EA042736B1 - ANTI-EGFR AND ANTI-CD3 BISPECIFIC ANTIBODY AND ITS APPLICATIONS - Google Patents

Description

Field of invention

This invention relates to the field of biotechnology and, in particular, to a method for constructing and obtaining a bispecific antibody against epidermal growth factor receptor (EGFR) and differentiation cluster 3 (CD3), and applications of this antibody in diseases.

Prior Art

A bispecific antibody (BiAb) is an artificial antibody composed of two different antibody fragments and is capable of specifically recognizing and binding to two different antigens or two different epitopes.

Monoclonal antibodies are widely used to treat cancer, inflammation, and other diseases. Because all these antibodies have a single target, many patients may not fully respond to monotherapy and often have drug resistance. A bispecific antibody is capable of simultaneously recognizing two different antigens or epitopes and can serve as a means to redirect immunological effector cells such as natural killer cells and T cells and enhance the killing effect of tumor cells. In addition, the bispecific antibody can be further located on two different antigens on the same cell to alter cellular signals, including a cancer spread signal or an inflammatory signal. As a result of long-term research and development, many forms of bispecific antibodies, such as a bispecific micro-antibody, a double-chain antibody, a single-chain divalent antibody, a multivalent bispecific antibody, and the like, have emerged. These bispecific antibodies are mainly divided into two main categories: Fc containing antibodies and Fc free antibodies. The former has excellent solubility, stability, and half-life, while Fc-antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may provide some of the additive effects required for treatment. In contrast, an Fc-deficient bispecific antibody is completely dependent on its ability to bind to an antigen in order to achieve a therapeutic effect. In addition, the Fc protein can extend the half-life of the drug protein (or peptide) in vivo, thereby extending the time of action of the active molecule in vivo.

Tumor cell surface antigen epidermal growth factor receptor (EGFR): epidermal growth factor receptor is widely distributed on the membrane of epithelial cells, except in vascular tissues. As a transmembrane protein of about 180 kDa, EGFR has ligand-inducible tyrosine protein kinase activity and is a member of the conserved ErbB receptor family. Other members of this family include HER2/Neu/ErbB2, HER3/ErbB3 and HER4/ErbB4. The general characteristics of ErbB receptors are as follows: each ErbB receptor includes an extracellular (EC) ligand-binding domain, one transmembrane domain consisting of two repetitive cysteine-rich regions, and an intracellular sequence including tyrosine protein kinase and autophosphorylation sites; after binding of ligands and receptors, receptor dimerization occurs, and homodimers or heterodimers are formed; dimerized receptors are cross-phosphorylated to activate a sub-region of TK in the intracellular region in such a way as to excite signal transduction of the next level, thereby causing cell proliferation and transformation. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of cell apoptosis, and the mechanisms include: increased subsequent signal transduction caused by high EGFR expression, prolonged EGFR activation caused by increased expression of a mutated EGFR receptor or ligand, increased autocrine loop action, disruption of the receptor downregulation mechanism, activation of an abnormal signal transduction pathway, and the like. The study shows that 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 cell apoptosis. Overexpression of EGFR plays an important role in tumor progression, and EGFR overexpression exists in tissues of spongiocytoma, lung cancer, prostate cancer, pancreatic cancer, and the like.

Immune cell surface antigen differentiation cluster 3 (CD3): The CD3 molecule is an important differentiation antigen on the T cell membrane, is a characteristic feature of mature T cells and consists of 6 peptide chains.

The TCR-CD3 complex consists of the CD3 T cell receptor (TCR) with non-covalent bonds. The CD3 molecule participates in the intracytoplasmic assembly of the TCR-CD3 complex and signals antigen stimulation through an immunoreceptor tyrosine-based activation motif (ITAM) of the cytoplasmic domain of each polypeptide chain. The main effects of the CD3 molecule include the stabilization of the TCR structure and the transmission of the T cell activation signal. After specific recognition and binding of TCR to antigens, CD3 is involved in signal transduction into the T cell cytoplasm. As the first signal to induce T cell activation, CD3 plays an important role in T cell antigen recognition and immune response generation processes.

