WO2018068652A1 - 抗egfr和抗cd3双特异抗体及其应用 - Google Patents

抗egfr和抗cd3双特异抗体及其应用 Download PDF

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WO2018068652A1
WO2018068652A1 PCT/CN2017/103896 CN2017103896W WO2018068652A1 WO 2018068652 A1 WO2018068652 A1 WO 2018068652A1 CN 2017103896 W CN2017103896 W CN 2017103896W WO 2018068652 A1 WO2018068652 A1 WO 2018068652A1
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antibody
bispecific antibody
seq
cells
bispecific
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周海平
张稳
徐萌
裴爽
昝琰璐
文圣梅
白义
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Beijing Jingyitaixiang Technology Development CoLtd
Beijing Dongfang Biotech Co Ltd
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Beijing Jingyitaixiang Technology Development CoLtd
Beijing Dongfang Biotech Co Ltd
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Priority to US16/321,438 priority Critical patent/US11396547B2/en
Priority to EA201990171A priority patent/EA201990171A1/ru
Priority to KR1020197003194A priority patent/KR102179674B1/ko
Priority to AU2017343414A priority patent/AU2017343414B2/en
Priority to JP2019505507A priority patent/JP6821006B2/ja
Priority to CA3032560A priority patent/CA3032560C/en
Priority to EP17860812.1A priority patent/EP3527590A4/en
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for constructing and preparing a bispecific antibody against EGFR and CD3, and the use of the antibody in diseases.
  • BiAb is an artificial antibody composed of two different antibody fragments that specifically recognize and bind two different antigens or two different antigenic epitopes.
  • Bispecific antibodies are widely used in the treatment of cancer, inflammation and other diseases. Since these antibodies target a single target, many patients are unable to respond adequately to a single therapy and often develop resistance. Bispecific antibodies can simultaneously recognize two different antigens or antigenic epitopes, and can be used as a medium to redirect immune effector cells, such as natural killer cells and T cells, to enhance the killing function of tumor cells. In addition, bispecific antibodies can also localize to two different antigens in the same cell, leading to changes in cellular signaling, including cancer spread signals or inflammatory signals. After long-term research and development, various forms of bispecific antibodies have emerged, such as bispecific microbodies, double-stranded antibodies, single-chain bivalent antibodies, and multivalent bispecific antibodies.
  • bispecific antibodies fall into two broad categories: Fc-containing and Fc-free.
  • the former has better solubility, stability and half-life, while Fc-mediated antibody-dependent cytotoxicity (ADCC) and complement-dependent cytolysis (CDC) can bring about some additional effects required for treatment.
  • a bispecific antibody lacking Fc relies entirely on its antigen-binding ability to exert its therapeutic effect; in addition, the Fc protein can prolong the half-life of the drug protein (or polypeptide) in vivo, thereby prolonging the action time of the active molecule in vivo.
  • Tumor cell surface antigen epidermal growth factor receptor epidermal growth factor receptor is widely distributed on epithelial membranes other than vascular tissue, is a transmembrane protein with a molecular weight of approximately l80KDa, with ligand-induced tyrosine protein kinase Active, it is a member of the conserved receptor family of ErbB, and other members of this family include HER2/Neu/ErbB2, HER3/ErbB3 and HER4/ErbB4.
  • ErbB receptor contains an extracellular (EC) ligand binding region, a single transmembrane region consisting of two repetitive cysteine-rich regions, and a tyrosine protein kinase and autophosphorus.
  • the intracellular sequence of the site when the ligand and the receptor bind, it causes dimerization of the receptor to form a homo- or heterodimer; the dimerized receptor undergoes cross-linking phosphorylation and activates the intracellular region.
  • the TK subregion thereby inducing the next level of signal transduction, leading to cell proliferation and transformation.
  • EGFR is involved in tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis.
  • the mechanism includes: high expression of EGFR leads to enhanced downstream signaling, and increased expression of mutant EGFR receptor or ligand leads to The sustained activation of EGFR, the enhancement of the role of the autocrine loop, the destruction of the receptor down-regulation mechanism, and the activation of abnormal signaling pathways.
  • EGFR is highly expressed or abnormally expressed in many solid tumors, and is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis.
  • Overexpression of EGFR plays an important role in the evolution of tumors, such as glioma, lung cancer, prostate cancer, pancreatic cancer and other tissues have overexpression of EGFR.
  • CD3 molecule is an important differentiation antigen on T cell membrane and is a characteristic marker of mature T cells. It consists of 6 peptide chains with non-covalent bonds and T cell antigen receptors. TCR) constitutes a TCR-CD3 complex that not only participates in the intracytoplasmic assembly of the TCR-CD3 complex, but also through the immunoreceptor tyrosine-based activation motif (ITAM) of the cytoplasmic region of each polypeptide chain. Delivery of antigenic stimulation signals. The main function of CD3 molecule is to stabilize the TCR structure and transmit T cell activation signal. When TCR specifically recognizes and binds antigen, CD3 participates in transduction of signal into T cell cytoplasm as the first signal to induce T cell activation. It plays an extremely important role in T cell antigen recognition and immune response production.
  • TCR immunoreceptor tyrosine-based activation motif
  • bispecific antibodies In medicine, especially in the immunotherapy of tumors, bispecific antibodies have good effects and prospects, and can simultaneously bind to specific antigen-anti-immunologically active cells CDl6 or CD3 on tumor cells and immune cells, with activated NK Cell or T cell action, and anti-tumor specific antigen part can bind to tumor cells, target immune cells to tumor cells, increase local NK cell or T cell concentration, so that immune effector cells can specifically kill tumor cells effect.
  • EGFR EGFR
  • CD3 bispecific antibody drug products this technology remains to be studied.
  • the present invention uses a tetravalent bispecific antibody to completely retain the biological activity of the anti-EGFR antibody; tetravalent bispecific can better recognize tumor antigens and effector cells (T cells and NK) Cells, etc., to better exert the biological activity of the bispecific antibody.
  • U.S. Patent No. 9249217 B2 uses the ScFv-ScFv (BITE) form, while BITE is bivalent and has no Fc fragment.
  • the present invention contains an Fc fragment in addition to the advantage of a tetravalent antibody; the Fc fragment can prolong the half-life of the effector molecule in vivo, thereby prolonging the action time of the effector molecule in vivo.
  • the present invention provides a bispecific antibody which increases the anti-CD3 ScFv sequence at the C-terminus of the anti-EFGR, which retains the intact molecular structure of the anti-EGFR antibody while increasing binding.
