TIGIT TsAb_D：二聚体形式的抗CD19/抗CD3/抗TIGIT三特异性抗体
CD19-CD3-BTLA TsAb_M：单体形式的抗CD19/抗CD3/抗BTLA三特异性抗体
CD19-CD3-BTLA TsAb_D：二聚体形式的抗CD19/抗CD3/抗BTLA三特异性抗体。
二、三功能分子
本发明的一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域。
进一步地，所述三功能分子能够在识别CD19的同时，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、第二功能域和第三功能域并无特殊限制，只要能够在识别CD19的同时，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CD19的抗体，所述第二功能域可以是抗CD3的抗体，所述第三功能域可以是抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2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，可以是所述第一功能域的C末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。本发明对于连接片段1和连接片段2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1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.23所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.25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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.27所示。所述连接片段2的氨基酸序列如SEQ ID NO.29所示。所述连接片段2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，所述三功能分子的结构示意图如图1-1所示。所述三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三功能分子的结构示意图如图1-1A所示，所述三功能分子的结构中含有一个与CD19抗原结合的第一功能域，一个与CD3抗原结合的第二功能域，一个与CD28抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与CD28抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的结构示意图如图1-1B所示，所述三功能分子的结构中含有两个与CD19抗原 结合的第一功能域，两个与CD3抗原结合的第二功能域，两个与CD28抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与CD28抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的抗原结合效价是单体形式的二倍。由于T细胞活化第一信号(CD3)和第二信号(CD28)的加倍，致使T细胞活化更为充分，对靶细胞的杀伤效果更强；CD19单链抗体结构域的加倍使其对靶细胞的识别也更为精准，因此二聚体较单体具有更好的使用效果。
具体地，所述第一功能域为抗CD19的单链抗体。所述抗CD19的单链抗体包括重链可变区和轻链可变区。所述抗CD19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CD19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CD19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.9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.10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.8所示。
所述第三功能域为抗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.12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.13所示。所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.11所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.1所示。二聚体形式的三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。
本发明的另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域。
进一步地，所述三功能分子能够在识别CD19的同时，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、第二功能域和第三功能域并无特殊限制，只要能够在识别CD19的同时，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CD19的抗体，所述第二功能域可以是抗CD3的抗体，所述第三功能域可以是抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2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，可以是所述第一功能域的C末端与所述第二功能域的N末端连接；所 述第二功能域的C末端与所述第三功能域的N末端连接。本发明对于连接片段1和连接片段2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1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.44所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.46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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.48所示。所述连接片段2的氨基酸序列如SEQ ID NO.50所示。所述连接片段2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，所述三功能分子的结构示意图如图2-1所示。所述三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三功能分子的结构示意图如图2-1A所示，所述三功能分子的结构中含有一个与CD19抗原结合的第一功能域，一个与CD3抗原结合的第二功能域，一个与T细胞正共刺激分子抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与T细胞正共刺激分子抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的结构示意图如图2-1B所示，所述三功能分子的结构中含有两个与CD19抗原结合的第一功能域，两个与CD3抗原结合的第二功能域，两个与T细胞正共刺激分子抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与T细胞正共刺激分子抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的抗原结合效价是单体形式的二倍。由于T细胞活化第一信号(CD3)和第二信号(T细胞正共刺激分子)的加倍，致使T细胞活化更为充分，对靶细胞的杀伤效果更强；CD19单链抗体结构域的加倍使其对靶细胞的识别也更为精准，因此二聚体较单体具有更好的使用效果。
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.52所示，具体为：
Figure PCTCN2017096594-appb-000037
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.53所示，具体为：
Figure PCTCN2017096594-appb-000038
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.54所示，具体为：
Figure PCTCN2017096594-appb-000039
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.55所示，具体为：
Figure PCTCN2017096594-appb-000040
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.56所示，具体为：
Figure PCTCN2017096594-appb-000041
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.57所示，具体为：
Figure PCTCN2017096594-appb-000042
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.58所示，具体为：
Figure PCTCN2017096594-appb-000043
具体地，所述第一功能域为抗CD19的单链抗体。所述抗CD19的单链抗体包括重链可变区和轻链可变区。所述抗CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.84所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.85所示。进一步地，所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.83所示。
所述第二功能域为抗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.87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.88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.86所示。
所述第三功能域为抗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.90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.91所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.89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.93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.94所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.92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.96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.97所示。所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.95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.99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.100所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.98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.102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.103所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.101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.105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.106所示。所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.104所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.59、SEQ ID NO.63、SEQ ID NO.67、SEQ ID NO.71、SEQ ID NO.75或SEQ ID NO.79之任一所示。二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.61、SEQ ID NO.65、SEQ ID NO.69、SEQ ID NO.73、SEQ ID NO.77或SEQ ID NO.81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。
本发明的另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域。
进一步地，所述三功能分子，能够在结合于CD19的同时，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4-1BB、ICOS、OX40、GITR、CD40L或CD27。
本发明对于第一功能域、第二功能域和第三功能域并无特殊限制，只要能够在识别CD19的同时，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CD19的抗体，所述第二功能域可以是抗CD3的抗体，所述第三功能域可以是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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2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，可以是所述第一功能域的C末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。本发明对于连接片段1和连接片段2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1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.159所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.161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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.163所示。所述连接片段2的氨基酸序列如SEQ ID NO.165所示。所述连接片段2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，所述三功能分子的结构示意图如图3-1所示。所述三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三功能分子的结构示意图如图3-1中A所示，所述三功能分子的结构中含有一个与CD19抗原结合的第一功能域，一个与CD3抗原结合的第二功能域，一个与T细胞正共刺激分子结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为T细胞正共刺激分子的配体胞外区结构域。本发明的二聚体形式的三功能分子的结构示意图如图3-1中B所示，所述三功能分子的结构中含有两个与CD19抗原结合的第一功能域，两个与CD3抗原结合的第二功能域，两个与T细胞正共刺激分子结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为T细胞正共刺激分子的配体胞外区结构域。本发明的二聚体形式的三功能分子的抗原结合效价是单体形式的二倍。由于T细胞活化第一信号(CD3)和第二信号(正共刺激信号)的加倍，致使T细胞活化更为充分，对靶细胞的杀伤效果更强；CD19 scFv结构域的加倍使其对靶细胞的识别也更为精准，因此二聚体较单体具有更好的使用效果。
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.167所示，其配体为人类4-1BBL(UniProt ID：P41273)，氨基酸序列如SEQ ID NO.168所示。
SEQ ID NO.167：
Figure PCTCN2017096594-appb-000044
SEQ ID NO.168：
Figure PCTCN2017096594-appb-000045
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.169所示，其配体为人类B7RP-1(UniProt ID：O75144)，氨基酸序列如SEQ ID NO.170所示。
SEQ ID NO.169：
Figure PCTCN2017096594-appb-000046
SEQ ID NO.170：
Figure PCTCN2017096594-appb-000047
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.171所示，其配体为人类OX40L(UniProt ID：P23510)，氨基酸序列如SEQ ID NO.172 所示。
SEQ ID NO.171：
Figure PCTCN2017096594-appb-000048
SEQ ID NO.172：
Figure PCTCN2017096594-appb-000049
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.173所示，其配体为人类GITRL(UniProt ID：Q9UNG2)，氨基酸序列如SEQ ID NO.174所示。
SEQ ID NO.173：
Figure PCTCN2017096594-appb-000050
SEQ ID NO.174：
Figure PCTCN2017096594-appb-000051
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.175所示，其配体为人类CD70(UniProt ID：P32970)，氨基酸序列如SEQ ID NO.176所示。
SEQ ID NO.175：
Figure PCTCN2017096594-appb-000052
SEQ ID NO.176：
Figure PCTCN2017096594-appb-000053
具体地，所述第一功能域为抗CD19的单链抗体。所述抗CD19的单链抗体包括重链可变区和轻链可变区。所述抗CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.198所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.199所示。进一步地，所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.197所示。
所述第二功能域为抗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.201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.202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.200所示。
所述第三功能域为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胞外区结构域之任一。
所述4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.203所示。
所述B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.204所示。
所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.205所示。
所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.206所示。
所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.207所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.177、SEQ ID NO.181、SEQ ID NO.185、SEQ ID NO.189或SEQ ID NO.193之任一所示。二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.179、SEQ ID NO.183、SEQ ID NO.187、SEQ ID NO.191或SEQ ID NO.195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。
本发明的另一种三功能分子，其结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺激分子的第三功能域。
进一步地，所述三功能分子能够在识别CD19的同时，结合并激活T细胞表面CD3分子、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、第二功能域和第三功能域并无特殊限制，只要能够在识别CD19的同时，结合并激活T细胞表面CD3分子、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CD19的抗体，所述第二功能域可以是抗CD3的抗体，所述第三功能域可以是抗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之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2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，可以是所述第一功能域的C末端与所述第二功能域的N末端连接；所述第二功能域的C末端与所述第三功能域的N末端连接。本发明对于连接片段1和连接片段2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1和连接片段2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1连接，第二功能域和第三功能域之间通过以G4S为单位的连接片段2连接。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.244所示。所述连接片段2含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.246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以G4S为单位的连接片段1连接，第二功能域和第三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。所述连接片段1含有一个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.248所示。所述连接片段2的氨基酸序列如SEQ ID NO.250所示。所述连接片段2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，所述三功能分子的结构示意图如图4-1所示。所述三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三功能分子的结构示意图如图4-1中A所示，所述三功能分子的结构中含有一个与CD19抗原结合的第一功能域，一个与CD3抗原结合的第二功能域，一个与T细胞负共刺激分子抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与T细胞负共刺激分子抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的结构示意图如图4-1中B所示，所述三功能分子的结构中含有两个与CD19抗原结合的第一功能域，两个与CD3抗原结合的第二功能域，两个与T细胞负共刺激分子抗原结合的第三功能域，所述第一功能域为与CD19抗原结合的单链抗体(scFv)，所述第二功能域为与CD3抗原结合的单链抗体(scFv)，所述第三功能域为与T细胞负共刺激分子抗原结合的单链抗体(scFv)。本发明的二聚体形式的三功能分子的抗原结合效价是单体形式的二倍。由于T细胞活化第一信号(CD3)和第二信号(负共刺激信号被阻断)的加倍，致使T细胞活化更为充分，对靶细胞的杀伤效果更强；CD19单链抗体结构域的加倍使其对靶细胞的识别也更为精准，因此二聚体较单体具有更好的使用效果。
所述T细胞负共刺激分子可以是PD-1、CTLA-4、LAG-3、TIM-3、TIGIT和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.252所示，具体为：
Figure PCTCN2017096594-appb-000054
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.253所示，具体为：
Figure PCTCN2017096594-appb-000055
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.254所示，具体为：
Figure PCTCN2017096594-appb-000056
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.255所示，具体为：
Figure PCTCN2017096594-appb-000057
Figure PCTCN2017096594-appb-000058
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.256所示，具体为：
Figure PCTCN2017096594-appb-000059
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.257所示，具体为：
Figure PCTCN2017096594-appb-000060
具体地，所述第一功能域为抗CD19的单链抗体。所述抗CD19的单链抗体包括重链可变区和轻链可变区。所述抗CD19的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.283所示。所述抗CD19的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.284所示。进一步地，所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.282所示。
所述第二功能域为抗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.286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.287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.285所示。
所述第三功能域为抗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。
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.289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.290所示。所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.288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.292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.293所示。所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.291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.295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.296所示。所述抗LAG-3的单链抗体的氨基酸序列如SEQ ID NO.294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.298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.299所示。所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.297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.301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.302所示。所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.300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.304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.305所示。所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.303所示。
在本案的较佳案例中，单体形式的三功能分子的氨基酸序列如SEQ ID NO.258、SEQ ID NO.262、SEQ ID NO.266、SEQ ID NO.270、SEQ ID NO.274或SEQ ID NO.278之任一所示。 二聚体形式的三功能分子的氨基酸序列如SEQ ID NO.260、SEQ ID NO.264、SEQ ID NO.268、SEQ ID NO.272、SEQ ID NO.276或SEQ ID NO.280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、编码三功能分子的多核苷酸
本发明的编码所述三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。
本发明的编码所述三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CD19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CD19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CD19的单链抗体的核苷酸序列如SEQ ID NO.14所示。
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.18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.19所示。
编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.17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.21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.22所示。
编码所述抗CD28的单链抗体的核苷酸序列如SEQ ID NO.20所示。
编码氨基酸序列如SEQ ID NO.23所示的连接片段1的核苷酸序列如SEQ ID NO.24所示。
编码氨基酸序列如SEQ ID NO.25所示的连接片段2的核苷酸序列如SEQ ID NO.26所示。
编码氨基酸序列如SEQ ID NO.27所示的连接片段1的核苷酸序列如SEQ ID NO.28所示。
编码氨基酸序列如SEQ ID NO.29所示的连接片段2的核苷酸序列如SEQ ID NO.30所示。
进一步地，编码单体形式的三功能分子的核苷酸序列如SEQ ID NO.2所示。编码二聚体形式的三功能分子的核苷酸序列如SEQ ID NO.4所示。
在本发明的另一些较佳实施例中，编码所述抗CD19的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.108所示。编码所述抗CD19的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.109所示。编码所述抗CD19的单链抗体的核苷酸序列如SEQ ID NO.107所示。
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.111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.112所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.110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.114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.115所示。编码所述抗4-1BB的单链抗体的核苷酸序列如SEQ ID NO.113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.117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.118所示。编码所述抗ICOS的单链抗体的核苷酸序列如SEQ ID NO.116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.120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.121所示。编码所述抗 OX40单链抗体的核苷酸序列如SEQ ID NO.119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.123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.124所示。编码所述抗GITR单链抗体的核苷酸序列如SEQ ID NO.122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.126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.127所示。编码所述抗CD40L单链抗体的核苷酸序列如SEQ ID NO.125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.129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.130所示。编码所述抗CD27单链抗体的核苷酸序列如SEQ ID NO.128所示。
编码氨基酸序列如SEQ ID NO.44所示的连接片段1的核苷酸序列如SEQ ID NO.45所示。
编码氨基酸序列如SEQ ID NO.46所示的连接片段2的核苷酸序列如SEQ ID NO.47所示。
编码氨基酸序列如SEQ ID NO.48所示的连接片段1的核苷酸序列如SEQ ID NO.49所示。
编码氨基酸序列如SEQ ID NO.50所示的连接片段2的核苷酸序列如SEQ ID NO.51所示。
进一步地，编码单体形式的三功能分子的核苷酸序列如SEQ ID NO.60、SEQ ID NO.64、SEQ ID NO.68、SEQ ID NO.72、SEQ ID NO.76或SEQ ID NO.80之任一所示。编码二聚体形式的三功能分子的核苷酸序列如SEQ ID NO.62、SEQ ID NO.66、SEQ ID NO.70、SEQ ID NO.74、SEQ ID NO.78或SEQ ID NO.82之任一所示。
在本发明的另一些较佳实施例中，编码所述抗CD19的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.209所示。编码所述抗CD19的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.210所示。编码所述抗CD19的单链抗体的核苷酸序列如SEQ ID NO.208所示。
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.212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.213所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.211所示。
编码所述4-1BBL胞外区结构域的核苷酸序列如SEQ ID NO.214所示。
编码所述B7RP-1胞外区结构域的核苷酸序列如SEQ ID NO.215所示。
编码所述OX40L胞外区结构域的核苷酸序列如SEQ ID NO.216所示。
编码所述GITRL胞外区结构域的核苷酸序列如SEQ ID NO.217所示。