In medicine, in particular in tumor immunotherapy, a bispecific antibody has excellent

- 1 042736 effects and prospects, able to simultaneously bind to parts of tumor cells and specific immune-active CD16 cells against antigen or CD3 on immune cells, and has the effect of activating NK cells (natural killer) or T cells. The anti-tumor-specific antigen moiety can specifically bind to tumor cells, immune cells target these tumor cells, and local NK cells or T cells are increased, thereby allowing the immunological effector cells to achieve tumor-specific killing effect. In the prior art, there is no successfully marketed anti-EGFR and CD3 bispecific antibody drug, and this technology should be investigated.

With respect to Chinese Patent CN 201510030519.9, the present invention adopts a tetravalent bispecific antibody, and the biological activity of the anti-EGFR antibody is fully preserved; this tetravalent bispecific antibody is able to recognize tumor antigen and effector cells (T cells, NK cells, and the like) well, thereby achieving the biological activity of the bispecific antibody well. US Pat. No. 9,249,217 B2 adopts the ScFv-ScFv single chain antibody (BITE) form, and BITE is bivalent and does not have any Fc moiety. Compared to BITE, in addition to the advantages of a tetravalent antibody, the present invention additionally contains Fc fragments; and these Fc fragments are capable of prolonging the half-life of the effector molecules in vivo, thereby extending the time of action of the effector molecules in vivo.

Brief summary of the invention

The present invention provides a bispecific antibody. This bispecific antibody has an anti-cluster of differentiation 3 (CD3) ScFv sequence added to the C-terminus of the anti-EGFR antibody. This bispecific antibody retains the overall molecular structure of the anti-EGFR antibody and increases the ability to bind to the CD3 antigen, thereby maintaining the biological activity of the parent anti-EGFR antibody in vivo while targeting immunological effector cells to tumor cells through the specific recognition of two different antigens, thereby increasing the influence of immunological effector cells on the death of tumor cells.

The invention provides an anti-EGFR and anti-CD3 bispecific antibody and its use.

This bispecific antibody includes: (a) a complete monoclonal antibody, (b) a single chain ScFv antibody, and (c) a linker; (a) binds specifically to the EGFR antigen and consists of two antibody heavy chains and two antibody light chains; (b) binds specifically to the CD3 antigen of an immune cell; and (b) refers to two single chain ScFv antibodies; moreover, two single-chain ScFv antibodies (b) are respectively linked to the C-terminus of the two heavy chains of the antibody (a) via a linker (c).

Here, the linker (c) in the bispecific antibody has the amino acid sequence (GGGGX) _n , X includes Ser or Ala, preferably Ser; and n is an integer 1-4, preferably 3.

Here, the full monoclonal antibody (a) in the bispecific antibody consists of two light chains and two heavy chains, each heavy chain and each light chain are disulfide bonded, and the two heavy chains are disulfide bonded at the hinge region. The variable regions of the heavy chains and light chains bind specifically to the EGFR antigen of tumor cells.

Here, the heavy chain constant region of the complete monoclonal antibody (a) is one of the constant regions of IgG1, IgG2, IgG3 or IgG4, preferably IgG2.

Here, the amino acid sequence of the heavy chain variable region of the complete monoclonal antibody (a) is one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and the amino acid sequence of the light chain variable region of the complete monoclonal antibody (a) is one of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The amino acid sequence of the heavy chain variable region of the single chain ScFv antibody (b) is SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the single chain ScFv antibody (b) is SEQ ID NO:9.

Here, both amino acid sequences of the two single chain ScFv antibodies (b) are SEQ ID NO:7.

Here, each of the ScFv single chain antibodies (b) consists of a heavy chain variable region, a linker (c) and a light chain variable region, the heavy chain variable region and the light chain variable region specifically bind to CD3 antigen on the surface of an immune cell; and the linker (c) has the amino acid sequence (GGGGS) _n , where n is an integer 1-4, preferably (GGGGS) ₃ .

Here, an expression vector of a bispecific antibody is constructed, here, a bispecific antibody can be constructed in a vector or, respectively, constructed in two different vectors.