  • the ability of CD3 antigen not only maintains the biological activity of the original EGFR antibody in vivo, but also enhances the effect of immune effector cells on killing tumor cells by specifically recognizing two different antigens and targeting immune effector cells to tumor cells. .
  • the present invention provides anti-EGFR and anti-CD3 bispecific antibodies and uses thereof,
  • a complete monoclonal antibody (b) a single chain antibody ScFv and (c) a linker; said (a) specifically binding to an EGFR antigen and consisting of two antibody heavy chains and two antibody light chains; The (b) specifically binds to the immune cell antigen CD3, the (b) is two single-chain antibodies ScFv; the (b) is two single-chain antibodies ScFv; the two single strands of the (b) The antibody ScFv is linked to the C-terminus of the two heavy chains of (a) via the (c) linker linker, respectively.
  • amino acid sequence of the (c) linker linker in the bispecific antibody is (GGGGX)n
  • X comprises Ser or Ala
  • X is preferably Ser
  • n is a natural number of 1-4
  • n is preferably 3.
  • the bispecific antibody (a) is a complete monoclonal antibody consisting of two light chains and two heavy chains, each of which is linked by a disulfide bond between the two heavy chains Connected by a disulfide bond in the hinge region.
  • Variable regions of the heavy and light chains Heterologous binding to tumor cell surface EGFR antigen.
  • the heavy chain constant region of the (a) intact monoclonal antibody is one of IgG1, IgG2, IgG3 or IgG4, preferably IgG2.
  • amino acid sequence of the heavy chain variable region of the (a) intact monoclonal antibody is one of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3; the (a) intact monoclonal antibody
  • amino acid sequence of the light chain variable region is one of SEQ ID NO 4, EQ ID NO 5 or SEQ ID NO 6.
  • amino acid sequence of the heavy chain variable region of the (b) single-chain antibody ScFv is SEQ ID NO: 8
  • amino acid sequence of the light chain variable region of the (b) single-chain antibody ScFv is SEQ ID NO 9.
  • amino acid sequence of the (b) two single-chain antibody ScFvs is SEQ ID NO 7.
  • each (b) single-chain antibody ScFv is composed of a heavy chain variable region, (c) a linker linker, and a light chain variable region, the heavy chain variable region and the light chain variable region specifically binding to the immune region
  • the cell surface CD3 antigen; the amino acid sequence of the (c) linker linker is (GGGGS)n, n is a natural number of 1-4, preferably (GGGGS) 3 .
  • an expression vector for constructing a bispecific antibody wherein the bispecific antibody can be constructed into one vector or separately constructed on two different vectors.
  • the constructed vector is transfected into a host cell by genetic engineering methods, the host cell comprising a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, a NSO cell or other mammalian cell, preferably a CHO cell.
  • the bispecific antibody is obtained by a conventional immunoglobulin method, including protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve method.
  • the bispecific antibody is used for the treatment of tumor tissues with high or abnormal expression of EGFR and other EGFR overexpression causes treatment of the disease.
  • the intact monoclonal antibody is a full length antibody.
  • the bispecific antibody of the present invention is in the form of a tetravalent antibody which completely retains the antibody sequence which binds to the tumor antigen and has a high affinity. At the same time, tetravalent bispecific antibodies can better connect tumor cells and effector cells, which is more advantageous. To play the biological function of bispecific antibodies.
  • the molecule of the present invention also includes an Fc fragment; the presence of the Fc fragment can prolong the half-life of the drug molecule in vivo, thereby enabling the drug molecule to function better and reducing the frequency of drug use. To alleviate the suffering of patients.
  • the present invention is useful for the treatment of high expression or abnormal expression of EGFR tumor tissue, as well as other EGFR overexpression causing treatment of the disease.
  • Figure 1 exemplarily shows a molecular schematic of a bispecific antibody
  • Figure 2 exemplarily shows a construction diagram of bispecific molecular plasmid expression
  • Figure 3 exemplarily shows a denatured SDS electropherogram of the purified bispecific molecule
  • Figure 4 exemplarily shows an ELISA method for detecting the binding ability of a bispecific molecule to an antigen
  • Figure 5 exemplarily shows the binding of a bispecific antibody to A431 cells
  • Figure 6 exemplarily shows the binding of a bispecific antibody to Jukart cells
  • Figure 7 exemplarily shows bispecific antibody-mediated binding of PBMC to A431
  • Figure 8 exemplarily shows the killing effect of bispecific antibody-mediated PBMC on H520.
  • the invention provides anti-EGFR and anti-CD3 bispecific antibodies and uses thereof, comprising (a) an intact monoclonal antibody, (b) a single chain antibody ScFv and (c) a linker; said (a) specific for EGFR antigen Binding and consisting of two antibody heavy chains and two antibody light chains; said (b) specifically binding to the immune cell antigen CD3, said (b) being two single chain antibodies ScFv; said (b) being two a single-chain antibody ScFv; the two single-chain antibodies ScFv of (b) are linked to the C-terminus of the two heavy chains of (a) by the (c) linker linker, respectively.
  • the amino acid sequence of the (c) linker linker is (GGGGX)n, X comprises Ser or Ala, X is preferably Ser; n is a natural number of 1-4, and n is preferably 3.
  • the bispecific antibody (a) is a complete monoclonal antibody consisting of two light chains and two heavy chains, each of which is disulfide bonded between the heavy and light chains. Then, the two heavy chains are connected by a disulfide bond in the hinge region. The variable regions of the heavy and light chains specifically bind to tumor cell surface EGFR antigens.
  • the heavy chain constant region of the (a) intact monoclonal antibody is one of IgG1, IgG2, IgG3 or IgG4, preferably IgG2.
  • amino acid sequence of the heavy chain variable region of the (a) intact monoclonal antibody is one of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3;
  • SEQ ID NO 1 (the heavy chain variable region amino acid sequence of a nicotine monoclonal antibody):
  • SEQ ID NO 2 (the heavy chain variable region amino acid sequence of a cetux monoclonal antibody):
  • SEQ ID NO 3 (the heavy chain variable region amino acid sequence of panitumumab monoclonal antibody):
  • amino acid sequence of the light chain variable region of the (a) intact monoclonal antibody is one of SEQ ID NO 4, EQ ID NO 5 or SEQ ID NO 6.