编码所述CD70胞外区结构域的核苷酸序列如SEQ ID NO.218所示。
编码氨基酸序列如SEQ ID NO.159所示的连接片段的核苷酸序列如SEQ ID NO.160所示。
编码氨基酸序列如SEQ ID NO.161所示的连接片段的核苷酸序列如SEQ ID NO.162所示。
编码氨基酸序列如SEQ ID NO.163所示的连接片段的核苷酸序列如SEQ ID NO.164所示。
编码氨基酸序列如SEQ ID NO.165所示的连接片段的核苷酸序列如SEQ ID NO.166所示。
进一步地，编码单体形式的三功能分子的核苷酸序列如SEQ ID NO.178、SEQ ID NO.182、SEQ ID NO.186、SEQ ID NO.190或SEQ ID NO.194之任一所示。编码二聚体形式的三功能分子的核苷酸序列如SEQ ID NO.180、SEQ ID NO.184、SEQ ID NO.188、SEQ ID  NO.192或SEQ ID NO.196之任一所示。
在本发明的另一些较佳实施例中，编码所述抗CD19的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.307所示。编码所述抗CD19的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.308所示。编码所述抗CD19的单链抗体的核苷酸序列如SEQ ID NO.306所示。
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.310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.311所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.309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.313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.314所示。编码所述抗PD-1的单链抗体的核苷酸序列如SEQ ID NO.312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.316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.317所示。编码所述抗CTLA-4的单链抗体的核苷酸序列如SEQ ID NO.315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.319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.320所示。编码所述抗LAG-3的单链抗体的核苷酸序列如SEQ ID NO.318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.322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.323所示。编码所述抗TIM-3的单链抗体的核苷酸序列如SEQ ID NO.321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.325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.326所示。编码所述抗TIGIT的单链抗体的核苷酸序列如SEQ ID NO.324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.328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.329所示。编码所述抗BTLA的单链抗体的核苷酸序列如SEQ ID NO.327所示。
编码氨基酸序列如SEQ ID NO.244所示的连接片段1的核苷酸序列如SEQ ID NO.245所示。
编码氨基酸序列如SEQ ID NO.246所示的连接片段2的核苷酸序列如SEQ ID NO.247所示。
编码氨基酸序列如SEQ ID NO.248所示的连接片段1的核苷酸序列如SEQ ID NO.249所示。
编码氨基酸序列如SEQ ID NO.250所示的连接片段2的核苷酸序列如SEQ ID NO.251所示。
进一步地，编码单体形式的三功能分子的核苷酸序列如SEQ ID NO.259、SEQ ID NO.263、SEQ ID NO.267、SEQ ID NO.271、SEQ ID NO.275或SEQ ID NO.279之任一所示。编码二聚体形式的三功能分子的核苷酸序列如如SEQ ID NO.261、SEQ ID NO.265、SEQ ID NO.269、SEQ ID NO.273、SEQ ID NO.277或SEQ ID NO.281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三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、制备三功能分子的方法
本发明的制备前述三功能分子的方法，包括：构建含有三功能分子基因序列的表达载体，然后将含三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三功能分子。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、三功能分子的用途
本发明的三功能分子可用于肿瘤治疗药物。所述肿瘤为细胞表面为CD19阳性的肿瘤。
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，用本发明所制备的上述单体形式的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域的三功能分子、二聚体形式的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域的三功能分子以及购买的抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+)。结果发现，在添加本发明的三功能分子后，CIK细胞对Raji细胞的杀伤效率均有显著的提高，对CD19阳性肿瘤细胞的靶向杀伤活性均优于抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)。
本发明另一些较佳实施方式中，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域的三功能分子均具有与CD19、CD3和相应T细胞正共刺激分子重组抗原的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且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，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域的三功能分子均具有与CD19重组抗原、CD3重组抗原和相应T细胞正共刺激分子重组蛋白的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且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，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺激分子的第三功能域的三功能分子均具有与CD19、CD3和相应T细胞负共刺激分子重组抗原的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且二聚体较单体具有更好的效果。
七、肿瘤治疗药物组合物
本发明的所述肿瘤治疗药物组合物，含有前述三功能分子及至少一种药学可接受的载体或赋形剂。所述肿瘤为细胞表面为CD19阳性的肿瘤。
本发明所提供的药物组合物可以多种剂型存在，如用于静脉注射等的注射剂，用于皮下注射、表皮外敷等的经皮吸收剂，用于喷鼻、喉、口腔、表皮、粘膜等的喷雾剂，用于滴鼻、眼、耳等的滴剂，用于肛肠等的栓剂、片剂、粉剂、粒剂、胶囊、口服液、膏剂、霜剂等多种形式，及肺部给药制剂及其他非肠道给药的组合物。上述各种剂型的药物均可以按照药学领域的常规方法制备。
所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。该药用组合物还可以加入香味剂、甜味剂 等。
如上所述的药物制剂可对哺乳动物临床使用，包括人和动物，可以经静脉注射或者口、鼻、皮肤、肺吸入等途径给药。上述药物的优选周剂量为0.1-5mg/kg体重，优选的疗程为10至30天。一次性给药，或分次给药。无论采用何种给药方法，个体人的最佳剂量应根据具体的治疗而定。
八、体外治疗肿瘤的方法
本发明的体外治疗肿瘤的方法，包括将前述三功能分子或肿瘤治疗药物组合物施用于于肿瘤患者。所述肿瘤为细胞表面为CD19阳性的肿瘤。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，用本发明所制备的上述单体形式的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域的三功能分子、二聚体形式的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞表面CD28分子的第三功能域的三功能分子以及购买的抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+)。结果发现，在添加本发明的三功能分子后，CIK细胞对Raji细胞的杀伤效率均有显著的提高，对CD19阳性肿瘤细胞的靶向杀伤活性均优于抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)。
本发明针对抗CD19/抗CD3 BiTE双特异性抗体以及靶向CD19的CAR-T技术的不足，通过基因工程和抗体工程的方法构建了能同时识别CD19，CD3及CD28的三功能分子。该分子在制备工艺和实际应用方面具有明显的优势：在赋予T细胞对CD19阳性细胞靶向性的同时进一步提高了活化T细胞的功效，单独添加时所介导的T细胞对CD19阳性靶细胞的结合与杀伤效果均优于抗CD19/抗CD3 BiTE双特异性抗体，在使用的方便性上优于靶向CD19的CAR-T技术。
本发明另一些较佳实施例中，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域的三功能分子均具有与CD19、CD3和相应T细胞正共刺激分子重组抗原的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且二聚体较单体具有更好的效果。
本发明针对抗CD19/抗CD3 BiTE双特异性抗体以及靶向CD19的CAR-T技术的不足，通过基因工程和抗体工程的方法构建了能同时识别CD19，CD3及任一T细胞正共刺激分子的三功能分子。该分子在制备工艺和实际应用方面具有明显的优势：在赋予T细胞对CD19阳性细胞靶向性的同时进一步提高了活化T细胞的功效，单独添加时所介导的T细胞对CD19阳性靶细胞的杀伤效果均优于抗CD19/抗CD3 BiTE双特异性抗体，在使用的方便性上优于靶向CD19的CAR-T技术。
本发明另一些较佳实施例中，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域的三功能分子均具有与CD19重组抗原、CD3重组抗原和相应T细胞正共刺激分子重组蛋白的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且二聚体较单体具有更好的效果。
本发明针对抗CD19/抗CD3 BiTE双特异性抗体以及靶向CD19的CAR-T技术的不足，通过基因工程和抗体工程的方法构建了能同时识别CD19，CD3及任一T细胞正共刺激分子的三特异性分子(Tri-specific Molecule，TsM)。该分子在制备工艺和实际应用方面具有明显的优势：在赋予T细胞对CD19阳性细胞靶向性的同时进一步提高了活化T细胞的功效， 单独添加时所介导的T细胞对CD19阳性靶细胞的杀伤效果均优于抗CD19/抗CD3 BiTE双特异性抗体，在使用的方便性上优于靶向CD19的CAR-T技术。
本发明另一些较佳实施例中，通过试验发现，本发明所制备的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺激分子的第三功能域的三功能分子均具有与CD19、CD3和相应T细胞负共刺激分子重组抗原的体外结合活性，可促进T细胞对CD19阳性靶细胞的靶向杀伤，并且二聚体较单体具有更好的效果。
本发明针对抗CD19/抗CD3 BiTE双特异性抗体以及靶向CD19的CAR-T技术的不足，通过基因工程和抗体工程的方法构建了能同时识别CD19，CD3及任一T细胞负共刺激分子的三特异性抗体分子(Tri-specific Antibody，TsAb)。该分子在制备工艺和实际应用方面具有明显的优势：在赋予T细胞对CD19阳性细胞靶向性的同时进一步提高了活化T细胞的功效，单独添加时所介导的T细胞对CD19阳性靶细胞的杀伤效果均优于抗CD19/抗CD3 BiTE双特异性抗体，在使用的方便性上优于靶向CD19的CAR-T技术。
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，1989 and Third edition，2001；Ausubel等，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(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 CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和CD28蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-CD28 TsAb。
一、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D构建方案设计
单体形式的CD19-CD3-CD28 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD28 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD28 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-CD28 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD28 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD28 scFv序列之间以IgD铰链区 (Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗CD28 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.15所示，具体为：
Figure PCTCN2017096594-appb-000061
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.16所示，具体为：
Figure PCTCN2017096594-appb-000062
抗CD19 scFv的核苷酸序列如SEQ ID NO.14所示，具体为：
Figure PCTCN2017096594-appb-000063
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.18所示，具体为：
Figure PCTCN2017096594-appb-000064
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.19所示，具体为：
Figure PCTCN2017096594-appb-000065
Figure PCTCN2017096594-appb-000066
抗CD3 scFv的核苷酸序列如SEQ ID NO.17所示，具体为：
Figure PCTCN2017096594-appb-000067
抗CD28 scFv的重链可变区的核苷酸序列如SEQ ID NO.21所示，具体为：
Figure PCTCN2017096594-appb-000068
抗CD28 scFv的轻链可变区的核苷酸序列如SEQ ID NO.22所示，具体为：
Figure PCTCN2017096594-appb-000069
抗CD28 scFv的核苷酸序列如SEQ ID NO.20所示，具体为：
Figure PCTCN2017096594-appb-000070
Figure PCTCN2017096594-appb-000071
单体形式的CD19-CD3-CD28 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.24所示，具体为：
Figure PCTCN2017096594-appb-000072
单体形式的CD19-CD3-CD28 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.26所示，具体为：
Figure PCTCN2017096594-appb-000073
二聚体形式的CD19-CD3-CD28 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.28所示，具体为：
Figure PCTCN2017096594-appb-000074
二聚体形式的CD19-CD3-CD28 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.30所示，具体为：
Figure PCTCN2017096594-appb-000075
为使三特异性抗体在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.31所示，具体为：
Figure PCTCN2017096594-appb-000076
该分泌表达信号肽的核苷酸序列如SEQ ID NO.32所示，具体为：
Figure PCTCN2017096594-appb-000077
二、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-CD28 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-CD28-F和pcDNA3.1-CD28-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗CD28 scFv的基因序列；针对CD19-CD3-CD28 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD28-F和pcDNA3.1-CD28-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗CD28 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000078
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三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，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CD19-CD3-CD28 TsAb和二聚体形式的CD19-CD3-CD28 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-CD28 TsAb_M的核苷酸序列如SEQ ID NO.2所示，具体为：
Figure PCTCN2017096594-appb-000079
二聚体形式的CD19-CD3-CD28 TsAb_D的核苷酸序列如SEQ ID NO.4所示，具体为：
Figure PCTCN2017096594-appb-000080
Figure PCTCN2017096594-appb-000081
表1-1.三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000082
实施例1-2 CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D的表达与纯化
一、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_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(CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_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、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_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CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_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，CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-CD28 TsAb_M重组蛋白的理论分子量为81.3kDa，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，因此该三特异性抗体为单体形式(图1-2A)；CD19-CD3-CD28 TsAb_D重组蛋白的理论分子量为89.1kDa，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(～180kDa)(图1-2B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-CD28 TsAb_M为单体形式，CD19-CD3-CD28 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-CD28 TsAb_M的氨基酸序列如SEQ ID NO.1所示，具体为：
Figure PCTCN2017096594-appb-000083
二聚体形式的CD19-CD3-CD28 TsAb_D的氨基酸序列如SEQ ID NO.3所示，具体为：
Figure PCTCN2017096594-appb-000084
Figure PCTCN2017096594-appb-000085
抗CD19 scFv的氨基酸序列如SEQ ID NO.5所示，具体为：
Figure PCTCN2017096594-appb-000086
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.6所示，具体为：
Figure PCTCN2017096594-appb-000087
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.7所示，具体为：
Figure PCTCN2017096594-appb-000088
抗CD3 scFv的氨基酸序列如SEQ ID NO.8所示，具体为：
Figure PCTCN2017096594-appb-000089
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.9所示，具体为：
Figure PCTCN2017096594-appb-000090
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.10所示，具体为：
Figure PCTCN2017096594-appb-000091
抗CD28 scFv的氨基酸序列如SEQ ID NO.11所示，具体为：
Figure PCTCN2017096594-appb-000092
抗CD28 scFv的重链可变区的氨基酸序列如SEQ ID NO.12所示，具体为：
Figure PCTCN2017096594-appb-000093
抗CD28 scFv的轻链可变区的氨基酸序列如SEQ ID NO.13所示，具体为：
Figure PCTCN2017096594-appb-000094
单体形式的CD19-CD3-CD28 TsAb_M中连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.23所示，具体为：GGGGS。
单体形式的CD19-CD3-CD28 TsAb_M中连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.25所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD19-CD3-CD28 TsAb_D中连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.27所示，具体为：GGGGS。
二聚体形式的CD19-CD3-CD28 TsAb_D中连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.29所示，具体为：
Figure PCTCN2017096594-appb-000095
实施例1-3：ELISA检测CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-CD28 TsAb_M或CD19-CD3-CD28 TsAb_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标记的抗His6融合标签抗体(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(OD450)。
ELISA结果如图1-3A和图1-3B所示：图1-3A说明CD19-CD3-CD28 TsAb_M与重组抗原CD19-hFc、CD3-hFc和CD28-hFc均具有体外结合活性，其中CD28结合活性活性最高，CD19结合活性次之，CD3结合活性较弱；图1-3B说明CD19-CD3-CD28 TsAb_D与重组抗原CD19-hFc、CD3-hFc和CD28-hFc同样具有体外结合活性，其中CD28结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例1-4：三特异抗体及双特异性抗体介导的细胞衔接实验
以CCL-86 Raji淋巴瘤细胞(购自ATCC)作为CD19阳性的靶细胞，TIB-152 Jurkat细胞(购自ATCC)作为CD3和CD28阳性的效应细胞，比较本发明所述单体形式的TiTE三特异抗体(CD19-CD3-CD28 TsAb_M)、二聚体形式的TiTE三特异性抗体(CD19-CD3-CD28 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)介导的细胞衔接活性差异。
细胞衔接实验步骤：
1、取Raji细胞～1×105个，设置高、中、低浓度3个实验组，分别添加终浓度为45、 0.45、0.0045ng/ml的CD19-CD3 BsAb、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D，静置5min；以未添加任何抗体的细胞作为空白对照；
2、取相同数量的Jurkat细胞，分别添加到上述Raji细胞样品中，37℃培养箱放置1h，取出细胞轻柔摇晃30s，静置2min，显微镜下观察细胞成团情况并拍照；
结果如图1-4所示：在未添加任何抗体的条件下，Raji细胞与Jurkat细胞无聚集成团(图1-4A)，说明两种细胞之间不存在非特异性衔接；在添加高浓度抗体(45ng/ml)的条件下，3个实验组中的Raji细胞与Jurkat细胞均明显成团(图1-4B-D)，说明两种形式的TiTE三特异抗体(CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D)与抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)在较高浓度下细胞衔接活性相差不大；在添加中浓度抗体(0.45ng/ml)的条件下，CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D仍可导致Raji细胞与Jurkat细胞明显成团，CD19-CD3 BsAb可导致两种细胞少量成团(图1-4E-G)，说明两种形式的TiTE三特异抗体在中等浓度下细胞衔接活性均优于BiTE双特异性抗体；在添加低浓度抗体(0.0045ng/ml)的条件下，CD19-CD3-CD28 TsAb_D仍可导致Raji细胞与Jurkat细胞明显成团，CD19-CD3-CD28 TsAb_M可导致两种细胞少量成团，而CD19-CD3 BsAb无法使细胞成团(图1-4H-J)，说明二聚体形式的TiTE三特异性抗体在低浓度下细胞衔接活性优于单体形式的TiTE三特异性抗体，而BiTE双特异性抗体在低浓度下无细胞衔接活性。
实施例1-5：三特异抗体及双特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体CD19-CD3-CD28 TsAb_M、二聚体形式的TiTE三特异抗体CD19-CD3-CD28 TsAb_D以及购买的抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的CIK细胞1×105个，加入Raji细胞5×105个(CIK效应细胞∶Raji靶细胞(E∶T比)为1∶5)或1×105个(E∶T比1∶1)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-CD28 TsAb_M和CD19-CD3-CD28 TsAb_D抗体样品，室温混匀3-5min，37℃共培养3h后，每孔添加10uL的CCK8，37度继续反应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 以未添加任何抗体的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000096
结果如图1-5所示：当CIK效应细胞∶Raji靶细胞(E∶T比)分别为1∶5和1∶1时，在未添加任何抗体的条件下，3h细胞杀伤效率约为17％(图1-5A)和21％(图1-5B)；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-CD28 TsAb_D所介导的细胞杀伤效果最好、当E∶T比为1∶5时，杀伤效率分别约为36％、29％和30％，当E∶T比为1∶1时，杀伤效率分别约为85％、90％和85％，CD19-CD3-CD28 TsAb_M的效果次之、当E∶T比为1∶5时，杀伤效率分别约为30％、23％和26％，当E∶T比为1∶1时，杀伤效率约为86％、82％和81％，CD19-CD3 BsAb的效果最弱、当E∶T比为1∶5时，杀伤效率分别约为23％、22％和22％，当E∶T比为1∶1时，杀伤效率分别约为80％、55％和56％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-CD28 TsAb_D与CD19-CD3-CD28 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高、当E∶T为1∶5时，杀伤效率分别约为23％和22％，当E∶T比为1∶1时，杀伤效率分别约为82％和70％，而CD19-CD3 BsAb与空白对照相比基本没有效果，上述结果说明两种形式的TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-1：CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子4-1BB蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-4-1BB TsAb。
一、CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D构建方案设计
单体形式的CD19-CD3-4-1BB TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗4-1BB scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗4-1BB scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-4-1BB TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗4-1BB scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗4-1BB scFv序列之间以IgD铰链区(Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗4-1BB scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示，具体为：
Figure PCTCN2017096594-appb-000097
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示，具体为：
Figure PCTCN2017096594-appb-000098
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示，具体为：
Figure PCTCN2017096594-appb-000099
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示，具体为：
Figure PCTCN2017096594-appb-000100
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示，具体为：
Figure PCTCN2017096594-appb-000101
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示，具体为：
Figure PCTCN2017096594-appb-000102
Figure PCTCN2017096594-appb-000103
抗4-1BB scFv的重链可变区的核苷酸序列如SEQ ID NO.114所示，具体为：
Figure PCTCN2017096594-appb-000104
抗4-1BB scFv的轻链可变区的核苷酸序列如SEQ ID NO.115所示，具体为：
Figure PCTCN2017096594-appb-000105
抗4-1BB scFv的核苷酸序列如SEQ ID NO.113所示，具体为：
Figure PCTCN2017096594-appb-000106
单体形式的CD19-CD3-4-1BB TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.45所示，具体为：GGTGGCGGAGGGTCC。
单体形式的CD19-CD3-4-1BB TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.47所示，具体为：
Figure PCTCN2017096594-appb-000107
二聚体形式的CD19-CD3-4-1BB TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示，具体为：GGTGGCGGAGGGTCC。
二聚体形式的CD19-CD3-4-1BB TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示，具体为：
Figure PCTCN2017096594-appb-000108
为使三特异性抗体在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.131所示，具体为：
Figure PCTCN2017096594-appb-000109
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示，具体为：
Figure PCTCN2017096594-appb-000110
二、CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-4-1BB TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-4-1BB-F和pcDNA3.1-4-1BB-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗4-1BB scFv的基因序列；针对CD19-CD3-4-1BB TsAb_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗4-1BB scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-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三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，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CD19-CD3-4-1BB TsAb_M和二聚体形式的CD19-CD3-4-1BB TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-4-1BB TsAb_M的核苷酸序列如SEQ ID NO.60所示，具体为：