- 2 042736

Here, the engineered vector is transfected into a host cell by a genetic engineering method, and the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell such as a CHO cell (Chinese Hamster Ovary cell), an NS0 cell (mouse myeloma cell), or a cell of another mammal , preferably a CHO cell.

Here, the bispecific antibody is prepared by a conventional method for preparing immunoglobulins, including protein A affinity chromatography and ion exchange, hydrophobic chromatography, or molecular sieve method.

Here, the bispecific antibody is used to treat tumor tissues overexpressing or abnormally expressing EGFR and other diseases caused by overexpression of EGFR.

Here, the complete monoclonal antibody is a full length antibody.

With the adoption of the above technical solutions, the present invention includes the following beneficial technical effects.

The bispecific antibody in the present invention is in the form of a tetravalent antibody, and this form of the tetravalent antibody completely retains the sequence of the antibody that binds to the tumor antigen and has high affinity. Meanwhile, the tetravalent bispecific antibody can better bind tumor cells and effector cells, thereby being more conducive to the biological functions of the bispecific antibody. Compared to the usual form of bispecific antibodies - BITE, the molecule in this invention further includes an Fc fragment; and the existing Fc fragment can prolong the half-life of the drug molecule in vivo, which allows the drug molecule to achieve better effects, reduce the frequency of administration in patients, and alleviate the pain of patients. The present invention is useful in the treatment of tumor tissues overexpressing or abnormally expressing EGFR and other diseases caused by overexpression of EGFR.

Brief description of graphic materials

In FIG. 1 is a schematic diagram of a bispecific antibody molecule;

in fig. 2 schematically shows a diagram of the construction of an expression plasmid for a bispecific molecule;

in fig. 3 is a schematic electropherogram of a purified bispecific molecule denatured with SDS (sodium dodecyl sulfate);

in fig. 4 schematically shows the detection of the ability to bind a bispecific molecule to an antigen by ELISA (enzyme-linked immunosorbent assay);

in fig. 5 schematically shows the binding of a bispecific antibody and an A431 cell;

in fig. 6 schematically shows the binding of a bispecific antibody to a Jurkat cell;

in fig. 7 schematically shows bispecific antibody mediated binding of PBMC (peripheral blood mononuclear cell) to A431;

in fig. 8 schematically shows the bispecific antibody-mediated killing effect of H520 by PBMC.

Detailed description of the invention

The present invention is further described in detail below with specific embodiments and in combination with the drawings.

The invention provides an anti-EGFR and anti-CD3 bispecific antibody and its use. This bispecific antibody includes: (a) a complete monoclonal antibody, (b) a single chain ScFv antibody, and (c) a linker; here (a) specifically binds to the EGFR antigen and consists of two heavy chains of the antibody and two light chains of the antibody, (b) specifically binds to the CD3 antigen of the immune cell and (b) refers to two single-chain ScFv antibodies; moreover, two single-chain ScFv antibodies (b) are respectively linked to the C-terminus of the two heavy chains of the antibody (a) via a linker (c).

In addition, preferably the linker (c) in the bispecific antibody has the amino acid sequence (GGGGX)n, X includes Ser or Ala, preferably Ser; and n is an integer 1-4, preferably 3.

Furthermore, preferably the full monoclonal antibody (a) in the bispecific antibody is composed of two light chains and two heavy chains, each heavy chain and each light chain are disulfide bonded, and the two heavy chains are disulfide bonded at the hinge region. The variable regions of the heavy chains and light chains bind specifically to the tumor cell surface EGFR antigen.

In addition, preferably the heavy chain constant region of the complete monoclonal antibody (a) is one of the constant regions of IgG1, IgG2, IgG3 or IgG4, preferably IgG2.