  • SEQ ID NO 4 (light chain variable region amino acid sequence of a nicotine monoclonal antibody):
  • SEQ ID NO 5 (light chain variable region amino acid sequence of cetux monoclonal antibody):
  • SEQ ID NO 6 (light chain variable region amino acid sequence of panitumumab monoclonal antibody):
  • amino acid sequence of the heavy chain variable region of the (b) single-chain antibody ScFv is SEQ ID NO 8 and the amino acid sequence of the light chain variable region of the (b) single-chain antibody ScFv is SEQ ID NO 9; among them,
  • SEQ ID NO 8 (heavy chain variable region amino acid sequence of anti-CD3 single chain antibody):
  • SEQ ID NO 9 (light chain variable region amino acid sequence of anti-CD3 single chain antibody):
  • amino acid sequence of the (b) two single-chain antibody ScFvs is SEQ ID NO 7; wherein
  • SEQ ID NO 7 (anti-CD3 single chain antibody amino acid sequence):
  • each (b) single chain antibody ScFv is composed of a heavy chain variable region, (c) a linker linker and a light chain variable region, said heavy chain variable region and light chain variable region specificity Binding to the immune cell surface CD3 antigen;
  • the amino acid sequence of the (c) linker linker is (GGGGS)n, n is a natural number from 1 to 4, preferably (GGGGS) 3 .
  • the constructed vector is transfected into a host cell comprising prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.
  • the bispecific antibody is used for the treatment of tumor tissue with high or abnormal expression of EGFR and other EGFR overexpression causes treatment of the disease.
  • PTSE was selected as an expression vector to clone and express the anti-EGFR light chain gene and the anti-EGFR heavy chain-CD3 ScFV fusion gene.
  • the light chain gene and the fusion gene were added to the Sall and BamHl restriction sites on both sides of the coding region, and the two genes were synthesized by Zhongmeitai Biotechnology (Beijing) Co., Ltd. and cloned into the PUC19 vector.
  • the two plasmids were separately transformed into TOP competent state (Huitian Dongfang, item number HT702-03), and the small endophore was used with the Kangwei Century Kit (Cat. No. CW0500).
  • the plasmid was digested with BamHI-HF and homologously recombined to obtain an expression vector containing the light chain and the fusion gene (see Fig. 2A and Fig. 2B).
  • the plasmids were named PTSE-JY016L-TetBiAb and PTSE-JY016H-TetBiAb, respectively.
  • PGN-2CMV was selected as an expression vector to clone and express the anti-EGFR light chain gene and the anti-EGFR heavy chain-CD3 ScFV fusion gene, which includes the selection markers Neomycin and GS, containing two CMV promoters and corresponding structural units.
  • the primers for the light chain and the fusion gene were designed and introduced into the Kozak sequence, the signal peptide and the corresponding restriction sites, and synthesized by Sino-Thailand Biotechnology (Beijing) Co., Ltd.
  • the plasmid was extracted by the endotoxin-free kit (Kangwei Century, CW2104), and the specific steps were carried out according to the instructions provided by the kit.
  • HEK293ES suspension cells Human embryonic kidney cells (HEK293ES suspension cells) were cultured in FreeStyle 293 Expression Medium (Gibco, 12338-026), and cells were subcultured every one to two days. After passage, the initial cell density was maintained at 0.2-0.6 ⁇ 10 6 /ml. The cell culture volume was 15 to 35% of the volume of the shake flask, and the cell culture flask was cultured in a shaker (shake speed: 135 rpm, temperature: 37 ° C, CO 2 : 5%).
  • HEK293ES cells in a logarithmic growth phase with good growth were passaged to a cell density of 0.5 ⁇ 10 6 /ml, and cultured overnight on a shaker (135 rpm, 37 ° C, 5% CO 2 ). Transfection was carried out for two days.
  • the prepared 1 ⁇ 10 6 /ml cell suspension was incubated for 2 h on a shaker (135 rpm, 39 ° C, 5% CO 2 ) before transfection.
  • PTSE-antiEGFR-H-TetBiAb was added in sequence (final concentration 0.5). Gg/ml), PTSE-antiEGFR-L (final concentration 0.5 ⁇ g/ml), polyethyleneimine PEI (Sigma) (final concentration 2 ug/ml), mixed, and co-transfected into HEK293ES suspension cells, then Incubate for 40 min on a shaker (135 rpm, 39 ° C, 5% CO 2 ).
  • the transfected cells were further cultured at 135 rpm, 37 ° C, 5% CO 2 shaker to express anti-EGFR x CD3 tetravalent antibody. After 96 hours of transfection, the expression supernatant was harvested by centrifugation.
  • the expression supernatant was filtered through a 0.22 uM filter, and an antibody having an Fc domain was obtained from the expression supernatant using an affinity chromatography column.
  • the equilibration buffer and elution buffer were 50 mM Tris-HCl 0.15 M NaCl pH 7.0 and 0.1 M citric acid-sodium citrate pH 3.0, respectively.
  • the target bispecific antibody was obtained by cation exchange chromatography.
  • the cation exchange column was HiTrap SP FF, and the SP column was equilibrated by an equilibration buffer of 20 mM PB PH6.3, and eluted with an elution buffer of 20 mMPB + 1 M NaCl (pH 6.3).
  • the EGFR antibody sequence of Bispecific-1 is the nimotuzumab sequence; the EGFR antibody sequence of Bispecific-2 is cetuximab; the EGFR antibody sequence of Bispecific-3 is panitumumab (Panitumumab) ).
  • the EGFR extracellular domain or the dimer of the CD3E and CD3G subunit extracellular domain was coated with a pH 9.6 carbonate buffer, 100 ng/well/100 ul, and coated overnight at 4 °C. Wash three times with 300 ul/well of 0.1% PBS (PBS-T) buffer, and then add 1% BSA-PBS solution for 2 h at room temperature. Add bispecific antibodies of different dilutions or corresponding antibodies. The highest concentration of various bispecific antibodies (or antibodies) was 1 uM, and 10 gradients were used to make 10 gradients. The last well was only diluted with PBS as a negative control and incubated at 37 ° C for 1 h.
  • the cells were washed five times with 300 ul/well PBS-T solution, and then incubated with Anti-Human Fc-HRP secondary antibody diluted 1:40,000 in 1% BSA-PBS solution for 1 h at 37 °C.
  • the results of the experiment are shown in Figure 4.
  • the ability of all bispecific antibodies to bind EGFR is similar to that of their corresponding monoclonal antibodies [ie, the ability of the bispecific antibody constructed by the nimotuzumab sequence to bind EGFR is similar to that of nimotuzumab. (Fig. 4A); the ability of the bispecific antibody constructed by the cetuximab sequence to bind EGFR is similar to cetuximab (Fig. 4B); the ability of the bispecific antibody constructed by the panitumumab sequence to bind EGFR The similarity of panitumumab (Fig. 4C)]; and the ability of various bispecific antibodies to bind to CD3E and CD3G dimers was similar (Fig. 4D). The results indicate that the form of the bispecific antibody used herein retains almost intact the ability of the original antibody to bind antigen; and that the ability of CD3 to bind CD3 is independent of the different antibodies to which it is linked (Fig. 4D).