Figure PCTCN2017096594-appb-000112
Figure PCTCN2017096594-appb-000113
二聚体形式的CD19-CD3-4-1BB TsAb_D的核苷酸序列如SEQ ID NO.62所示，具体为：
Figure PCTCN2017096594-appb-000114
Figure PCTCN2017096594-appb-000115
表2-1.CD19-CD3-4-1BB三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000116
Figure PCTCN2017096594-appb-000117
实施例2-2：CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D的表达与纯化
一、CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_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(CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_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、CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_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CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_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，CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D重组蛋白的纯 度均＞95％：其中CD19-CD3-4-1BB TsAb_M重组蛋白的理论分子量为80.6kDa，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，因此该三特异性抗体为单体形式(图2-2A)；CD19-CD3-4-1BB TsAb_D重组蛋白的理论分子量为88.4kDa，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(约180kDa)(图2-2B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-4-1BB TsAb_M为单体形式，CD19-CD3-4-1BB TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-4-1BB TsAb_M的氨基酸序列如SEQ ID NO.59所示，具体为：
Figure PCTCN2017096594-appb-000118
二聚体形式的CD19-CD3-4-1BB TsAb_D的氨基酸序列如SEQ ID NO.61所示，具体为：
Figure PCTCN2017096594-appb-000119
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示，具体为：
Figure PCTCN2017096594-appb-000120
Figure PCTCN2017096594-appb-000121
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示，具体为：
Figure PCTCN2017096594-appb-000122
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示，具体为：
Figure PCTCN2017096594-appb-000123
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示，具体为：
Figure PCTCN2017096594-appb-000124
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示，具体为：
Figure PCTCN2017096594-appb-000125
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示，具体为：
Figure PCTCN2017096594-appb-000126
抗4-1BB scFv的氨基酸序列如SEQ ID NO.89所示，具体为：
Figure PCTCN2017096594-appb-000127
抗4-1BB scFv的重链可变区的氨基酸序列如SEQ ID NO.90所示，具体为：
Figure PCTCN2017096594-appb-000128
抗4-1BB scFv的轻链可变区的氨基酸序列如SEQ ID NO.91所示，具体为：
Figure PCTCN2017096594-appb-000129
单体形式的CD19-CD3-4-1BB TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示，具体为：GGGGS。
单体形式的CD19-CD3-4-1BB TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD19-CD3-4-1BB TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示，具体为：GGGGS。
二聚体形式的CD19-CD3-4-1BB TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示，具体为：
Figure PCTCN2017096594-appb-000130
实施例2-3：ELISA检测CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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 Na2HPO4，0.24gNaH2PO4，0.2g 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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-4-1BB TsAb_M或CD19-CD3-4-1BB TsAb_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(OD450)。
ELISA结果如图2-3A和图2-3B所示：图2-3A说明CD19-CD3-4-1BB TsAb_M与重组抗原CD19-hFc、CD3-hFc和4-1BB-hFc均具有体外结合活性，其中4-1BB结合活性最高，CD19结合活性次之，CD3结合活性较弱；图2-3B说明CD19-CD3-4-1BB TsAb_D与重组抗原CD19-hFc、CD3-hFc和4-1BB-hFc同样具有体外结合活性，其中4-1BB结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例2-4：CD19-CD3-4-1BB三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-4-1BB TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-4-1BB TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率 差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-4-1BB TsAb_M和CD19-CD3-4-1BB TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000131
结果如图2-4所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-4-1BB TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为96％、96％和92％，CD19-CD3-4-1BB TsAb_M的效果次之，杀伤效率约为92％、90％和86％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-4-1BB TsAb_D与CD19-CD3-4-1BB TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为87％和80％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-4-1BB TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-5：CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子ICOS蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-ICOS TsAb。
一、CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D构建方案设计
单体形式的CD19-CD3-ICOS TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗ICOS scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗ICOS scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-ICOS TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗ICOS scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗ICOS scFv序列之间以IgD铰链区(Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗ICOS scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示。
抗ICOS scFv的重链可变区的核苷酸序列如SEQ ID NO.117所示，具体为：
Figure PCTCN2017096594-appb-000132
抗ICOS scFv的轻链可变区的核苷酸序列如SEQ ID NO.118所示，具体为：
Figure PCTCN2017096594-appb-000133
抗ICOS scFv的核苷酸序列如SEQ ID NO.116所示，具体为：
Figure PCTCN2017096594-appb-000134
单体形式的CD19-CD3-ICOS TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.45所示。
单体形式的CD19-CD3-ICOS TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.47所示。
二聚体形式的CD19-CD3-ICOS TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示。
二聚体形式的CD19-CD3-ICOS TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示。
为使三特异性抗体在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.131所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示。
二、CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-ICOS TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗ICOS scFv的基因序列；针对CD19-CD3-ICOS TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗ICOS scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000135
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三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，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CD19-CD3-ICOS TsAb_M和二聚体形式的CD19-CD3-ICOS TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-ICOS TsAb_M的核苷酸序列如SEQ ID NO.64所示，具体为：
Figure PCTCN2017096594-appb-000136
Figure PCTCN2017096594-appb-000137
二聚体形式的CD19-CD3-ICOS TsAb_D的核苷酸序列如SEQ ID NO.66所示，具体为：
Figure PCTCN2017096594-appb-000138
Figure PCTCN2017096594-appb-000139
表2-2.CD19-CD3-ICOS三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000140
实施例2-6：CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D的表达与纯化
一、CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_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(CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-5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、CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_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CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-5所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-ICOS TsAb_M重组蛋白的理论分子量为80.7kDa，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，因此该三特异性抗体为单体形式(图2-5A)；CD19-CD3-ICOS TsAb_D重组蛋白的理论分子量为88.6kDa，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(约180kDa)(图2-5B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-ICOS TsAb_M为单体形式，CD19-CD3-ICOS TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-ICOS TsAb_M的氨基酸序列如SEQ ID NO.63所示，具体为：
Figure PCTCN2017096594-appb-000141
Figure PCTCN2017096594-appb-000142
二聚体形式的CD19-CD3-ICOS TsAb_D的氨基酸序列如SEQ ID NO.65所示，具体为：
Figure PCTCN2017096594-appb-000143
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示。
抗ICOS scFv的氨基酸序列如SEQ ID NO.92所示，具体为：
Figure PCTCN2017096594-appb-000144
抗ICOS scFv的重链可变区的氨基酸序列如SEQ ID NO.93所示，具体为：
Figure PCTCN2017096594-appb-000145
Figure PCTCN2017096594-appb-000146
抗ICOS scFv的轻链可变区的氨基酸序列如SEQ ID NO.94所示，具体为：
Figure PCTCN2017096594-appb-000147
单体形式的CD19-CD3-ICOS TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示。
单体形式的CD19-CD3-ICOS TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示。
二聚体形式的CD19-CD3-ICOS TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示。
二聚体形式的CD19-CD3-ICOS TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示。
实施例2-7：ELISA检测CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-ICOS TsAb_M或CD19-CD3-ICOS TsAb_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(OD450)。
ELISA结果如图2-6A和图2-6B所示：图2-6A说明CD19-CD3-ICOS TsAb_M与重组抗原CD19-hFc、CD3-hFc和ICOS-hFc均具有体外结合活性，其中ICOS结合活性最高，CD19结合活性次之，CD3结合活性较弱；图2-6B说明CD19-CD3-ICOS TsAb_D与重组抗原CD19-hFc、CD3-hFc和ICOS-hFc同样具有体外结合活性，其中ICOS结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例2-8：CD19-CD3-ICOS三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-ICOS TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-ICOS TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率 差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-ICOS TsAb_M和CD19-CD3-ICOS TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000148
结果如图2-7所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-ICOS TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为94％、94％和82％，CD19-CD3-ICOS TsAb_M的效果次之，杀伤效率约为92％、86％和84％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-ICOS TsAb_D与CD19-CD3-ICOS TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为76％和71％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-ICOS TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-9：CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子OX40蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-OX40 TsAb。
一、CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D构建方案设计
单体形式的CD19-CD3-OX40 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗OX40 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗OX40 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-OX40 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗OX40 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗OX40 scFv序列之间以IgD铰链区(Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗OX40 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示。
抗OX40 scFv的重链可变区的核苷酸序列如SEQ ID NO.120所示，具体为：
Figure PCTCN2017096594-appb-000149
抗OX40 scFv的轻链可变区的核苷酸序列如SEQ ID NO.121所示，具体为：
Figure PCTCN2017096594-appb-000150
抗OX40 scFv的核苷酸序列如SEQ ID NO.119所示，具体为：
Figure PCTCN2017096594-appb-000151
单体形式的CD19-CD3-OX40 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.45所示。
单体形式的CD19-CD3-OX40 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.47所示。
二聚体形式的CD19-CD3-OX40 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示。
二聚体形式的CD19-CD3-OX40 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示。
为使三特异性抗体在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.131所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示。
二、CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D真核表达载体构建
本发明三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三特异性抗体，分别设计了如表2-3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CD19-CD3-OX40 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-OX40-F和pcDNA3.1-OX40-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗OX40 scFv的基因序列；针对CD19-CD3-OX40 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-OX40-F和pcDNA3.1-OX40-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗OX40 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000152
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三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，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CD19-CD3-OX40 TsAb_M和二聚体形式的CD19-CD3-OX40 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-OX40 TsAb_M的核苷酸序列如SEQ ID NO.68所示，具体为：
Figure PCTCN2017096594-appb-000153
Figure PCTCN2017096594-appb-000154
二聚体形式的CD19-CD3-OX40 TsAb_D的核苷酸序列如SEQ ID NO.70所示，具体为：
Figure PCTCN2017096594-appb-000155
Figure PCTCN2017096594-appb-000156
表2-3.CD19-CD3-OX40三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000157
实施例2-10：CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D的表达与纯化
一、CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_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(CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-9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、CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_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CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-8所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-OX40 TsAb_M重组蛋白的理论分子量为80.1kDa，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，因此该三特异性抗体为单体形式(图2-8A)；CD19-CD3-OX40 TsAb_D重组蛋白的理论分子量为88.0kDa，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(约180kDa)(图2-8B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-OX40 TsAb_M为单体形式，CD19-CD3-OX40 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-OX40 TsAb_M的氨基酸序列如SEQ ID NO.67所示，具体为：
Figure PCTCN2017096594-appb-000158
Figure PCTCN2017096594-appb-000159
二聚体形式的CD19-CD3-OX40 TsAb_D的氨基酸序列如SEQ ID NO.69所示，具体为：
Figure PCTCN2017096594-appb-000160
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示。
抗OX40 scFv的氨基酸序列如SEQ ID NO.95所示，具体为：
Figure PCTCN2017096594-appb-000161
抗OX40 scFv的重链可变区的氨基酸序列如SEQ ID NO.96所示，具体为：
Figure PCTCN2017096594-appb-000162
抗OX40 scFv的轻链可变区的氨基酸序列如SEQ ID NO.97所示，具体为：
Figure PCTCN2017096594-appb-000163
单体形式的CD19-CD3-OX40 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示。
单体形式的CD19-CD3-OX40 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示。
二聚体形式的CD19-CD3-OX40 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示。
二聚体形式的CD19-CD3-OX40 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示。
实施例2-11：ELISA检测CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-OX40 TsAb_M或CD19-CD3-OX40 TsAb_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(OD450)。
ELISA结果如图2-9A和图2-9B所示：图2-9A说明CD19-CD3-OX40 TsAb_M与重组抗原CD19-hFc、CD3-hFc和OX40-hFc均具有体外结合活性，其中CD19结合活性最高，OX40结合活性次之，CD3结合活性较弱；图2-9B说明CD19-CD3-OX40 TsAb_D与重组抗原CD19-hFc、CD3-hFc和OX40-hFc同样具有体外结合活性，其中CD19结合活性最高，OX40结合活性次之，CD3结合活性较弱。
实施例2-12：CD19-CD3-OX40三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-OX40 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-OX40 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-OX40 TsAb_M和CD19-CD3-OX40 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000164
结果如图2-10所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-OX40 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为96％、92％和86％，CD19-CD3-OX40 TsAb_M的效果次之，杀伤效率约为89％、82％和80％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-OX40 TsAb_D与CD19-CD3-OX40 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为72％和68％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-OX40 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-13：CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子GITR蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-GITR TsAb。
一、CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D构建方案设计
单体形式的CD19-CD3-GITR TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗GITR scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗GITR scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-GITR TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv 和抗GITR scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗GITR scFv序列之间以IgD铰链区(Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗GITR scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示。
抗GITR scFv的重链可变区的核苷酸序列如SEQ ID NO.123所示，具体为：
Figure PCTCN2017096594-appb-000165
抗GITR scFv的轻链可变区的核苷酸序列如SEQ ID NO.124所示，具体为：
Figure PCTCN2017096594-appb-000166
抗GITR scFv的核苷酸序列如SEQ ID NO.122所示，具体为：
Figure PCTCN2017096594-appb-000167
单体形式的CD19-CD3-GITR TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO. 45所示。
单体形式的CD19-CD3-GITR TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.47所示。
二聚体形式的CD19-CD3-GITR TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示。
二聚体形式的CD19-CD3-GITR TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示。
为使三特异性抗体在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.131所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示。
二、CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-GITR TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-GITR-F和pcDNA3.1-GITR-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗GITR scFv的基因序列；针对CD19-CD3-GITR TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-GITR-F和pcDNA3.1-GITR-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗GITR scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000168
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三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，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CD19-CD3-GITR TsAb_M和二聚体形式的CD19-CD3-GITR TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-GITR TsAb_M的核苷酸序列如SEQ ID NO.72所示，具体为：
Figure PCTCN2017096594-appb-000169
Figure PCTCN2017096594-appb-000170
二聚体形式的CD19-CD3-GITR TsAb_D的核苷酸序列如SEQ ID NO.74所示，具体为：
Figure PCTCN2017096594-appb-000171
Figure PCTCN2017096594-appb-000172
表2-4.CD19-CD3-GITR三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000173
实施例2-14：CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D的表达与纯化