  • a tumor cell line (A431) overexpressing EGFR is used to detect the binding of different bispecific antibodies to cell surface EGFR, and a corresponding antibody is used as a positive control, and human IgG (hIgG) is used as an isotype control.
  • A431 cells were collected by centrifugation with 0.25% trypsin. Diluted various antibodies at the same time, the highest concentration was 1 uM, 3 times gradient dilution. The collected cells were washed three times with PBS+1% BSA, and the cells were resuspended with PBS+1% BSA, and then the cells were plated in 96-well plates at 1 ⁇ 10 5 cells per well, and 100 ⁇ l of diluted dispecific was added.
  • the antibody was incubated for 1 hour at room temperature; the supernatant was removed by centrifugation, the cells were washed three times with PBS, and the cells were resuspended with diluted Alexa488-labeled anti-human IgG-Fc antibody, incubated at room temperature for 1 hour in the dark, and washed three times with PBS.
  • the cells were resuspended in 100 ul PBS and the fluorescence intensity was measured by flow cytometry.
  • the results were analyzed using Graphpad Prism. The results show (Fig. 5) that the binding ability of each bispecific antibody to A431 is comparable to its corresponding antibody, while the binding capacity of hIgG to A431 is weak. It was demonstrated that the bispecific antibody preferably retains the specific binding activity of the parent antibody to the cell surface EGFR-specific binding.
  • Jurkat cells overexpress CD3 and can be used to detect the binding of bispecific antibodies to cell surface CD3.
  • the binding process of the bispecific antibody to Jurkat cells was similar to that of Example 4a except that the Jurkat cells were suspension cells and A431 was adherent cells. Jurkat cells were collected by centrifugation, and various antibodies were added, and the rest of the procedures were the same as those of the A431 experiment.
  • the results showed (Fig. 6) that the ability of the isotype control to bind Jurkat was weak; different bispecific antibodies were able to bind well to the cells, and the ability of the three bispecific antibodies to bind Jurkat cells was consistent, indicating the bispecific antibody of Figure 1.
  • the ability of the anti-CD3 ScFv to bind to cell surface antigens is not affected by the antibodies to which it is linked.
  • A431 is a tumor cell line that overexpresses EGFR; while H520 does not.
  • A431 was used as the experimental cell line; H520 was used as a negative control.
  • the reaction was stopped by adding an equal volume of 0.5% BSA-PBS solution to the solution in the tube, and the cells were diluted by adding 1 ml of the corresponding complete medium, and the cell pellet was collected by centrifugation at 1500 rpm for 5 minutes. After resuspending in complete medium, culture in a cell culture flask for use.
  • PBS Ph7.2, containing 0.5% BSA, 2 mM EDTA
  • Platelets were removed by centrifugation (20 degrees, 200 g, 10-15 minutes), and then a 30 ⁇ m filter was used to remove cell clumps.
  • PBMC cells were collected: centrifugation at 200 g for 10 minutes, and the supernatant was discarded.
  • 2x10 7 cells were resuspended in 60 ⁇ l buffer + 20 ⁇ l FcR Blocking Reagent. Add 20 ⁇ l of magnetic beads, mix and incubate at 2-8 ° C for 15 minutes; add 1-2 ml of buffer, 300 g, centrifuge for 10 minutes, discard the supernatant; resuspend 500 ⁇ l.
  • the sorting column was placed on a sorting rack, 500 ⁇ l was rinsed; the cell suspension was added, and the unbound cells were collected; after the liquid at the top of the column was drained, 500 ⁇ l was washed three times; the sorting column was removed from the sorting rack, and placed. In the collection tube, 1 ml was quickly rinsed, cells were collected, counted and the proportion of viable cells was observed.
  • the cells were cultured for 18 hours, and after the incubation, the TO-PRO3 dye was added at a ratio of 1:50,000, and incubated at 37 ° C for 10 min in the dark; and a flow-controlled 4TO-PRO3 labeled cell was set up. After centrifugation at 3000 rpm for 5 min, part of the medium supernatant was discarded, resuspended in 0.5% BSA-PBS solution, and flow-detected.
  • Cell death rate% (1-PKH26 + TOPRO3 - cell number (drug action group) / PKH26 + TOPRO3 - cell number (no drug action group)) X100
  • the bispecific antibody-mediated PBMC has a very good killing effect on A431 effector cells overexpressing EGFR; while their corresponding antibodies and hIgG have a weak killing effect.
  • all bispecific antibodies had a very weak killing effect on H520 cells that did not express EGFR, and were similar to the isotype control hIgG (Fig. 8). This indicates that the killing effect of all constructed bispecific antibodies on target cells is specific; the ability of three different bispecific antibodies to kill specific target cells is similar, and the cell death rate is about 80%. The above indicates that the bispecific antibody of Figure 1 has good biological activity.

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Abstract

一种抗EGFR和抗CD3的双特异抗体及其应用，所述双特异性抗体能够特异性结合肿瘤细胞表面抗原表皮生长因子受体EGFR和免疫细胞表面抗原分化簇3即CD3分子；其中抗CD3抗体的单链抗体ScFv位于抗EGFR抗体恒定区的C末端。还提供了该双特异性抗体的制备方法及其临床用途。所述双特异性抗体具有较高的亲和力，用于治疗EGFR高表达或非正常表达所引起的肿瘤疾病，以及其他EGFR过表达引起的疾病。

Description

抗EGFR和抗CD3双特异抗体及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种针对EGFR和CD3的双特异性抗体的构建和制备方法，以及该抗体在疾病中的应用。
背景技术
双特异性抗体(bispecific antibody,BiAb)是由两种不同的抗体片段构成的人工抗体，能够特异性识别和结合两个不同的抗原或两个不同的抗原表位。
单克隆抗体广泛应用于癌症、炎症和其它疾病的治疗，由于这些抗体所针对的都是单一的靶标，很多患者不能充分响应单一的疗法，时常出现抗药性。双特异性抗体能够同时识别两个不同的抗原或抗原表位，可以作为一种媒介重新定向免疫效应细胞，如自然杀伤细胞和T细胞，加强对肿瘤细胞的杀伤功能。此外，双特异性抗体还可以定位于同一个细胞的两个不同的抗原，导致细胞信号的改变，包括癌症扩散信号或炎症信号。经过长时间的研究和发展，出现了多种形式的双特异性抗体，如双特异性微抗体、双链抗体、单链双价抗体和多价双特异性抗体等。这些双特异性抗体基本上分为两大类：含有Fc的和不含有Fc的。前者具有较好的溶解性、稳定性和半衰期，而Fc介导的抗体依赖的细胞毒性作用(ADCC)和补体依赖性细胞溶解效应(CDC)可以带来一些治疗所需的附加效应。相比之下，缺乏Fc的双特异性抗体完全依赖其抗原结合能力发挥其治疗作用；另外Fc蛋白可以延长药物蛋白(或多肽)在体内的半衰期，从而延长活性分子在体内的作用时间。
肿瘤细胞表面抗原表皮生长因子受体(EGFR)：表皮生长因子受体广泛分布于除血管组织外的上皮细胞膜上，是一个跨膜蛋白,分子量约为l80KDa，具有配体诱导酪氨酸蛋白激酶活性，它是ErbB这个保守的受体家族中的一员,这个家族的其他成员包括 HER2/Neu/ErbB2,HER3/ErbB3和HER4/ErbB4。ErbB受体的共同特征是：包含一个胞外(EC)配体结合区，由两个重复的富含半胱氨酸的区域组成的单一跨膜区，以及含有酪氨酸蛋白激酶和自身磷酸化位点的胞内序列；配体和受体结合后，引起受体的二聚化作用，形成同型或异型二聚体；二聚化的受体发生交联磷酸化，激活胞内区的TK亚区，从而激发下一级信号传导，导致细胞增殖、转化。EGFR与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关，其机制包含有：EGFR的高表达引起下游信号传导的增强，突变型EGFR受体或配体表达的增加导致EGFR的持续活化，自分泌环的作用增强，受体下调机制的破坏，异常信号传导通路的激活等。研究表明EGFR在很多实体瘤中高表达或异常表达，与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关。EGFR的过表达在肿瘤的演进中发挥着重要作用，如胶质细胞瘤、肺癌、前列腺癌、胰腺癌等组织中都有EGFR的过度表达。
免疫细胞表面抗原分化簇3(CD3)：CD3分子是T细胞膜上的重要分化抗原,是成熟T细胞的特征性标志，由6条肽链组成，以非共价键与T细胞抗原受体(TCR)组成TCR-CD3复合体，不仅参与TCR-CD3复合体的胞浆内组装，而且通过各多肽链胞浆区的免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif，ITAM)传递抗原刺激信号。CD3分子的主要功能为：稳定TCR结构，传递T细胞活化信号,当TCR特异性识别并结合抗原后,CD3参与将信号转导到T细胞胞浆内,作为诱导T细胞活化的第一信号，在T细胞抗原识别和免疫应答产生过程中具有极其重要的作用。
在医学中，特别是肿瘤的免疫治疗中，双特异性抗体具有良好的效果和前景，能够同时结合肿瘤细胞和免疫细胞上的特异性抗原-抗免疫活性细胞CDl6或CD3的部分，具有激活NK细胞或T细胞作用，而抗肿瘤的特异性抗原部分可以结合肿瘤细胞，将免疫细胞靶向到肿瘤细胞，提高局部NK细胞或T细胞浓度，从而使免疫效应细胞对肿瘤细胞起到特异性杀伤作用。现有技术中，还没有成功上市的EGFR 和CD3双特异性抗体药物产品，该项技术有待研究。
相对于中国专利CN201510030519.9，本发明是采用四价双特异性抗体，完整的保留了抗EGFR抗体的生物学活性；四价双特异能够更好的识别肿瘤抗原和效应细胞(T细胞和NK细胞等)，从而能够更好地发挥双特异抗体的生物学活性。美国专利US9249217 B2则采用ScFv-ScFv(BITE)形式，而BITE是二价的且没有Fc片段。与BITE相比，本发明除了具有四价抗体的优势外，还含有Fc片段；Fc片段可以延长效应分子在体内的半衰期，从而延长了效应分子在体内的作用时间。
发明内容
本发明中提供了一种双特异性抗体，该双特异性抗体在抗EFGR的C-端增加了抗CD3的ScFv序列，这种双特异抗体保留了抗EGFR抗体完整分子结构，同时增加了结合CD3抗原的能力，这样既保持了原有EGFR抗体在体内生物学活性，同时通过特异性识别两种不同的抗原，靶向免疫效应细胞到肿瘤细胞，从而增加了免疫效应细胞杀伤肿瘤细胞的效果。
本发明提供了抗EGFR和抗CD3双特异抗体及其应用，
包含(a)完整的单克隆抗体，(b)单链抗体ScFv和(c)连接子；所述(a)与EGFR抗原特异性结合并且由两条抗体重链和两条抗体轻链组成；所述(b)与免疫细胞抗原CD3特异性结合，所述(b)为两条单链抗体ScFv；所述(b)为两条单链抗体ScFv；所述(b)的两条单链抗体ScFv分别通过所述(c)连接子linker与所述(a)的两条重链的C末端连接。
其中，所述双特异抗体中所述(c)连接子linker的氨基酸序列为(GGGGX)n,X包含Ser或Ala，X优选Ser；n为1-4的自然数，n优选3。
其中，所述双特异性抗体(a)完整的单克隆抗体是由两条轻链和两条重链组成，每条重链和轻链之间通过二硫键连接，两条重链之间通过铰链区的二硫键连接。所述重链和轻链的可变区特 异性结合肿瘤细胞表面EGFR抗原。
其中，所述(a)完整的单克隆抗体的重链恒定区为IgG1、IgG2、IgG3或IgG4中的一种，优选IgG2。