一、CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_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(CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-13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、CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_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CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2-11所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-GITR TsAb_M重组蛋白的理论分子量为80.1kDa，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，因此该三特异性抗体为单体形式(图2-11A)；CD19-CD3-GITR TsAb_D重组蛋白的理论分子量为88.0kDa，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(约180kDa)(图2-11B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-GITR TsAb_M为单体形式，CD19-CD3-GITR TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-GITR TsAb_M的氨基酸序列如SEQ ID NO.71所示，具体为：
Figure PCTCN2017096594-appb-000174
Figure PCTCN2017096594-appb-000175
二聚体形式的CD19-CD3-GITR TsAb_D的氨基酸序列如SEQ ID NO.73所示，具体为：
Figure PCTCN2017096594-appb-000176
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示。
抗GITR scFv的氨基酸序列如SEQ ID NO.98所示，具体为：
Figure PCTCN2017096594-appb-000177
抗GITR scFv的重链可变区的氨基酸序列如SEQ ID NO.99所示，具体为：
Figure PCTCN2017096594-appb-000178
抗GITR scFv的轻链可变区的氨基酸序列如SEQ ID NO.100所示，具体为：
Figure PCTCN2017096594-appb-000179
单体形式的CD19-CD3-GITR TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示。
单体形式的CD19-CD3-GITR TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示。
二聚体形式的CD19-CD3-GITR TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示。
二聚体形式的CD19-CD3-GITR TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示。
实施例2-15：ELISA检测CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-GITR TsAb_M或CD19-CD3-GITR TsAb_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(OD450)。
ELISA结果如图2-12A和图2-12B所示：图2-12A说明CD19-CD3-GITR TsAb_M与重组抗原CD19-hFc、CD3-hFc和GITR-hFc均具有体外结合活性，其中GITR和CD19结合活性均较高，CD3结合活性较弱；图2-12B说明CD19-CD3-GITR TsAb_D与重组抗原CD19-hFc、CD3-hFc和GITR-hFc同样具有体外结合活性，其中GITR和CD19结合活性均较高，CD3结合活性较弱。
实施例2-16：CD19-CD3-GITR三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-GITR TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-GITR TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-GITR TsAb_M和CD19-CD3-GITR TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
结果如图2-13所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著的提高，其中CD19-CD3-GITR TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为93％、77％和73％，CD19-CD3-GITR TsAb_M的效果次之，杀伤效率约为88％、83％和66％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-GITR TsAb_D与CD19-CD3-GITR TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为57％和49％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-GITR TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-17：CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子CD40L蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-CD40L TsAb。
一、CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D构建方案设计
单体形式的CD19-CD3-CD40L TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD40L scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD40L scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-CD40L TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD40L scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv 之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD40L scFv序列之间以IgD铰链区(Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗CD40L scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示。
抗CD40L scFv的重链可变区的核苷酸序列如SEQ ID NO.126所示，具体为：
Figure PCTCN2017096594-appb-000181
抗CD40L scFv的轻链可变区的核苷酸序列如SEQ ID NO.127所示，具体为：
Figure PCTCN2017096594-appb-000182
抗CD40L scFv的核苷酸序列如SEQ ID NO.125所示，具体为：
Figure PCTCN2017096594-appb-000183
单体形式的CD19-CD3-CD40L TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.45所示。
单体形式的CD19-CD3-CD40L TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.47所示。
二聚体形式的CD19-CD3-CD40L TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示。
二聚体形式的CD19-CD3-CD40L TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示。
为使三特异性抗体在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.131所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示。
二、CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-CD40L TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗CD40L scFv的基因序列；针对CD19-CD3-CD40L TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗CD40L scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-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三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，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CD19-CD3-CD40L TsAb_M和二聚体形式的CD19-CD3-CD40L TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-CD40L TsAb_M的核苷酸序列如SEQ ID NO.76所示，具体为：
Figure PCTCN2017096594-appb-000185
Figure PCTCN2017096594-appb-000186
二聚体形式的CD19-CD3-CD40L TsAb_D的核苷酸序列如SEQ ID NO.78所示，具体为：
Figure PCTCN2017096594-appb-000187
Figure PCTCN2017096594-appb-000188
表2-5.CD19-CD3-CD40L三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000189
实施例2-18：CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D的表达与纯化
一、CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_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(CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-17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、CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_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CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_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，CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-CD40L TsAb_M重组蛋白的理论分子量为79.2kDa，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，因此该三特异性抗体为单体形式(图2-14A)；CD19-CD3-CD40L TsAb_D重组蛋白的理论分子量为87.1kDa，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(约180kDa)(图2-14B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-CD40L TsAb_M为单体形式，CD19-CD3-CD40L TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-CD40L TsAb_M的氨基酸序列如SEQ ID NO.75所示，具体为：
Figure PCTCN2017096594-appb-000190
Figure PCTCN2017096594-appb-000191
二聚体形式的CD19-CD3-CD40L TsAb_D的氨基酸序列如SEQ ID NO.77所示，具体为：
Figure PCTCN2017096594-appb-000192
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示。
抗CD40L scFv的氨基酸序列如SEQ ID NO.101所示，具体为：
Figure PCTCN2017096594-appb-000193
抗CD40L scFv的重链可变区的氨基酸序列如SEQ ID NO.102所示，具体为：
Figure PCTCN2017096594-appb-000194
抗CD40L scFv的轻链可变区的氨基酸序列如SEQ ID NO.103所示，具体为：
Figure PCTCN2017096594-appb-000195
Figure PCTCN2017096594-appb-000196
单体形式的CD19-CD3-CD40L TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示。
单体形式的CD19-CD3-CD40L TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示。
二聚体形式的CD19-CD3-CD40L TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示。
二聚体形式的CD19-CD3-CD40L TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示。
实施例2-19：ELISA检测CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-CD40L TsAb_M或CD19-CD3-CD40L TsAb_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(OD450)。
ELISA结果如图2-15A和图2-15B所示：图2-15A说明CD19-CD3-CD40L TsAb_M与重组抗原CD19-hFc、CD3-hFc和CD40L-hFc均具有体外结合活性，其中CD40L结合活性最高，CD19结合活性次之，CD3结合活性较弱；图2-15B说明CD19-CD3-CD40L TsAb_D与重组抗原CD19-hFc、CD3-hFc和CD40L-hFc同样具有体外结合活性，其中CD40L结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例2-20：CD19-CD3-CD40L三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-CD40L TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-CD40L TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-CD40L TsAb_M和CD19-CD3-CD40L TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000197
结果如图2-16所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-CD40L TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为94％、90％和82％，CD19-CD3-CD40L TsAb_M的效果次之，杀伤效率约为91％、88％和73％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-CD40L TsAb_D与CD19-CD3-CD40L TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为68％和61％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-CD40L TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例2-21：CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞正共刺激分子CD27蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-CD27 TsAb。
一、CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D构建方案设计
单体形式的CD19-CD3-CD27 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD27 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD27 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-CD27 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CD27 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CD27 scFv序列之间以IgD铰链区 (Ala 90-Val 170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗CD27 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.108所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.109所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.107所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.111所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.112所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.110所示。
抗CD27 scFv的重链可变区的核苷酸序列如SEQ ID NO.129所示，具体为：
Figure PCTCN2017096594-appb-000198
抗CD27 scFv的轻链可变区的核苷酸序列如SEQ ID NO.130所示，具体为：
Figure PCTCN2017096594-appb-000199
抗CD27 scFv的核苷酸序列如SEQ ID NO.128所示，具体为：
Figure PCTCN2017096594-appb-000200
单体形式的CD19-CD3-CD27 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.45所示。
单体形式的CD19-CD3-CD27 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO. 47所示。
二聚体形式的CD19-CD3-CD27 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.49所示。
二聚体形式的CD19-CD3-CD27 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.51所示。
为使三特异性抗体在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.131所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.132所示。
二、CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D真核表达载体构建
本发明三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三特异性抗体，分别设计了如表2-6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CD19-CD3-CD27 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-CD27-F和pcDNA3.1-CD27-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗CD27 scFv的基因序列；针对CD19-CD3-CD27 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD27-F和pcDNA3.1-CD27-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗CD27 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000201
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三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，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CD19-CD3-CD27 TsAb_M和二聚体形式的CD19-CD3-CD27 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-CD27 TsAb_M的核苷酸序列如SEQ ID NO.80所示，具体为：
Figure PCTCN2017096594-appb-000202
Figure PCTCN2017096594-appb-000203
二聚体形式的CD19-CD3-CD27 TsAb_D的核苷酸序列如SEQ ID NO.82所示，具体为：
Figure PCTCN2017096594-appb-000204
Figure PCTCN2017096594-appb-000205
表2-6.CD19-CD3-CD27三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000206
实施例2-22：CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D的表达与纯化
一、CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_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(CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例2-21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、CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_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CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_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，CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-CD27 TsAb_M重组蛋白的理论分子量为80.1kDa，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，因此该三特异性抗体为单体形式(图2-17A)；CD19-CD3-CD27 TsAb_D重组蛋白的理论分子量为88.0kDa，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(约180kDa)(图2-17B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-CD27 TsAb_M为单体形式，CD19-CD3-CD27 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-CD27 TsAb_M的氨基酸序列如SEQ ID NO.79所示，具体为：
Figure PCTCN2017096594-appb-000207
Figure PCTCN2017096594-appb-000208
二聚体形式的CD19-CD3-CD27 TsAb_D的氨基酸序列如SEQ ID NO.81所示，具体为：
Figure PCTCN2017096594-appb-000209
抗CD19 scFv的氨基酸序列如SEQ ID NO.83所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.84所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.85所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.86所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.87所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.88所示。
抗CD27 scFv的氨基酸序列如SEQ ID NO.104所示，具体为：
Figure PCTCN2017096594-appb-000210
抗CD27 scFv的重链可变区的氨基酸序列如SEQ ID NO.105所示，具体为：
Figure PCTCN2017096594-appb-000211
抗CD27 scFv的轻链可变区的氨基酸序列如SEQ ID NO.106所示，具体为：
Figure PCTCN2017096594-appb-000212
Figure PCTCN2017096594-appb-000213
单体形式的CD19-CD3-CD27 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.44所示。
单体形式的CD19-CD3-CD27 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.46所示。
二聚体形式的CD19-CD3-CD27 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.48所示。
二聚体形式的CD19-CD3-CD27 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.50所示。
实施例2-23：ELISA检测CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-CD27 TsAb_M或CD19-CD3-CD27 TsAb_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(OD450)。
ELISA结果如图2-18A和图2-18B所示：图2-18A说明CD19-CD3-CD27 TsAb_M与重组抗原CD19-hFc、CD3-hFc和CD27-hFc均具有体外结合活性，其中CD27结合活性最高，CD19结合活性次之，CD3结合活性较弱；图2-18B说明CD19-CD3-CD27 TsAb_D与重组抗原CD19-hFc、CD3-hFc和CD27-hFc同样具有体外结合活性，其中CD27结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例2-24：CD19-CD3-CD27三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-CD27 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-CD27 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-CD27 TsAb_M和CD19-CD3-CD27 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000214
结果如图2-19所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-CD27 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为89％、84％和74％，CD19-CD3-CD27 TsAb_M的效果次之，杀伤效率约为89％、84％和67％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-CD27 TsAb_D与CD19-CD3-CD27 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为55％和49％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-CD27 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例3-1：CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D真核表达载体的构建
在本发明中，一种融合了1)抗淋巴瘤B细胞表面人类CD19蛋白scFv结构域、2)抗T细胞表面人类CD3蛋白scFv结构域和3)T细胞正共刺激分子配体4-1BBL胞外区结构域的TiTE三特异性分子被命名为CD19-CD3-4-1BBL TsM。
一、CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D构建方案设计
单体形式的CD19-CD3-4-1BBL TsM_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和4-1BBL胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和4-1BBL胞外区序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-4-1BBL TsM_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和4-1BBL胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv 之间通过连接片段1(Linker 1)相连，抗CD3 scFv和4-1BBL胞外区序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性分子在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，4-1BBL胞外区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.209所示，具体为：
Figure PCTCN2017096594-appb-000215
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.210所示，具体为：
Figure PCTCN2017096594-appb-000216
抗CD19 scFv的核苷酸序列如SEQ ID NO.208所示，具体为：
Figure PCTCN2017096594-appb-000217
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.212所示，具体为：
Figure PCTCN2017096594-appb-000218
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.213所示，具体为：
Figure PCTCN2017096594-appb-000219
Figure PCTCN2017096594-appb-000220
抗CD3 scFv的核苷酸序列如SEQ ID NO.211所示，具体为：
Figure PCTCN2017096594-appb-000221
4-1BBL胞外区的核苷酸序列如SEQ ID NO.214所示，具体为：
Figure PCTCN2017096594-appb-000222
单体形式的CD19-CD3-4-1BBL TsM_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.160所示，具体为：
Figure PCTCN2017096594-appb-000223
单体形式的CD19-CD3-4-1BBL TsM_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.162所示，具体为：
Figure PCTCN2017096594-appb-000224
二聚体形式的CD19-CD3-4-1BBL TsM_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.164所示，具体为：
Figure PCTCN2017096594-appb-000225
二聚体形式的CD19-CD3-4-1BBL TsM_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.166所示，具体为：
Figure PCTCN2017096594-appb-000226
Figure PCTCN2017096594-appb-000227
为使三特异性分子在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.219所示，具体为：
Figure PCTCN2017096594-appb-000228
该分泌表达信号肽的核苷酸序列如SEQ ID NO.220所示，具体为：
Figure PCTCN2017096594-appb-000229
二、CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D真核表达载体构建
本发明三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三特异性分子，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CD19-CD3-4-1BBL TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-4-1BBL-F和pcDNA3.1-4-1BBL-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+4-1BBL胞外区的基因序列；针对CD19-CD3-4-1BBL TsM_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、4-1BBL胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000230
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三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，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CD19-CD3-4-1BBL TsM_M和二聚体形式的CD19-CD3-4-1BBL TsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-4-1BBL TsM_M的核苷酸序列如SEQ ID NO.178所示，具体为：
Figure PCTCN2017096594-appb-000231
Figure PCTCN2017096594-appb-000232
二聚体形式的CD19-CD3-4-1BBL TsM_D的核苷酸序列如SEQ ID NO.180所示，具体为：
Figure PCTCN2017096594-appb-000233
Figure PCTCN2017096594-appb-000234
表3-1.CD19-CD3-4-1BBL三特异性分子基因克隆中使用的引物
Figure PCTCN2017096594-appb-000235
Figure PCTCN2017096594-appb-000236
实施例3-2：CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D的表达与纯化