其中，所述(a)完整的单克隆抗体的重链可变区氨基酸序列为SEQ ID NO 1、SEQ ID NO 2或SEQ ID NO 3中的一种；所述(a)完整的单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO 4、EQ ID NO 5或SEQ ID NO 6中的一种。所述(b)单链抗体ScFv的重链可变区的氨基酸序列为SEQ ID NO 8，所述(b)单链抗体ScFv的轻链可变区的氨基酸序列为SEQ ID NO 9。
其中，所述(b)两条单链抗体ScFv的氨基酸序列均为SEQ ID NO 7。
其中，每个(b)单链抗体ScFv是由重链可变区、(c)连接子linker和轻链可变区组成，所述重链可变区和轻链可变区特异性结合免疫细胞表面CD3抗原；所述(c)连接子linker的氨基酸序列为(GGGGS)n，n为1-4的自然数，优选为(GGGGS)3。
其中，构建双特异抗体的表达载体，其中所述双特异抗体可以构建到一个载体中，或者分别构建到两个不同的载体上。
其中，通过基因工程方法将构建好的载体转染到宿主细胞中，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其他哺乳细胞，优选为CHO细胞。
其中，通过常规的免疫球蛋白方法，包含蛋白质A亲和层析和离子交换、疏水层析或分子筛方法获得所述双特异性抗体。
其中，所述双特异性抗体用于EGFR高表达或非正常表达的肿瘤组织的治疗及其他EGFR过表达引起疾病的治疗。
其中，所述完整的单克隆抗体为全长抗体。
采用上述技术方案，包括以下有益技术效果：
本发明的双特异性抗体是采用四价抗体的形式，这种形式的四价抗体完整的保留了结合肿瘤抗原的抗体序列，具有较高的亲和力。同时四价双特异抗体可以更好的连接肿瘤细胞和效应细胞，从而更有利 于发挥双特异抗体的生物学功能。相对于现在常用的BITE双特异抗体形式，本发明中的分子还包括了Fc片段；Fc片段的存在可以延长药物分子在体内的半衰期，从而能够使药物分子更好的发挥作用，降低病人用药频率，减轻病人的痛苦。本发明用于高表达或非正常表达EGFR肿瘤组织的治疗，以及其他EGFR过表达引起疾病的治疗。
附图说明
图1示例性示出了双特异抗体的分子示意图；
图2示例性示出了双特异分子质粒表达的构建图；
图3示例性示出了纯化后的双特异分子的变性SDS电泳图；
图4示例性示出了ELISA法检测双特异分子与抗原的结合能力；
图5示例性示出了双特异抗体与A431细胞的结合；
图6示例性示出了双特异抗体与Jukart细胞的结合；
图7示例性示出了双特异抗体介导的PBMC对A431的结合；
图8示例性示出了双特异抗体介导的PBMC对H520的杀伤效果。
具体实施方式
下面通过具体的实施例并结合附图对本发明做进一步的详细描述。
本发明提供了抗EGFR和抗CD3双特异抗体及其应用，包含(a)完整的单克隆抗体，(b)单链抗体ScFv和(c)连接子；所述(a)与EGFR抗原特异性结合并且由两条抗体重链和两条抗体轻链组成；所述(b)与免疫细胞抗原CD3特异性结合，所述(b)为两条单链抗体ScFv；所述(b)为两条单链抗体ScFv；所述(b)的两条单链抗体ScFv分别通过所述(c)连接子linker与所述(a)的两条重链的C末端连接。
进一步优选地，所述双特异抗体中所述(c)连接子linker的氨基酸序列为(GGGGX)n,X包含Ser或Ala，X优选Ser；n为1-4的自然数，n优选3。
进一步优选地，所述双特异性抗体(a)完整的单克隆抗体是由两条轻链和两条重链组成，每条重链和轻链之间通过二硫键连 接，两条重链之间通过铰链区的二硫键连接。所述重链和轻链的可变区特异性结合肿瘤细胞表面EGFR抗原。
进一步优选地，所述(a)完整的单克隆抗体的重链恒定区为IgG1、IgG2、IgG3或IgG4中的一种，优选IgG2。
进一步优选地，所述(a)完整的单克隆抗体的重链可变区氨基酸序列为SEQ ID NO 1、SEQ ID NO 2或SEQ ID NO 3中的一种；其中，
SEQ ID NO 1(尼妥珠单克隆抗体的重链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000001
SEQ ID NO 2(西妥昔单克隆抗体的重链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000002
SEQ ID NO 3(帕尼单抗单克隆抗体的重链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000003
其中，所述(a)完整的单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO 4、EQ ID NO 5或SEQ ID NO 6中的一种，
SEQ ID NO 4(尼妥珠单克隆抗体的轻链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000004
SEQ ID NO 5(西妥昔单克隆抗体的轻链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000005
SEQ ID NO 6(帕尼单抗单克隆抗体的轻链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000006
进一步优选地，所述(b)单链抗体ScFv的重链可变区的氨基酸序列为SEQ ID NO 8，所述(b)单链抗体ScFv的轻链可变区的氨基酸序列为SEQ ID NO 9；其中，
SEQ ID NO 8(抗CD3单链抗体的重链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000007
SEQ ID NO 9(抗CD3单链抗体的轻链可变区氨基酸序列)：
Figure PCTCN2017103896-appb-000008
进一步优选地，所述(b)两条单链抗体ScFv的氨基酸序列均为SEQ ID NO 7；其中，
SEQ ID NO 7(抗CD3单链抗体氨基酸序列)：
Figure PCTCN2017103896-appb-000009
进一步优选地，每个(b)单链抗体ScFv是由重链可变区、(c)连接子linker和轻链可变区组成，所述重链可变区和轻链可变区特异性结合免疫细胞表面CD3抗原；所述(c)连接子linker的氨基酸序列为(GGGGS)n，n为1-4的自然数，优选为(GGGGS)3。
进一步优选地，将构建好的载体转染到宿主细胞中，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其他哺乳细胞，优选为CHO细胞。
进一步优选地，所述双特异性抗体用于EGFR高表达或非正常表达的肿瘤组织的治疗及其他EGFR过表达引起疾病的治疗。