一、CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_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(CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_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、CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_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CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_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，CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D重组蛋白的纯度均＞95％：其中CD19-CD3-4-1BBL TsM_M重组蛋白的理论分子量为75.6kDa，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，因此该三特异性分子为单体形式(图3-2A)；CD19-CD3-4-1BBL TsM_D重组蛋白的理论分子量为83.5kDa，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3-2B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-4-1BBL TsM_M为单体形式，CD19-CD3-4-1BBL TsM_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-4-1BBL TsM_M的氨基酸序列如SEQ ID NO.177所示，具体为：
Figure PCTCN2017096594-appb-000237
二聚体形式的CD19-CD3-4-1BBL TsM_D的氨基酸序列如SEQ ID NO.179所示，具体为：
Figure PCTCN2017096594-appb-000238
抗CD19 scFv的氨基酸序列如SEQ ID NO.197所示，具体为：
Figure PCTCN2017096594-appb-000239
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.198所示，具体为：
Figure PCTCN2017096594-appb-000240
Figure PCTCN2017096594-appb-000241
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.199所示，具体为：
Figure PCTCN2017096594-appb-000242
抗CD3 scFv的氨基酸序列如SEQ ID NO.200所示，具体为：
Figure PCTCN2017096594-appb-000243
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.201所示，具体为：
Figure PCTCN2017096594-appb-000244
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.202所示，具体为：
Figure PCTCN2017096594-appb-000245
4-1BBL胞外区的氨基酸序列如SEQ ID NO.203所示，具体为：
Figure PCTCN2017096594-appb-000246
单体形式的CD19-CD3-4-1BBL TsM_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.159所示，具体为：
Figure PCTCN2017096594-appb-000247
单体形式的CD19-CD3-4-1BBL TsM_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.161所示，具体为：
Figure PCTCN2017096594-appb-000248
二聚体形式的CD19-CD3-4-1BBL TsM_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.163所示，具体为：
Figure PCTCN2017096594-appb-000249
二聚体形式的CD19-CD3-4-1BBL TsM_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.165所示，具体为：
Figure PCTCN2017096594-appb-000250
实施例3-3：ELISA检测CD19-CD3-4-1BBL TsM_M和CD19-CD3-4-1BBL TsM_D的CD19抗原、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CD19-hFc、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，分别加入纯化的三特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-4-1BBL TsM_M或CD19-CD3-4-1BBL TsM_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(OD450)。
ELISA结果如图3-3A和图3-3B所示：图3-3A说明CD19-CD3-4-1BBL TsM_M与抗原CD19-hFc、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结合活性最高，CD19结合活性次之，CD3结合活性较弱；图3-3B说明CD19-CD3-4-1BBL TsM_D与抗原CD19-hFc、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结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例3-4：CD19-CD3-4-1BBL三特异性分子介导的细胞杀伤实验
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TiTE三特异分子(CD19-CD3-4-1BBL TsM_M)、二聚体形式的TiTE三特异分子(CD19-CD3-4-1BBL TsM_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种蛋白介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-4-1BBLTsM_M和CD19-CD3-4-1BBLTsM_D蛋白样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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；同时以未添加任何蛋白的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000251
结果如图3-4所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何蛋白的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度蛋白(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-4-1BBL TsM_D所介导的细胞杀伤效果最好，杀伤效率分别约为96％、92％和87％，CD19-CD3-4-1BBL TsM_M的效果次之，杀伤效率约为93％、88％和83％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度蛋白(3.125ng/ml)的条件下，CD19-CD3-4-1BBL TsM_D与CD19-CD3-4-1BBL TsM_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为82％和72％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-4-1BBL TiTE三特异分子所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例3-5：CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D真核表达载体的构建
在本发明中，一种融合了1)抗淋巴瘤B细胞表面人类CD19蛋白scFv结构域、2)抗T细胞表面人类CD3蛋白scFv结构域和3)T细胞正共刺激分子配体B7RP-1胞外区结构域的TiTE三特异性分子被命名为CD19-CD3-B7RP-1 TsM。
一、CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D构建方案设计
单体形式的CD19-CD3-B7RP-1 TsM_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和B7RP-1胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和B7RP-1胞外区序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-B7RP-1 TsM_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和B7RP-1胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和B7RP-1胞外区序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性分子在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，B7RP-1胞外区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.209所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.210所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.208所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.212所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.213所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.211所示。
B7RP-1胞外区的核苷酸序列如SEQ ID NO.215所示，具体为：
Figure PCTCN2017096594-appb-000252
Figure PCTCN2017096594-appb-000253
单体形式的CD19-CD3-B7RP-1 TsM_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.160所示。
单体形式的CD19-CD3-B7RP-1 TsM_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.162所示。
二聚体形式的CD19-CD3-B7RP-1 TsM_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.164所示。
二聚体形式的CD19-CD3-B7RP-1 TsM_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.166所示。
为使三特异性分子在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.219所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.220所示。
二、CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D真核表达载体构建
本发明三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三特异性分子，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CD19-CD3-B7RP-1 TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-B7RP-1-F和pcDNA3.1-B7RP-1-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+B7RP-1胞外区的基因序列；针对CD19-CD3-B7RP-1 TsM_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、B7RP-1胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000254
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三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，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CD19-CD3-B7RP-1 TsM_M和二聚体形式的CD19-CD3-B7RP-1 TsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-B7RP-1 TsM_M的核苷酸序列如SEQ ID NO.182所示，具体为：
Figure PCTCN2017096594-appb-000255
Figure PCTCN2017096594-appb-000256
二聚体形式的CD19-CD3-B7RP-1 TsM_D的核苷酸序列如SEQ ID NO.184所示，具体为：
Figure PCTCN2017096594-appb-000257
Figure PCTCN2017096594-appb-000258
表3-2.CD19-CD3-B7RP-1三特异性分子基因克隆中使用的引物
Figure PCTCN2017096594-appb-000259
Figure PCTCN2017096594-appb-000260
实施例3-6：CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D的表达与纯化
一、CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_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(CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_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、CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_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CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_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，CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D重组蛋白的纯度均＞95％：其中CD19-CD3-B7RP-1 TsM_M重组蛋白的理论分子量为80.6kDa，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，该三特异性分子为糖基化的单体形式(图3-5A)；CD19-CD3-B7RP-1 TsM_D重组蛋白的理论分子量为88.5kDa，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IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-B7RP-1 TsM_M为单体形式，CD19-CD3-B7RP-1 TsM_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-B7RP-1 TsM_M的氨基酸序列如SEQ ID NO.181所示，具体为：
Figure PCTCN2017096594-appb-000261
二聚体形式的CD19-CD3-B7RP-1 TsM_D的氨基酸序列如SEQ ID NO.183所示，具体为：
Figure PCTCN2017096594-appb-000262
抗CD19 scFv的氨基酸序列如SEQ ID NO.197所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.198所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.199所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.200所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.201所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.202所示。
B7RP-1胞外区的氨基酸序列如SEQ ID NO.204所示，具体为：
Figure PCTCN2017096594-appb-000263
单体形式的CD19-CD3-B7RP-1 TsM_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO. 159所示。
单体形式的CD19-CD3-B7RP-1 TsM_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.161所示。
二聚体形式的CD19-CD3-B7RP-1 TsM_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.163所示。
二聚体形式的CD19-CD3-B7RP-1 TsM_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.165所示。
实施例3-7：ELISA检测CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D的CD19抗原、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CD19-hFc、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，分别加入纯化的三特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-B7RP-1 TsM_M或CD19-CD3-B7RP-1 TsM_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(OD450)。
ELISA结果如图3-6A和图3-6B所示：图3-6A说明CD19-CD3-B7RP-1 TsM_M与抗原CD19-hFc、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ICOS和CD19结合活性均较高，CD3结合活性较弱；图3-6B说明CD19-CD3-B7RP-1 TsM_D与抗原CD19-hFc、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ICOS和CD19结合活性均较高，CD3结合活性较弱。
实施例3-8：CD19-CD3-B7RP-1三特异性分子介导的细胞杀伤实验
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TiTE三特异分子(CD19-CD3-B7RP-1 TsM_M)、二聚体形式的TiTE三特异分子(CD19-CD3-B7RP-1 TsM_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种蛋白介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-B7RP-1 TsM_M和CD19-CD3-B7RP-1 TsM_D蛋白样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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；同时以未添加任何蛋白的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000264
结果如图3-7所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何蛋白的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度蛋白(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-B7RP-1 TsM_D所介导的细胞杀伤效果最好，杀伤效率分别约为92％、88％和84％，CD19-CD3-B7RP-1 TsM_M的效果次之，杀伤效率约为89％、85％和78％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度蛋白(3.125ng/ml)的条件下，CD19-CD3-B7RP-1 TsM_D与CD19-CD3-B7RP-1 TsM_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为79％和68％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-B7RP-1 TiTE三特异分子所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例3-9：CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D真核表达载体的构建
在本发明中，一种融合了1)抗淋巴瘤B细胞表面人类CD19蛋白scFv结构域、2)抗T细胞表面人类CD3蛋白scFv结构域和3)T细胞正共刺激分子配体OX40L胞外区结构域的TiTE三特异性分子被命名为CD19-CD3-OX40L TsM。
一、CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D构建方案设计
单体形式的CD19-CD3-OX40L TsM_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和OX40L胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和OX40L胞外区序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-OX40L TsM_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和OX40L胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和OX40L胞外区序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性分子在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，OX40L胞 外区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.209所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.210所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.208所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.212所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.213所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.211所示。
OX40L胞外区的核苷酸序列如SEQ ID NO.216所示，具体为：
Figure PCTCN2017096594-appb-000265
单体形式的CD19-CD3-OX40L TsM_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.160所示。
单体形式的CD19-CD3-OX40L TsM_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.162所示。
二聚体形式的CD19-CD3-OX40L TsM_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.164所示。
二聚体形式的CD19-CD3-OX40L TsM_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.166所示。
为使三特异性分子在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.219所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.220所示。
二、CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D真核表达载体构建
本发明三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三特异性分子，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CD19-CD3-OX40L TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-OX40L-F和pcDNA3.1-OX40L-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+OX40L胞外区的基因序列；针对CD19-CD3-OX40L TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-OX40L-F和pcDNA3.1-OX40L-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、OX40L胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000266
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三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，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CD19-CD3-OX40L TsM_M和二聚体形式的CD19-CD3-OX40L TsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-OX40L TsM_M的核苷酸序列如SEQ ID NO.186所示，具体为：
Figure PCTCN2017096594-appb-000267
二聚体形式的CD19-CD3-OX40L TsM_D的核苷酸序列如SEQ ID NO.188所示，具体为：
Figure PCTCN2017096594-appb-000268
Figure PCTCN2017096594-appb-000269
表3-3.CD19-CD3-OX40L三特异性分子基因克隆中使用的引物
Figure PCTCN2017096594-appb-000270
Figure PCTCN2017096594-appb-000271
实施例3-10：CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D的表达与纯化
一、CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_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(CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_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、CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_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CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_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，CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D重组蛋白的纯度均＞95％：其中CD19-CD3-OX40L TsM_M重组蛋白的理论分子量为69.6kDa，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，该三特异性分子为糖基化的单体形式(图3-8A)；CD19-CD3-OX40L TsM_D重组蛋白的理论分子量为77.5kDa，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IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-OX40L TsM_M 为单体形式，CD19-CD3-OX40L TsM_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-OX40L TsM_M的氨基酸序列如SEQ ID NO.185所示，具体为：
Figure PCTCN2017096594-appb-000272
二聚体形式的CD19-CD3-OX40L TsM_D的氨基酸序列如SEQ ID NO.187所示，具体为：
Figure PCTCN2017096594-appb-000273
抗CD19 scFv的氨基酸序列如SEQ ID NO.197所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.198所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.199所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.200所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.201所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.202所示。
OX40L胞外区的氨基酸序列如SEQ ID NO.205所示，具体为：
Figure PCTCN2017096594-appb-000274
单体形式的CD19-CD3-OX40L TsM_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.159所示。
单体形式的CD19-CD3-OX40L TsM_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.161所示。
二聚体形式的CD19-CD3-OX40L TsM_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.163所示。
二聚体形式的CD19-CD3-OX40L TsM_D连接片段2(Linker 2)的氨基酸序列如SEQ ID  NO.165所示。
实施例3-11：ELISA检测CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D的CD19抗原、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CD19-hFc、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 Na2HPO4，0.24gNaH2PO4，0.2g 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，分别加入纯化的三特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-OX40L TsM_M或CD19-CD3-OX40L TsM_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(OD450)。
ELISA结果如图3-9A和图3-9B所示：图3-9A说明CD19-CD3-OX40L TsM_M与抗原CD19-hFc、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CD19结合活性最高，CD3结合活性次之，OX40结合活性较弱；图3-9B说明CD19-CD3-OX40L TsM_D与抗原CD19-hFc、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CD19结合活性最高，CD3结合活性次之，OX40结合活性较弱。
实施例3-12：CD19-CD3-OX40L三特异性分子介导的细胞杀伤实验
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TiTE三特异分子(CD19-CD3-OX40L TsM_M)、二聚体形式的TiTE三特异分子(CD19-CD3-OX40L TsM_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种蛋白介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-OX40L TsM_M和CD19-CD3-OX40L TsM_D蛋白样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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；同时以未添加任何蛋白的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000275
结果如图3-10所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何蛋白的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度蛋白(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-OX40L TsM_D所介导的细胞杀伤效果最好，杀伤效率分别约为96％、92％和87％，CD19-CD3-OX40L TsM_M的效果次之，杀伤效率约为93％、88％和82％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度蛋白(3.125ng/ml)的条件下，CD19-CD3-OX40L TsM_D与CD19-CD3-OX40L TsM_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为82％和72％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-OX40L TiTE三特异分子所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例3-13：CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D真核表达载体的构建
在本发明中，一种融合了1)抗淋巴瘤B细胞表面人类CD19蛋白scFv结构域、2)抗T细胞表面人类CD3蛋白scFv结构域和3)T细胞正共刺激分子配体GITRL胞外区结构域的TiTE三特异性分子被命名为CD19-CD3-GITRL TsM。
一、CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D构建方案设计
单体形式的CD19-CD3-GITRL TsM_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和GITRL胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和GITRL胞外区序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-GITRL TsM_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和GITRL胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和GITRL胞外区序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性分子在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，GITRL胞外区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.209所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.210所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.208所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.212所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.213所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.211所示。