具体实施例1、双特异抗体分子的表达载体的构建
a)双特异瞬时表达载体构建
按照图1中双特异抗体的形式设计相应的基因序列。选择PTSE作为表达载体去克隆和表达抗EGFR的轻链基因和抗EGFR重链—CD3的ScFV融合基因。轻链基因和融合基因分别在编码区序列两侧加入Sall、BamHl酶切位点，两条基因由中美泰和生物技术(北京)有限公司合成并分别克隆到PUC19载体中。将两个质粒分别转化到TOP感受态(汇天东方，货号HT702-03)中，用康为世纪小提试剂盒(货号CW0500)进行小提后与载体分别用限制性内切酶Sall-HF和BamHI-HF酶切，进行同源重组，分别得到含有轻链及融合基因的表达载体(示意图见图2A和图2B)，质粒分别命名为：PTSE-JY016L-TetBiAb、PTSE-JY016H-TetBiAb。
b)双特异稳转表达载体构建
选择PGN-2CMV作为表达载体去克隆和表达抗EGFR的轻链基因和抗EGFR重链-CD3的ScFV融合基因，该载体包括筛选标记Neomycin和GS，含有两个CMV启动子及相应的结构单元。设计轻链及融合基因的引物并引入Kozak序列、信号肽及相应的酶切位点，由中美泰和生物技术(北京)有限公司进行合成。以质粒PTSE-JY016L-TetBiAb、PTSE-JY016H-TetBiAb为模板，PCR扩增出相应的条带并与载体进行同源重组(质粒图谱见图2C)。
具体实施例2、双特异抗体分子的表达与纯化
a)四价抗体的表达
利用无内毒素大提试剂盒(康为世纪，CW2104)进行质粒大提，具体操作步骤按照试剂盒提供的说明书进行操作。
人胚肾细胞(HEK293ES悬浮细胞)在FreeStyle 293 Expression Medium(Gibco，12338-026)中培养，细胞每隔一到两天传代一次，传代后细胞起始密度维持在0.2～0.6×106/ml，细胞培养体积为摇瓶容积的15～35％，细胞培养瓶放在摇床(摇床 转速:135rpm，温度:37℃，CO2:5％)中培养。转染前一天，将处于对数生长期，生长状态良好的HEK293ES细胞，传代到细胞密度为0.5×106/ml，放摇床(135rpm，37℃，5％CO2)培养过夜，待第二天进行转染。
转染前将准备好的1×106/ml细胞悬液在摇床(135rpm，39℃，5％CO2)培养2h，转染时，依次加入PTSE-antiEGFR-H-TetBiAb(终浓度0.5μg/ml)、PTSE-antiEGFR-L(终浓度0.5μg/ml)、聚乙烯亚胺PEI(Sigma)(终浓度2ug/ml)，混匀，一起共转染到HEK293ES悬浮细胞中，之后，置于摇床(135rpm，39℃，5％CO2)培养40min。转染后的细胞继续在135rpm，37℃，5％CO2摇床中培养，表达抗EGFR×CD3的四价抗体。转染96小时后离心收获表达上清。
b)四价双特异抗体的纯化
表达上清用0.22uM滤膜过滤，利用亲和层析柱从表达上清中获得带有Fc结构域的抗体。平衡缓冲液和洗脱缓冲液分别为50mM Tris-HCl 0.15M NaCl PH7.0和0.1M柠檬酸-柠檬酸钠PH3.0。通过阳离子交换层析获得目标双特异抗体，阳离子交换柱为HiTrap SP FF，通过平衡缓冲20mM PB PH6.3平衡SP层析柱，用洗脱缓冲液20mMPB+1M NaCl(PH6.3)进行洗脱，最后用PBS缓冲液进行换液浓缩。纯化后的双特异分子还原SDS-PAGE如图3所示。其中Bispecific-1的EGFR抗体序列为尼妥珠单抗(nimotuzumab)序列；Bispecific-2的EGFR抗体序列为西妥昔单抗(Cetuximab)；Bispecific-3的EGFR抗体序列为帕尼单抗(Panitumumab)。
具体实施例3：双特异抗体分子与CD3和EGFR分子结合情况
用pH9.6的碳酸盐缓冲液包被EGFR胞外区或者CD3E与CD3G亚基胞外区的二聚体，100ng/孔/100ul，4℃包被过夜。用300ul/孔含0.1％的PBS(PBS-T)缓冲液洗五次，再加入1％BSA-PBS 溶液在室温封闭2h。加入不同稀释度的双特异抗体或者是对应的抗体。各种双特异抗体(或抗体)最高浓度是1uM，3倍稀释做10个梯度，最后一个孔只加稀释液PBS作为阴性对照，37℃孵育1h。用300ul/孔PBS-T溶液洗五次，再加入用1％BSA-PBS溶液1:40000稀释的Anti-Human Fc-HRP二抗37℃孵育1h。用100ul/孔的TMB显色试剂盒显色，室温显色8min，然后用2M H2SO4终止显色，50ul/孔,450nm/630nm读数。实验结果如图4所示，所有双特异抗体结合EGFR的能力均与其对应的单抗能力相似[即由尼妥珠单抗序列构建的双特异抗体结合EGFR的能力与尼妥珠单抗的相似(图4A)；由西妥昔单抗序列构建的双特异抗体结合EGFR的能力与西妥昔单抗的相似(图4B)；由帕尼单抗序列构建的双特异抗体结合EGFR的能力与帕尼单抗的相似(图4C)]；而各种双特异抗体结合CD3E和CD3G二聚体的能力相近(图4D)。结果表明了文中采用的双特异抗体的形式几乎完整的保留了原先抗体结合抗原的能力；而且CD3的ScFv结合CD3的能力与其连接的不同抗体无关(图4D)。
具体实施例4、双特异抗体分子与过表达EGFR或CD3细胞结合情况
a)双特异性抗体与A431细胞的结合情况
本发明采用过表达EGFR的肿瘤细胞系(A431)来检测不同的双特异抗体与细胞表面EGFR的结合情况，采用相对应的抗体做阳性对照，用人的IgG(hIgG)作为同型对照。用0.25％胰酶消化、离心收集A431细胞。同时稀释各种抗体，最高浓度为1uM，3倍梯度稀释。将收集的细胞用PBS+1％BSA洗三遍，再加PBS+1％BSA重悬细胞,然后铺细胞于96孔板中，每孔1×105个细胞，加入100ul稀释好的双特异性抗体，室温孵育1小时；离心去上清，用PBS洗细胞三遍，再用稀释好的Alexa488标记的抗人IgG-Fc抗体重悬细胞，室温避光孵育1小时，PBS洗三遍，再用100ul  PBS重悬，用流式细胞仪检测荧光强度。结果用Graphpad Prism分析。结果显示(图5)，每种双特异抗体与A431的结合能力与其对应的抗体具有可比性，而hIgG与A431的结合力很弱。证明双特异抗体较好的保持了其母本抗体的特异结合细胞表面EGFR特异结合的活性。
b)双特异抗体与Jurkat细胞结合情况
Jurkat细胞过表达CD3，可以用来检测双特异抗体与细胞表面CD3的结合情况。双特异抗体与Jurkat细胞结合情况实验过程与实施例4a类似，区别在于Jurkat细胞是悬浮细胞，而A431是贴壁细胞。离心收集Jurkat细胞，加入各种抗体，其余操作与A431实验相同。结果显示(图6)，同型对照结合Jurkat的能力很弱；不同的双特异抗体均能够很好的结合细胞，而且三种双特异抗体结合Jurkat细胞的能力一致，表明了图1的双特异抗体中抗CD3的ScFv结合细胞表面抗原的能力不受与它连接的抗体影响。