GITRL胞外区的核苷酸序列如SEQ ID NO.217所示，具体为：
Figure PCTCN2017096594-appb-000276
单体形式的CD19-CD3-GITRL TsM_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.160所示。
单体形式的CD19-CD3-GITRL TsM_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.162所示。
二聚体形式的CD19-CD3-GITRL TsM_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.164所示。
二聚体形式的CD19-CD3-GITRL TsM_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.166所示。
为使三特异性分子在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.219所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.220所示。
二、CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D真核表达载体构建
本发明三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三特异性分子，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CD19-CD3-GITRL TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-GITRL-F和pcDNA3.1-GITRL-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+GITRL胞外区的基因序列；针对CD19-CD3-GITRL TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-GITRL-F和pcDNA3.1-GITRL-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、GITRL胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000277
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三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，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CD19-CD3-GITRL TsM_M和二聚体形式的CD19-CD3-GITRL TsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-GITRL TsM_M的核苷酸序列如SEQ ID NO.190所示，具体为：
Figure PCTCN2017096594-appb-000278
Figure PCTCN2017096594-appb-000279
二聚体形式的CD19-CD3-GITRL TsM_D的核苷酸序列如SEQ ID NO.192所示，具体为：
Figure PCTCN2017096594-appb-000280
Figure PCTCN2017096594-appb-000281
表3-4.CD19-CD3-GITRL三特异性分子基因克隆中使用的引物
Figure PCTCN2017096594-appb-000282
实施例3-14：CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D的表达与纯化
一、CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_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(CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_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、CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_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CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_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，CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D重组蛋白的纯度均＞95％：其中CD19-CD3-GITRL TsM_M重组蛋白的理论分子量为68.7kDa，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，该三特异性分子为糖基化的单体形式(图3-11A)；CD19-CD3-GITRL TsM_D重组蛋白的理论分子量为76.6kDa，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3-11B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-GITRL TsM_M为单体形式，CD19-CD3-GITRL TsM_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-GITRL TsM_M的氨基酸序列如SEQ ID NO.189所示，具体为：
Figure PCTCN2017096594-appb-000283
Figure PCTCN2017096594-appb-000284
二聚体形式的CD19-CD3-GITRL TsM_D的氨基酸序列如SEQ ID NO.191所示，具体为：
Figure PCTCN2017096594-appb-000285
抗CD19 scFv的氨基酸序列如SEQ ID NO.197所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.198所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.199所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.200所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.201所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.202所示。
GITRL胞外区的氨基酸序列如SEQ ID NO.206所示，具体为：
Figure PCTCN2017096594-appb-000286
单体形式的CD19-CD3-GITRL TsM_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.159所示。
单体形式的CD19-CD3-GITRL TsM_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.161所示。
二聚体形式的CD19-CD3-GITRL TsM_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.163所示。
二聚体形式的CD19-CD3-GITRL TsM_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.165所示。
实施例3-15：ELISA检测CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D的CD19抗原、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CD19-hFc、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，分别加入纯化的三特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-GITRL TsM_M或CD19-CD3-GITRL TsM_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(OD450)。
ELISA结果如图3-12A和图3-12B所示：图3-12A说明CD19-CD3-GITRL TsM_M与抗原CD19-hFc、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结合活性最高，CD19结合活性次之，CD3结合活性较弱；图3-12B说明CD19-CD3-GITRL TsM_D与抗原CD19-hFc、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结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例3-16：CD19-CD3-GITRL三特异性分子介导的细胞杀伤实验
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TiTE三特异分子(CD19-CD3-GITRL TsM_M)、二聚体形式的TiTE三特异分子(CD19-CD3-GITRL TsM_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种蛋白介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-GITRL TsM_M和CD19-CD3-GITRL TsM_D蛋白样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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；同时以未添加任何蛋白的细胞 杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000287
结果如图3-13所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何蛋白的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度蛋白(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-GITRL TsM_D所介导的细胞杀伤效果最好，杀伤效率分别约为92％、88％和84％，CD19-CD3-GITRL TsM_M的效果次之，杀伤效率约为89％、85％和78％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度蛋白(3.125ng/ml)的条件下，CD19-CD3-GITRL TsM_D与CD19-CD3-GITRL TsM_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为78％和68％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-GITRL TiTE三特异分子所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例3-17：CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D真核表达载体的构建
在本发明中，一种融合了1)抗淋巴瘤B细胞表面人类CD19蛋白scFv结构域、2)抗T细胞表面人类CD3蛋白scFv结构域和3)T细胞正共刺激分子配体CD70胞外区结构域的TiTE三特异性分子被命名为CD19-CD3-CD70 TsM。
一、CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D构建方案设计
单体形式的CD19-CD3-CD70 TsM_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和CD70胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和CD70胞外区序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-CD70 TsM_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和CD70胞外区的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和CD70胞外区序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性分子在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，CD70胞外区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.209所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.210所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.208所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.212所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.213所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.211所示。
CD70胞外区的核苷酸序列如SEQ ID NO.218所示，具体为：
Figure PCTCN2017096594-appb-000288
Figure PCTCN2017096594-appb-000289
单体形式的CD19-CD3-CD70 TsM_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.160所示。
单体形式的CD19-CD3-CD70 TsM_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.162所示。
二聚体形式的CD19-CD3-CD70 TsM_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.164所示。
二聚体形式的CD19-CD3-CD70 TsM_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.166所示。
为使三特异性分子在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.219所示
该分泌表达信号肽的核苷酸序列如SEQ ID NO.220所示
二、CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D真核表达载体构建
本发明三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三特异性分子，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CD19-CD3-CD70 TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-CD70-F和pcDNA3.1-CD70-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+CD70胞外区的基因序列；针对CD19-CD3-CD70 TsM_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD70-F和pcDNA3.1-CD70-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、CD70胞外区的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000290
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三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，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CD19-CD3-CD70 TsM_M和二聚体形式的CD19-CD3-CD70 TsM_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-CD70 TsM_M的核苷酸序列如SEQ ID NO.194所示，具体为：
Figure PCTCN2017096594-appb-000291
Figure PCTCN2017096594-appb-000292
二聚体形式的CD19-CD3-CD70 TsM_D的核苷酸序列如SEQ ID NO.196所示，具体为：
Figure PCTCN2017096594-appb-000293
Figure PCTCN2017096594-appb-000294
表3-5.CD19-CD3-CD70三特异性分子基因克隆中使用的引物
Figure PCTCN2017096594-appb-000295
实施例3-18：CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D的表达与纯化
一、CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_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(CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_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、CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_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CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_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Protein L亲和层析柱纯化后，CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D重组蛋白的纯度均＞95％：其中CD19-CD3-CD70 TsM_M重组蛋白的理论分子量为71.3kDa，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，该三特异性分子为糖基化的单体形式(图3-14A)；CD19-CD3-CD70 TsM_D重组蛋白的理论分子量为79.2kDa，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IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-CD70 TsM_M为单体形式，CD19-CD3-CD70 TsM_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-CD70 TsM_M的氨基酸序列如SEQ ID NO.193所示，具体为：
Figure PCTCN2017096594-appb-000296
Figure PCTCN2017096594-appb-000297
二聚体形式的CD19-CD3-CD70 TsM_D的氨基酸序列如SEQ ID NO.195所示，具体为：
Figure PCTCN2017096594-appb-000298
抗CD19 scFv的氨基酸序列如SEQ ID NO.197所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.198所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.199所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.200所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.201所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.202所示。
CD70胞外区的氨基酸序列如SEQ ID NO.207所示，具体为：
Figure PCTCN2017096594-appb-000299
单体形式的CD19-CD3-CD70 TsM_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.159所示。
单体形式的CD19-CD3-CD70 TsM_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.161所示。
二聚体形式的CD19-CD3-CD70 TsM_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.163所示。
二聚体形式的CD19-CD3-CD70 TsM_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.165所示。
实施例3-19：ELISA检测CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D的CD19抗原、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CD19-hFc、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，分别加入纯化的三特异性分子样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-CD70 TsM_M或CD19-CD3-CD70 TsM_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(OD450)。
ELISA结果如图3-15A和图3-15B所示：图3-15A说明CD19-CD3-CD70 TsM_M与抗原CD19-hFc、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和CD19结合活性均较高，CD3结合活性较弱；图3-15B说明CD19-CD3-CD70 TsM_D与抗原CD19-hFc、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和CD19结合活性均较高，CD3结合活性较弱。
实施例3-20：CD19-CD3-CD70三特异性分子介导的细胞杀伤实验
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TiTE三特异分子(CD19-CD3-CD70 TsM_M)、二聚体形式的TiTE三特异分子(CD19-CD3-CD70 TsM_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种蛋白介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-CD70 TsM_M和CD19-CD3-CD70 TsM_D蛋白样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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；同时以未添加任何蛋白的细胞 杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000300
结果如图3-16所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何蛋白的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度蛋白(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-CD70 TsM_D所介导的细胞杀伤效果最好，杀伤效率分别约为96％、92％和87％，CD19-CD3-CD70 TsM_M的效果次之，杀伤效率约为93％、88％和83％，CD19-CD3BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度蛋白(3.125ng/ml)的条件下，CD19-CD3-CD70 TsM_D与CD19-CD3-CD70 TsM_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为82％和72％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-CD70 TiTE三特异分子所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-1：CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子PD-1蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-PD-1 TsAb。
一、CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D构建方案设计
单体形式的CD19-CD3-PD-1 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗PD-1 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗PD-1 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-PD-1 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗PD-1 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗PD-1 scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗PD-1 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示，具体为：
Figure PCTCN2017096594-appb-000301
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示，具体为：
Figure PCTCN2017096594-appb-000302
Figure PCTCN2017096594-appb-000303
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示，具体为：
Figure PCTCN2017096594-appb-000304
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示，具体为：
Figure PCTCN2017096594-appb-000305
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示，具体为：
Figure PCTCN2017096594-appb-000306
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示，具体为：
Figure PCTCN2017096594-appb-000307
Figure PCTCN2017096594-appb-000308
抗PD-1 scFv的重链可变区的核苷酸序列如SEQ ID NO.313所示，具体为：
Figure PCTCN2017096594-appb-000309
抗PD-1 scFv的轻链可变区的核苷酸序列如SEQ ID NO.314所示，具体为：
Figure PCTCN2017096594-appb-000310
抗PD-1 scFv的核苷酸序列如SEQ ID NO.312所示，具体为：
Figure PCTCN2017096594-appb-000311
单体形式的CD19-CD3-PD-1 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示，具体为：
Figure PCTCN2017096594-appb-000312
单体形式的CD19-CD3-PD-1 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示，具体为：
Figure PCTCN2017096594-appb-000313
二聚体形式的CD19-CD3-PD-1 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示，具体为：
Figure PCTCN2017096594-appb-000314
二聚体形式的CD19-CD3-PD-1 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示，具体为：
Figure PCTCN2017096594-appb-000315
Figure PCTCN2017096594-appb-000316
为使三特异性抗体在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.330所示，具体为：
Figure PCTCN2017096594-appb-000317
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示，具体为：
Figure PCTCN2017096594-appb-000318
二、CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-PD-1 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-PD-1-F和pcDNA3.1-PD-1-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗PD-1 scFv的基因序列；针对CD19-CD3-PD-1 TsAb_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗PD-1 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000319
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三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，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CD19-CD3-PD-1 TsAb_M和二聚体形式的CD19-CD3-PD-1 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-PD-1 TsAb_M的核苷酸序列如SEQ ID NO.259所示，具体为：
Figure PCTCN2017096594-appb-000320
Figure PCTCN2017096594-appb-000321
二聚体形式的CD19-CD3-PD-1 TsAb_D的核苷酸序列如SEQ ID NO.261所示，具体为：
Figure PCTCN2017096594-appb-000322
Figure PCTCN2017096594-appb-000323
表4-1.CD19-CD3-PD-1三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000324
Figure PCTCN2017096594-appb-000325
实施例4-2：CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D的表达与纯化
一、CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_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(CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_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、CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_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CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_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，CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-PD-1 TsAb_M重组蛋白的理论分子量为79.4kDa，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，因此该三特异性抗体为单体形式(图4-2A)；CD19-CD3-PD-1 TsAb_D重组蛋白的理论分子量为87.3kDa，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(约180kDa) (图4-2B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-PD-1 TsAb_M为单体形式，CD19-CD3-PD-1 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-PD-1 TsAb_M的氨基酸序列如SEQ ID NO.258所示，具体为：
Figure PCTCN2017096594-appb-000326
二聚体形式的CD19-CD3-PD-1 TsAb_D的氨基酸序列如SEQ ID NO.260所示，具体为：
Figure PCTCN2017096594-appb-000327
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示，具体为：
Figure PCTCN2017096594-appb-000328
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示，具体为：
Figure PCTCN2017096594-appb-000329
Figure PCTCN2017096594-appb-000330
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示，具体为：
Figure PCTCN2017096594-appb-000331
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示，具体为：
Figure PCTCN2017096594-appb-000332
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示，具体为：
Figure PCTCN2017096594-appb-000333
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示，具体为：
Figure PCTCN2017096594-appb-000334
抗PD-1 scFv的氨基酸序列如SEQ ID NO.288所示，具体为：
Figure PCTCN2017096594-appb-000335
抗PD-1 scFv的重链可变区的氨基酸序列如SEQ ID NO.289所示，具体为：
Figure PCTCN2017096594-appb-000336
抗PD-1 scFv的轻链可变区的氨基酸序列如SEQ ID NO.290所示，具体为：
Figure PCTCN2017096594-appb-000337
单体形式的CD19-CD3-PD-1 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示，具体为：
Figure PCTCN2017096594-appb-000338
单体形式的CD19-CD3-PD-1 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示，具体为：
Figure PCTCN2017096594-appb-000339
二聚体形式的CD19-CD3-PD-1 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示，具体为：
Figure PCTCN2017096594-appb-000340
二聚体形式的CD19-CD3-PD-1 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示，具体为：
Figure PCTCN2017096594-appb-000341
实施例4-3：ELISA检测CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-PD-1 TsAb_M或CD19-CD3-PD-1 TsAb_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(OD450)。
ELISA结果如图4-3A和图4-3B所示：图4-3A说明CD19-CD3-PD-1 TsAb_M与重组抗原CD19-hFc、CD3-hFc和PD-1-hFc均具有体外结合活性，其中PD-1结合活性最高，CD19结合活性次之，CD3结合活性较弱；图4-3B说明CD19-CD3-PD-1 TsAb_D与重组抗原CD19-hFc、CD3-hFc和PD-1-hFc同样具有体外结合活性，其中PD-1结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例4-4：CD19-CD3-PD-1三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-PD-1 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-PD-1 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-PD-1 TsAb_M和CD19-CD3-PD-1 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000342
结果如图4-4所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-PD-1 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为97％、94％和93％，CD19-CD3-PD-1 TsAb_M的效果次之，杀伤效率约为92％、89％和83％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-PD-1 TsAb_D与CD19-CD3-PD-1 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为86％和75％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-PD-1 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-5：CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子CTLA-4蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-CTLA-4TsAb。
一、CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D构建方案设计
单体形式的CD19-CD3-CTLA-4 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CTLA-4 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CTLA-4 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-CTLA-4 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗CTLA-4 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗CTLA-4 scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗CTLA-4 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示。
抗CTLA-4 scFv的重链可变区的核苷酸序列如SEQ ID NO.316所示，具体为：
Figure PCTCN2017096594-appb-000343
Figure PCTCN2017096594-appb-000344
抗CTLA-4 scFv的轻链可变区的核苷酸序列如SEQ ID NO.317所示，具体为：
Figure PCTCN2017096594-appb-000345
抗CTLA-4 scFv的核苷酸序列如SEQ ID NO.315所示，具体为：
Figure PCTCN2017096594-appb-000346
单体形式的CD19-CD3-CTLA-4 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示。
单体形式的CD19-CD3-CTLA-4 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示。
二聚体形式的CD19-CD3-CTLA-4 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示。
二聚体形式的CD19-CD3-CTLA-4 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示。
为使三特异性抗体在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.330所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示。
二、CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-CTLA-4 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-CTLA-4-F和pcDNA3.1-CTLA-4-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗CTLA-4 scFv的基因序列；针对CD19-CD3-CTLA-4 TsAb_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗CTLA-4 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000347
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三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，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CD19-CD3-CTLA-4 TsAb_M和二聚体形式的CD19-CD3-CTLA-4 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-CTLA-4 TsAb_M的核苷酸序列如SEQ ID NO.263所示，具体为：
Figure PCTCN2017096594-appb-000348
Figure PCTCN2017096594-appb-000349
二聚体形式的CD19-CD3-CTLA-4 TsAb_D的核苷酸序列如SEQ ID NO.265所示，具体为：
Figure PCTCN2017096594-appb-000350
Figure PCTCN2017096594-appb-000351
表4-2.CD19-CD3-CTLA-4三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000352
实施例4-6：CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D的表达与纯化
一、CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_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(CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-5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、CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_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CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-5所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-CTLA-4 TsAb_M重组蛋白的理论分子量为80.1kDa，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，因此该三特异性抗体为单体形式(图4-5A)；CD19-CD3-CTLA-4 TsAb_D重组蛋白的理论分子量为88.0kDa，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(约180kDa)(图4-5B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-CTLA-4 TsAb_M为单体形式，CD19-CD3-CTLA-4 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-CTLA-4 TsAb_M的氨基酸序列如SEQ ID NO.262所示，具体为：
Figure PCTCN2017096594-appb-000353
二聚体形式的CD19-CD3-CTLA-4 TsAb_D的氨基酸序列如SEQ ID NO.264所示，具体为：
Figure PCTCN2017096594-appb-000354
Figure PCTCN2017096594-appb-000355
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示。
抗CTLA-4 scFv的氨基酸序列如SEQ ID NO.291所示，具体为：
Figure PCTCN2017096594-appb-000356
抗CTLA-4 scFv的重链可变区的氨基酸序列如SEQ ID NO.292所示，具体为：
Figure PCTCN2017096594-appb-000357
抗CTLA-4 scFv的轻链可变区的氨基酸序列如SEQ ID NO.293所示，具体为：
Figure PCTCN2017096594-appb-000358
单体形式的CD19-CD3-CTLA-4 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示。
单体形式的CD19-CD3-CTLA-4 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示。
二聚体形式的CD19-CD3-CTLA-4 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示。
二聚体形式的CD19-CD3-CTLA-4 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示。
实施例4-7：ELISA检测CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-CTLA-4 TsAb_M或CD19-CD3-CTLA-4 TsAb_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(OD450)。
ELISA结果如图4-6A和图4-6B所示：图4-6A说明CD19-CD3-CTLA-4 TsAb_M与重组抗原CD19-hFc、CD3-hFc和CTLA-4-hFc均具有体外结合活性，其中CTLA-4结合活性最高，CD19结合活性次之，CD3结合活性较弱；图4-6B说明CD19-CD3-CTLA-4 TsAb_D与重组抗原CD19-hFc、CD3-hFc和CTLA-4-hFc同样具有体外结合活性，其中CTLA-4结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例4-8：CD19-CD3-CTLA-4三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-CTLA-4 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-CTLA-4 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-CTLA-4 TsAb_M和CD19-CD3-CTLA-4 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的 细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000359
结果如图4-7所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-CTLA-4 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为94％、91％和89％，CD19-CD3-CTLA-4 TsAb_M的效果次之，杀伤效率约为86％、82％和76％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件，CD19-CD3-CTLA-4 TsAb_D与CD19-CD3-CTLA-4 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有明显地提高，杀伤效率分别约为82％和71％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-CTLA-4 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-9：CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子LAG-3蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-LAG-3 TsAb。
一、CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D构建方案设计
单体形式的CD19-CD3-LAG-3 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗LAG-3 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗LAG-3 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-LAG-3 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗LAG-3 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗LAG-3 scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗LAG-3 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示。
抗LAG-3 scFv的重链可变区的核苷酸序列如SEQ ID NO.319所示，具体为：
Figure PCTCN2017096594-appb-000360
抗LAG-3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.320所示，具体为：
Figure PCTCN2017096594-appb-000361
抗LAG-3 scFv的核苷酸序列如SEQ ID NO.318所示，具体为：
Figure PCTCN2017096594-appb-000362
单体形式的CD19-CD3-LAG-3 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示。
单体形式的CD19-CD3-LAG-3 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示。
二聚体形式的CD19-CD3-LAG-3 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示。
二聚体形式的CD19-CD3-LAG-3 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示。
为使三特异性抗体在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.330所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示。
二、CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-LAG-3 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-LAG-3-F和pcDNA3.1-LAG-3-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗LAG-3 scFv的基因序列；针对CD19-CD3-LAG-3 TsAb_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗LAG-3 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000363
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三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，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CD19-CD3-LAG-3 TsAb_M和二聚体形式的CD19-CD3-LAG-3 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-LAG-3 TsAb_M的核苷酸序列如SEQ ID NO.267所示，具体为：
Figure PCTCN2017096594-appb-000364
Figure PCTCN2017096594-appb-000365
二聚体形式的CD19-CD3-LAG-3 TsAb_D的核苷酸序列如SEQ ID NO.269所示，具体为：
Figure PCTCN2017096594-appb-000366
Figure PCTCN2017096594-appb-000367
表4-3.CD19-CD3-LAG-3三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000368
实施例4-10：CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D的表达与纯化
一、CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_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(CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-9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、CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_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CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-8所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-LAG-3 TsAb_M重组蛋白的理论分子量为80.4kDa，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，因此该三特异性抗体为单体形式(图4-8A)；CD19-CD3-LAG-3 TsAb_D重组蛋白的理论分子量为88.3kDa，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(约180kDa)(图4-8B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-LAG-3 TsAb_M为单体形式，CD19-CD3-LAG-3 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-LAG-3 TsAb_M的氨基酸序列如SEQ ID NO.266所示，具体为：
Figure PCTCN2017096594-appb-000369
二聚体形式的CD19-CD3-LAG-3 TsAb_D的氨基酸序列如SEQ ID NO.268所示，具体为：
Figure PCTCN2017096594-appb-000370
Figure PCTCN2017096594-appb-000371
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示。
抗LAG-3 scFv的氨基酸序列如SEQ ID NO.294所示，具体为：
Figure PCTCN2017096594-appb-000372
抗LAG-3 scFv的重链可变区的氨基酸序列如SEQ ID NO.295所示，具体为：
Figure PCTCN2017096594-appb-000373
抗LAG-3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.296所示，具体为：
Figure PCTCN2017096594-appb-000374
单体形式的CD19-CD3-LAG-3 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示。
单体形式的CD19-CD3-LAG-3 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示。
二聚体形式的CD19-CD3-LAG-3 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示。
二聚体形式的CD19-CD3-LAG-3 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示。
实施例4-11：ELISA检测CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-LAG-3 TsAb_M或CD19-CD3-LAG-3 TsAb_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(OD450)。
ELISA结果如图4-9A和图4-9B所示：图4-9A说明CD19-CD3-LAG-3 TsAb_M与重组抗原CD19-hFc、CD3-hFc和LAG-3-hFc均具有体外结合活性，其中LAG-3结合活性最高，CD19结合活性次之，CD3结合活性较弱；图4-9B说明CD19-CD3-LAG-3 TsAb_D与重组抗原CD19-hFc、CD3-hFc和LAG-3-hFc同样具有体外结合活性，其中LAG-3结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例4-12：CD19-CD3-LAG-3三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-LAG-3 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-LAG-3 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-LAG-3 TsAb_M和CD19-CD3-LAG-3 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000375
结果如图4-10所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-LAG-3 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为95％、92％和83％，CD19-CD3-LAG-3 TsAb_M的效果次之，杀伤效率约为89％、86％和73％，CD19-CD3  BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-LAG-3 TsAb_D与CD19-CD3-LAG-3 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为72％和61％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-LAG-3 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-13：CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子TIM-3蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-TIM-3 TsAb。
一、CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D构建方案设计
单体形式的CD19-CD3-TIM-3 TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗TIM-3 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗TIM-3 scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-TIM-3 TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗TIM-3 scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗TIM-3 scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗TIM-3 scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示。
抗TIM-3 scFv的重链可变区的核苷酸序列如SEQ ID NO.322所示，具体为：
Figure PCTCN2017096594-appb-000376
抗TIM-3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.323所示，具体为：
Figure PCTCN2017096594-appb-000377
抗TIM-3 scFv的核苷酸序列如SEQ ID NO.321所示，具体为：
Figure PCTCN2017096594-appb-000378
Figure PCTCN2017096594-appb-000379
单体形式的CD19-CD3-TIM-3 TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示。
单体形式的CD19-CD3-TIM-3 TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示。
二聚体形式的CD19-CD3-TIM-3 TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示。
二聚体形式的CD19-CD3-TIM-3 TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示。
为使三特异性抗体在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.330所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示。
二、CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-TIM-3 TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-TIM-3-F和pcDNA3.1-TIM-3-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗TIM-3 scFv的基因序列；针对CD19-CD3-TIM-3 TsAb_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-CD19-F和CD19-R、CD19-G4S-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CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗TIM-3 scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000380
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三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，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CD19-CD3-TIM-3 TsAb_M和二聚体形式的CD19-CD3-TIM-3 TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-TIM-3 TsAb_M的核苷酸序列如SEQ ID NO.271所示，具体为：
Figure PCTCN2017096594-appb-000381
二聚体形式的CD19-CD3-TIM-3 TsAb_D的核苷酸序列如SEQ ID NO.273所示，具体为：
Figure PCTCN2017096594-appb-000382
表4-4.CD19-CD3-TIM-3三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000383
实施例4-14：CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D的表达与纯化
一、CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_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(CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-13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、CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_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CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-11所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-TIM-3 TsAb_M重组蛋白的理论分子量为80.1kDa，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，因此该三特异性抗体为单体 形式(图4-11A)；CD19-CD3-TIM-3 TsAb_D重组蛋白的理论分子量为88.0kDa，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(约180kDa)(图4-11B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-TIM-3 TsAb_M为单体形式，CD19-CD3-TIM-3 TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-TIM-3 TsAb_M的氨基酸序列如SEQ ID NO.270所示，具体为：
Figure PCTCN2017096594-appb-000384
二聚体形式的CD19-CD3-TIM-3 TsAb_D的氨基酸序列如SEQ ID NO.272所示，具体为：
Figure PCTCN2017096594-appb-000385
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示。
抗TIM-3 scFv的氨基酸序列如SEQ ID NO.297所示，具体为：
Figure PCTCN2017096594-appb-000386
抗TIM-3 scFv的重链可变区的氨基酸序列如SEQ ID NO.298所示，具体为：
Figure PCTCN2017096594-appb-000387
抗TIM-3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.299所示，具体为：
Figure PCTCN2017096594-appb-000388
单体形式的CD19-CD3-TIM-3 TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示。
单体形式的CD19-CD3-TIM-3 TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示。
二聚体形式的CD19-CD3-TIM-3 TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示。
二聚体形式的CD19-CD3-TIM-3 TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示。
实施例4-15：ELISA检测CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-TIM-3 TsAb_M或CD19-CD3-TIM-3 TsAb_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(OD450)。
ELISA结果如图4-12A和图4-12B所示：图4-12A说明CD19-CD3-TIM-3 TsAb_M与重组抗原CD19-hFc、CD3-hFc和TIM-3-hFc均具有体外结合活性，其中TIM-3结合活性最高，CD19结合活性次之，CD3结合活性较弱；图4-12B说明CD19-CD3-TIM-3 TsAb_D与重组抗原CD19-hFc、CD3-hFc和TIM-3-hFc同样具有体外结合活性，其中TIM-3结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例4-16：CD19-CD3-TIM-3三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-TIM-3 TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-TIM-3 TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-TIM-3 TsAb_M和CD19-CD3-TIM-3 TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000389
结果如图4-13所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-TIM-3 TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为98％、97％和86％，CD19-CD3-TIM-3 TsAb_M的效果次之，杀伤效率约为92％、89％和76％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-TIM-3 TsAb_D与CD19-CD3-TIM-3 TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为76％和68％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-TIM-3 TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-17：CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子TIGIT蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-TIGIT TsAb。
一、CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D构建方案设计