具体实施例5、双特异抗体分子介导的PBMC对效应细胞的杀伤作用
a)A431或H520细胞PKH26标记
A431是一种过表达EGFR的肿瘤细胞株；而H520则不表达EGFR。本实验采用A431作为实验细胞株；而H520作为阴性对照。
取2×106个细胞于1.5ml离心管中1500rpm离心5min，弃完全培养基，用无血清的培养基分别清洗细胞两次；用PKH26试剂盒中的Diluent C溶液重悬细胞，然后加入等体积的2×P KH26染料液(配制比例：0.4μl染料原液溶于100μl的Diluent C)，混匀后室温放置1min。反应结束后，立即加入与管中溶液等体积的0.5％BSA-PBS溶液终止反应，再加入1ml相应的完全培养基稀释重悬细胞，1500rpm离心5min收集细胞沉淀。用完全培养基重 悬后与细胞培养瓶中培养待用。
b)PBMC分离
在50ml管中加入20ml单个核细胞分离液；用全血稀释液将采集到的血液按1:1进行稀释处理，混匀后沿康宁管内壁匀速缓慢的铺至分离液上层，每管内加入稀释后全血体积为20ml；待各管加液完毕后放入提前预冷至22℃的离心机内，600g水平离心15min(加减速设置为1)；离心完成后取出离心管，用移液器小心吸取置于分离液和血清间呈圆弧状分布的细胞层—单个核细胞(PBMC)，置于新的50ml管中；按照1:5比例在细胞液中加入细胞洗涤液，充分混匀后离心，弃上清，再重复洗涤一次，收集细胞沉淀，用RPMI-1640培养基重悬，培养于细胞瓶中待用。
c)CD3+T细胞磁珠分选
PBS(Ph7.2,含0.5％BSA，2mM EDTA),过滤除菌，同时避免产生气泡。
离心去血小板(20度，200g，10-15分钟)，然后30μm滤膜去除细胞团块。收集PBMC细胞：200g离心10分钟，弃上清。2x107细胞重悬于60μl buffer+20μl FcR Blocking Reagent中。加入20μl磁珠，混匀，2-8℃孵育15分钟；加入1-2ml buffer，300g，离心10分钟，弃尽上清；500μl重悬。将分选柱置于分选架上，500μl润洗；加入细胞悬液，收集未结合细胞；柱体顶端液体流尽后，500μl洗三次；将分选柱从分选架上取下，置于收集管中，1ml快速冲洗，收集细胞，计数并观察活细胞比例。
d)双特异抗体效应功能检测
按照终浓度1uM起，1:3倍比稀释，10梯度，2复孔；同时设立2个无药物对照孔，用培养基补足体积,将标记后的A431或H520细胞(2×104个/孔/50μl)与CD3+T细胞(2×105个/孔/50μl)，按照所需用量混匀后分至V-bottom 96孔板各孔，同时设立以下三个流式对照孔：①无标记的Raji细胞(2×104个/孔)②PKH26标记后的细胞(2×104个/孔)；③CD3+T细胞(2×105个/孔)。 培养18小时，孵育结束后按照1:50000的比例加入TO-PRO3染料，37℃避光孵育10min；同时设立一个流式对照④TO-PRO3标记后的细胞。3000rpm离心5min后弃部分培养基上清，用0.5％BSA-PBS溶液重悬，流式检测。
计算公式：
细胞死亡率％＝(1-PKH26+TOPRO3-细胞数目(药物作用组)/PKH26+TOPRO3-细胞数目(无药物作用组))X100
e)结果分析
由图7可知，双特异抗体分子介导的PBMC对过表达EGFR的A431效应细胞有非常好的杀伤作用；而他们对应的抗体和hIgG则杀伤效果很弱。同时所有的双特异抗体对不表达EGFR的H520细胞杀伤效果都非常弱，且与同型对照hIgG类似(图8)。这表明了所有构建的双特异抗体对靶细胞的杀伤作用是特异的；三种不同的双特异抗体杀伤特异性靶细胞的能力相似，而且细胞死亡率在80％左右。以上表明了图1中双特异抗体具有良好的生物学活性。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (13)

  1. 一种双特异性抗体，其特征在于，包含(a)完整的单克隆抗体、(b)单链抗体ScFv和(c)连接子；所述(a)与EGFR抗原特异性结合并且由两条抗体重链和两条抗体轻链组成；所述(b)与免疫细胞抗原CD3特异性结合，所述(b)为两条单链抗体ScFv；所述(b)的两条单链抗体ScFv分别通过所述(c)连接子linker与所述(a)的两条重链的C末端连接。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述(c)连接子linker的氨基酸序列为(GGGGX)n，其中X为Gly或Ser；n为1-4的自然数。
  3. 根据权利要求2所述的双特异性抗体，其特征在于，所述X为Ser；所述n为3。
  4. 根据权利要求1所述的双特异性抗体，其特征在于，所述(a)完整的单克隆抗体的重链可变区和轻链可变区的氨基酸序列为SEQ ID NO 1和SEQ ID NO 4、SEQ ID NO 2和SEQ ID NO 5或SEQ ID NO 3和SEQ ID NO 6三种组合中的一种。
  5. 根据权利要求1所述的双特异性抗体，其特征在于，所述(b)两条单链抗体ScFv的氨基酸序列均为SEQ ID NO 7。
  6. 根据权利要求1所述的双特异性抗体，其特征在于，所述(b)单链抗体ScFv的重链可变区的氨基酸序列为SEQ ID NO 8，所述(b)单链抗体ScFv的轻链可变区的氨基酸序列为SEQ ID NO 9。
  7. 根据权利要求1所述的双特异性抗体，其特征在于，所述(a)完整的单克隆抗体的重链恒定区为IgG1、IgG2、IgG3或IgG4中的一种。
  8. 一种抗体、多肽或蛋白，其特征在于，所述抗体、多肽或蛋白包含权利要求1-7任一所述的双特异性抗体。
  9. 一种多核苷酸序列或其组合，其特征在于，所述多核苷酸序列或其组合编码包含权利要求1-7任一所述的双特异性抗体。
  10. 一种重组DNA表达载体，其特征在于，所述重组DNA表达载体包含权利要求9所述的多核苷酸序列或其组合。
  11. 一种宿主细胞，其特征在于，所述宿主细胞在转染如权利要求10所述的重组DNA表达载体时用到；所述宿主细胞包含原 核细胞、酵母或哺乳动物细胞。
  12. 一种药物或药物组合物，其特征在于，所述药物或药物组合物包含如权利要求1-7任一所述的双特异性抗体。
  13. 根据权利要求1-7任一所述的双特异性抗体在制备治疗EGFR抗原特异性表达引起的肿瘤疾病或其他疾病的药物中的用途。
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