单体形式的CD19-CD3-TIGIT TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗TIGIT scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗TIGIT scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-TIGIT TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗TIGIT scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗TIGIT scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗TIGIT scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示。
抗TIGIT scFv的重链可变区的核苷酸序列如SEQ ID NO.325所示，具体为：
Figure PCTCN2017096594-appb-000390
抗TIGIT scFv的轻链可变区的核苷酸序列如SEQ ID NO.326所示，具体为：
Figure PCTCN2017096594-appb-000391
抗TIGIT scFv的核苷酸序列如SEQ ID NO.324所示，具体为：
Figure PCTCN2017096594-appb-000392
Figure PCTCN2017096594-appb-000393
单体形式的CD19-CD3-TIGIT TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示。
单体形式的CD19-CD3-TIGIT TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示。
二聚体形式的CD19-CD3-TIGIT TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示。
二聚体形式的CD19-CD3-TIGIT TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示。
为使三特异性抗体在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.330所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示。
二、CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-TIGIT TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-TIGIT-F和pcDNA3.1-TIGIT-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗TIGIT scFv的基因序列；针对CD19-CD3-TIGIT TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-TIGIT-F和pcDNA3.1-TIGIT-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗TIGIT scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000394
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三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，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CD19-CD3-TIGIT TsAb_M和二聚体形式的CD19-CD3-TIGIT TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-TIGIT TsAb_M的核苷酸序列如SEQ ID NO.275所示，具体为：
Figure PCTCN2017096594-appb-000395
Figure PCTCN2017096594-appb-000396
二聚体形式的CD19-CD3-TIGIT TsAb_D的核苷酸序列如SEQ ID NO.277所示，具体为：
Figure PCTCN2017096594-appb-000397
Figure PCTCN2017096594-appb-000398
表4-5.CD19-CD3-TIGIT三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000399
Figure PCTCN2017096594-appb-000400
实施例4-18：CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D的表达与纯化
一、CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_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(CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例4-17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、CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_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CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_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，CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-TIGIT TsAb_M重组蛋白的理论分子量为80.9kDa，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，因此该三特异性抗体为单体形式(图4-14A)；CD19-CD3-TIGIT TsAb_D重组蛋白的理论分子量为88.8kDa，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(约180kDa)(图4-14B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-TIGIT TsAb_M为单体形式，CD19-CD3-TIGIT TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-TIGIT TsAb_M的氨基酸序列如SEQ ID NO.274 所示，具体为：
Figure PCTCN2017096594-appb-000401
二聚体形式的CD19-CD3-TIGIT TsAb_D的氨基酸序列如SEQ ID NO.276所示，具体为：
Figure PCTCN2017096594-appb-000402
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示。
抗TIGIT scFv的氨基酸序列如SEQ ID NO.300所示，具体为：
Figure PCTCN2017096594-appb-000403
抗TIGIT scFv的重链可变区的氨基酸序列如SEQ ID NO.301所示，具体为：
Figure PCTCN2017096594-appb-000404
Figure PCTCN2017096594-appb-000405
抗TIGIT scFv的轻链可变区的氨基酸序列如SEQ ID NO.302所示，具体为：
Figure PCTCN2017096594-appb-000406
单体形式的CD19-CD3-TIGIT TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示。
单体形式的CD19-CD3-TIGIT TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示。
二聚体形式的CD19-CD3-TIGIT TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示。
二聚体形式的CD19-CD3-TIGIT TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示。
实施例4-19：ELISA检测CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGITTsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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/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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-TIGIT TsAb_M或CD19-CD3-TIGIT TsAb_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(OD450)。
ELISA结果如图4-15A和图4-15B所示：图4-15A说明CD19-CD3-TIGIT TsAb_M与重组抗原CD19-hFc、CD3-hFc和TIGIT-hFc均具有体外结合活性，其中TIGIT和CD19结合活性均较高，CD3结合活性较弱；图4-15B说明CD19-CD3-TIGIT TsAb_D与重组抗原CD19-hFc、CD3-hFc和TIGIT-hFc同样具有体外结合活性，其中TIGIT和CD19结合活性均较高，CD3结合活性较弱。
实施例4-20：CD19-CD3-TIGIT三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-TIGIT TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-TIGIT TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-TIGIT TsAb_M和CD19-CD3-TIGIT TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000407
结果如图4-16所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-TIGIT TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为88％、86％和76％，CD19-CD3-TIGIT TsAb_M的效果次之，杀伤效率约为82％、79％和66％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-TIGIT TsAb_D与CD19-CD3-TIGIT TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为66％和55％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-TIGIT TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
实施例4-21：CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D真核表达载体的构建
在本发明中，以淋巴瘤B细胞表面的人类CD19蛋白，T细胞表面人类CD3和T细胞负共刺激分子BTLA蛋白为靶点的TiTE三特异性抗体被命名为CD19-CD3-BTLA TsAb。
一、CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D构建方案设计
单体形式的CD19-CD3-BTLA TsAb_M具体构建方案为：抗CD19 scFv，抗CD3 scFv和抗BTLA scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗BTLA scFv序列之间则通过连接片段2(Linker 2)相连。
二聚体形式的CD19-CD3-BTLA TsAb_D具体构建方案为：抗CD19 scFv，抗CD3 scFv 和抗BTLA scFv的序列通过连接片段(Linker)相连，具体地，抗CD19 scFv和抗CD3 scFv之间通过连接片段1(Linker 1)相连，抗CD3 scFv和抗BTLA scFv序列之间以IgD铰链区(Ala90-Val170)作为连接片段2(Linker 2)相连。
为使三特异性抗体在哺乳细胞中进行表达，针对抗CD19 scFv，抗CD3 scFv，抗BTLA scFv序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD19 scFv的重链可变区的核苷酸序列如SEQ ID NO.307所示。
抗CD19 scFv的轻链可变区的核苷酸序列如SEQ ID NO.308所示。
抗CD19 scFv的核苷酸序列如SEQ ID NO.306所示。
抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.310所示。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.311所示。
抗CD3 scFv的核苷酸序列如SEQ ID NO.309所示。
抗BTLA scFv的重链可变区的核苷酸序列如SEQ ID NO.328所示，具体为：
Figure PCTCN2017096594-appb-000408
抗BTLA scFv的轻链可变区的核苷酸序列如SEQ ID NO.329所示，具体为：
Figure PCTCN2017096594-appb-000409
抗BTLA scFv的核苷酸序列如SEQ ID NO.327所示，具体为：
Figure PCTCN2017096594-appb-000410
单体形式的CD19-CD3-BTLA TsAb_M连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.245所示。
单体形式的CD19-CD3-BTLA TsAb_M连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.247所示。
二聚体形式的CD19-CD3-BTLA TsAb_D连接片段1(Linker 1)的核苷酸序列如SEQ ID NO.249所示。
二聚体形式的CD19-CD3-BTLA TsAb_D连接片段2(Linker 2)的核苷酸序列如SEQ ID NO.251所示。
为使三特异性抗体在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.330所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.331所示。
二、CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D真核表达载体构建
本发明三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三特异性抗体，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CD19-CD3-BTLA TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-(GGGGS)3-BTLA-F和pcDNA3.1-BTLA-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、(GGGGS)3 Linker 2+抗BTLA scFv的基因序列；针对CD19-CD3-BTLA TsAb_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-CD19-F和CD19-R、CD19-G4S-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-BTLA-F和pcDNA3.1-BTLA-R扩增出抗CD19 scFv、GGGGS Linker 1+抗CD3 scFv、IgD铰链区Linker 2、抗BTLA scFv的基因序列。扩增完毕后，利用
Figure PCTCN2017096594-appb-000411
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三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，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CD19-CD3-BTLA TsAb_M和二聚体形式的CD19-CD3-BTLA TsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD19-CD3-BTLA TsAb_M的核苷酸序列如SEQ ID NO.279所示，具体为：
Figure PCTCN2017096594-appb-000412
Figure PCTCN2017096594-appb-000413
二聚体形式的CD19-CD3-BTLA TsAb_D的核苷酸序列如SEQ ID NO.281所示，具体为：
Figure PCTCN2017096594-appb-000414
Figure PCTCN2017096594-appb-000415
表4-6.CD19-CD3-BTLA三特异性抗体基因克隆中使用的引物
Figure PCTCN2017096594-appb-000416
实施例4-22：CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D的表达与纯化
一、CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_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(CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_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、CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_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CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图4-17所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D重组蛋白的纯度均＞95％：其中CD19-CD3-BTLA TsAb_M重组蛋白的理论分子量为80.0kDa，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，因此该三特异性抗体为单体形式(图4-17A)；CD19-CD3-BTLA TsAb_D重组蛋白的理论分子量为87.9kDa，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(约180kDa)(图4-17B)，说明两个蛋白分子可通过IgD铰链区形成二硫键相互连接，因此该三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CD19-CD3-BTLA TsAb_M为单体形式，CD19-CD3-BTLA TsAb_D为二聚体形式。
因此，可得知，单体形式的CD19-CD3-BTLA TsAb_M的氨基酸序列如SEQ ID NO.278所示，具体为：
Figure PCTCN2017096594-appb-000417
Figure PCTCN2017096594-appb-000418
二聚体形式的CD19-CD3-BTLA TsAb_D的氨基酸序列如SEQ ID NO.280所示，具体为：
Figure PCTCN2017096594-appb-000419
抗CD19 scFv的氨基酸序列如SEQ ID NO.282所示。
抗CD19 scFv的重链可变区的氨基酸序列如SEQ ID NO.283所示。
抗CD19 scFv的轻链可变区的氨基酸序列如SEQ ID NO.284所示。
抗CD3 scFv的氨基酸序列如SEQ ID NO.285所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.286所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.287所示。
抗BTLA scFv的氨基酸序列如SEQ ID NO.303所示，具体为：
Figure PCTCN2017096594-appb-000420
抗BTLA scFv的重链可变区的氨基酸序列如SEQ ID NO.304所示，具体为：
Figure PCTCN2017096594-appb-000421
抗BTLA scFv的轻链可变区的氨基酸序列如SEQ ID NO.305所示，具体为：
Figure PCTCN2017096594-appb-000422
单体形式的CD19-CD3-BTLA TsAb_M连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.244所示。
单体形式的CD19-CD3-BTLA TsAb_M连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.246所示。
二聚体形式的CD19-CD3-BTLA TsAb_D连接片段1(Linker 1)的氨基酸序列如SEQ ID NO.248所示。
二聚体形式的CD19-CD3-BTLA TsAb_D连接片段2(Linker 2)的氨基酸序列如SEQ ID NO.250所示。
实施例4-23：ELISA检测CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD19-hFc、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/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，分别加入纯化的三特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD19-CD3-BTLA TsAb_M或CD19-CD3-BTLA TsAb_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(OD450)。
ELISA结果如图4-18A和图4-18B所示：图4-18A说明CD19-CD3-BTLA TsAb_M与重组抗原CD19-hFc、CD3-hFc和BTLA-hFc均具有体外结合活性，其中BTLA结合活性最高，CD19结合活性次之，CD3结合活性较弱；图4-18B说明CD19-CD3-BTLA TsAb_D与重组抗原CD19-hFc、CD3-hFc和BTLA-hFc同样具有体外结合活性，其中BTLA结合活性最高，CD19结合活性次之，CD3结合活性较弱。
实施例4-24：CD19-CD3-BTLA三特异性抗体介导的细胞杀伤实验
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TiTE三特异抗体(CD19-CD3-BTLA TsAb_M)、二聚体形式的TiTE三特异抗体(CD19-CD3-BTLA TsAb_D)以及抗CD19/抗CD3 BiTE双特异抗体(CD19-CD3 BsAb，购自吴江近岸蛋白质科技有限公司)分别作用于同一供体来源的人血PBMC制备的CIK细胞(CD3+CD56+)与CCL-86 Raji淋巴瘤细胞(CD19+，购自ATCC)，检测细胞死亡情况，比较三种抗体介导的CIK效应细胞对CCL-86 Raji靶细胞的杀伤效率差异。
细胞杀伤实验步骤：
1.PBMC的分离：使用新抽取的志愿者抗凝血液，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)(购自 Gbico公司)重悬，调整细胞密度为1×106/ml，添加到全长抗体Anti-CD3(5ug/ml)、全长抗体Anti-CD28(5ug/ml)和NovoNectin(25ug/ml)包被的T25培养瓶中(全长抗体与NovoNectin均购自吴江近岸蛋白质科技有限公司)，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；
3.CIK细胞对Raji细胞的杀伤效率：在96孔板中进行细胞杀伤实验，反应体积为100uL，取上述培养的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)，分别添加不同终浓度(25、12.5、6.25、3.125ng/ml)的CD19-CD3 BsAb、CD19-CD3-BTLA TsAb_M和CD19-CD3-BTLA TsAb_D抗体样品，室温混匀3～5min，37℃共培养3h后，每孔添加10μl的CCK-8，37℃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的细胞杀伤效率作为空白对照。
Figure PCTCN2017096594-appb-000423
结果如图4-19所示：当CIK效应细胞∶Raji靶细胞(E∶T比)为1∶1时，在未添加任何抗体的条件下，CIK细胞对Raji细胞3h的杀伤效率约为23％；在添加较高浓度抗体(25、12.5、6.25ng/ml)的条件下，CIK细胞对Raji细胞的杀伤效率均有显著地提高，其中CD19-CD3-BTLA TsAb_D所介导的细胞杀伤效果最好，杀伤效率分别约为90％、87％和86％，CD19-CD3-BTLA TsAb_M的效果次之，杀伤效率约为86％、82％和76％，CD19-CD3 BsAb的效果最弱，杀伤效率分别约为80％、54％和54％；在添加较低浓度抗体(3.125ng/ml)的条件下，CD19-CD3-BTLA TsAb_D与CD19-CD3-BTLA TsAb_M所介导的CIK细胞对Raji细胞的杀伤效率仍有一定程度地提高，杀伤效率分别约为79％和68％，而CD19-CD3 BsAb与空白对照相比基本没有效果。上述结果说明两种形式的CD19-CD3-BTLA TiTE三特异抗体所介导的T细胞对CD19阳性肿瘤细胞的靶向杀伤活性均优于CD19-CD3 BiTE双特异性抗体，其中二聚体形式较单体形式具有更好的效果。
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. 一种三功能分子，所述三功能分子选自以下之任一：(1)所述三功能分子的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并激活T细胞正共刺激分子的第三功能域；(2)所述三功能分子的结构中包括能够结合于CD19的第一功能域、能够结合并激活T细胞表面CD3分子的第二功能域和能够结合并阻断T细胞负共刺激分子的第三功能域。
  2. 根据权利要求1所述的三功能分子，其特征在于，所述三功能分子能够在识别CD19的同时，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
    所述三功能分子能够在识别CD19的同时，结合并激活T细胞表面CD3分子、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所述的三功能分子，其特征在于，所述第一功能域为抗CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为抗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CD19的抗体，所述第二功能域为抗CD3的抗体，所述第三功能域为抗T细胞负共刺激分子的抗体。
  4. 根据权利要求3所述的三功能分子，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所述的三功能分子，其特征在于，所述第一功能域和所述第二功能域之间通过连接片段1连接，所述第二功能域和所述第三功能域之间通过连接片段2连接。
  6. 根据权利要求5所述的三功能分子，其特征在于，所述连接片段1和连接片段2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所述的三功能分子，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.23、SEQ ID NO.25、SEQ ID NO.27之任一所示；免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.29所示。
  8. 根据权利要求1所述的三功能分子，其特征在于，所述第一功能域为抗CD19的单链抗体，所述第二功能域为抗CD3的单链抗体，所述第三功能域为抗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CD19的单链抗体，所述第二功能域为抗CD3的单链抗体，所述第三功能域为抗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所述的三功能分子，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所述的三功能分子，其特征在于，所述抗CD19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CD19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CD3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CD3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的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.12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.13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.90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.91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.93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.94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.96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.97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.99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.100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.102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.103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.105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.106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.289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.290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.292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.293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.295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.296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.298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.299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.301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.302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.304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.305所示。
  11. 根据权利要求9所述的三功能分子，其特征在于，所述抗CD19的单链抗体的氨基酸序列如SEQ ID NO.5所示；所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.8所示；所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.11所示；所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.89所示；所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.92所示；所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.95所示；所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.98所示；所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.101所示；所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.104所示；所述4-1BBL胞外区结构域的氨基酸序列如SEQ ID NO.203所示；所述B7RP-1胞外区结构域的氨基酸序列如SEQ ID NO.204所示；所述OX40L胞外区结构域的氨基酸序列如SEQ ID NO.205所示；所述GITRL胞外区结构域的氨基酸序列如SEQ ID NO.206所示；所述CD70胞外区结构域的氨基酸序列如SEQ ID NO.207所示；所述抗PD-1的单链抗体的氨基酸序列如SEQ ID NO.288所示；所述抗CTLA-4的单链抗体的氨基酸序列如SEQ ID NO.291所示；所述抗LAG-3的单链抗体的氨基酸序列如SEQ ID NO.294所示；所述抗TIM-3的单链抗体的氨基酸序列如SEQ ID NO.297所示；所述抗TIGIT的单链抗体的氨基酸序列如SEQ ID NO.300所示；所述抗BTLA的单链抗体的氨基酸序列如SEQ ID NO.303所示。
  12. 根据权利要求1所述的三功能分子，其特征在于，所述三功能分子的氨基酸序列如SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.59、SEQ ID NO.61、SEQ ID NO.63、SEQ ID  NO.65、SEQ ID NO.67、SEQ ID NO.69、SEQ ID NO.71、SEQ ID NO.73、SEQ ID NO.75、SEQ ID NO.77、SEQ ID NO.79、SEQ ID NO.81、SEQ ID NO.177、SEQ ID NO.179、SEQ ID NO.181、SEQ ID NO.183、SEQ ID NO.185、SEQ ID NO.187、SEQ ID NO.189、SEQ ID NO.191、SEQ ID NO.193、SEQ ID NO.195、SEQ ID NO.258、SEQ ID NO.260、SEQ ID NO.262、SEQ ID NO.264、SEQ ID NO.266、SEQ ID NO.268、SEQ ID NO.270、SEQ ID NO.272、SEQ ID NO.274、SEQ ID NO.276、SEQ ID NO.278或SEQ ID NO.280之任一所示。
  13. 一种多核苷酸，其编码如权利要求1～12任一项所述三功能分子。
  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任一项所述三功能分子的制备方法，包括：构建含有三功能分子基因序列的表达载体，然后将含三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三功能分子。
  17. 如权利要求1～12任一项所述三功能分子用于制备肿瘤治疗药物的用途。
  18. 一种肿瘤治疗药物组合物，含有如权利要求1～12任一项所述三功能分子及至少一种药学可接受的载体或赋形剂。
  19. 一种体外治疗肿瘤的方法，包括将如权利要求1～12任一项所述三功能分子或如权利要求18所述肿瘤治疗药物组合物施用于肿瘤患者。
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