In addition, preferably, the amino acid sequence of the heavy chain variable region of the complete monoclonal antibody (a) is one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

- 3 042736 here SEQ ID NO: 1 (Nimotuzumab Heavy Chain Variable Region Amino Acid Sequence):

QVQLQQPGAELVKPGASVKLSCKASGYTFTNYYIYWVKQRPGQGLEWIGGI

NPTSGGSNFNEKFKTKATLTVDESSTTAYMQLSSLTSEDSAVYYCTRQGLWFDSDG

RGFDFWGQGTTLTVSS

SEQ ID NO: 2 (amino acid sequence of cetuximab heavy chain variable region):

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI

WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFA

YWGQGTLVTVSA

SEQ ID NO: 3 (amino acid sequence of the variable region of the heavy chain of panitu- mumaba): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYYWTWIRQSPGKGLEWIG HIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIW GQGTMVTVSS

Here, the amino acid sequence of the light chain variable region of the complete monoclonal antibody (a) is one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6,

SEQ ID NO: 4 (Nimotuzu-light chain variable region amino acid sequence)

maba): DVLMTQIPLSLPVSLGDQASISCRSSQNIVHSNGNTYLDWYLQKPGQSPNLLI YKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYYCFQYSHVPWTFGGGTKL

EIK

SEQ ID NO: 5 (amino acid sequence of cetuximab light chain variable region):

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI

SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

SEQ ID NO: 6 (panitumumab light chain variable region amino acid sequence):

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASN

LETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK

In addition, preferably, the amino acid sequence of the variable region of the heavy chain of the single chain ScFv antibody (b) is SEQ ID NO: 8, and the amino acid sequence of the variable region of the light chain of the single chain ScFv antibody (b) is SEQ ID NO: 9, here SEQ ID NO: 8 (amino acid sequence of the heavy chain variable region of a single chain anti-CD3 antibody):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGY

INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCL

DYWGQGTTTLTVSS

SEQ ID NO: 9 (anti-CD3 single chain antibody light chain variable amino acid sequence):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSK

VASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

Further, preferably, the amino acid sequences of the two single chain ScFv antibodies (b) are shown here as SEQ ID NO: 7,

SEQ ID NO: 7 (anti-CD3 single chain antibody amino acid sequence):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGY

INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCL

DYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASS SVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAA TYYCQQWSSNPLTFGAGTKLELK

In addition, preferably, each of the single chain ScFv antibodies (b) consists of a heavy chain variable region, a linker (c) and a light chain variable region, wherein the heavy chain variable region and the light chain variable region specifically bind to an immune cell surface CD3 antigen; and the linker (c) has the amino acid sequence (GGGGS)n, where n is an integer 1-4, preferably (GGGGS) ₃ .

In addition, preferably, the engineered vector is transfected into a host cell, which is a prokaryotic cell, a yeast cell, or a mammalian cell such as CHO cells, NS0 cells, or other mammalian cells, preferably CHO cells.

Further, preferably, the bispecific antibody is used to treat tumor tissues overexpressing or abnormally expressing EGFR and other diseases caused by overexpression of EGFR.

- 4 042736

Specific embodiment 1: construction of an expression vector of a bispecific antibody molecule.

a) Construction of a bispecific vector for transient expression

Appropriate gene sequences are constructed according to the shape of the bispecific antibody in FIG. 1. Select PTSE as an expression vector for cloning and expression of anti-EGFR light chain genes and anti-EGFR-ScFv anti-CD3 heavy chain fusion genes. Sall and BamHI restriction sites were added to light chain genes and fusion genes on both sides of the coding region sequences, respectively, and these two genes were synthesized by Taihe Biotechnology (Beijing) Co., Ltd. and cloned accordingly into the PUC19 vector. Transform two plasmids into TOP competence (Huitian Dongfang, item no. HT702-03), prepare a miniprep using the CWBio miniprep kit (item no. CW0500), perform restriction digestion with restriction enzymes Sall-HF and BamHI-HF, perform homologous recombination, respectively, to obtain expression vectors (schematic diagrams are shown in Figs. 2A and 2B) containing the light chain gene and the fused gene, and with the name of the plasmids PTSE-JY016L-TetBiAb and PTSE-JY016H-TetBiAb, respectively.

b) Construction of a bispecific expression vector for stable transformation

PGN-2CMV serving as an expression vector for cloning and expression of anti-EGFR light chain genes and anti-CD3 EGFR-ScFv heavy chain fusion genes is selected, which vector includes selectable markers for neomycin and GS, two CMV (cytomegalovirus) promoters, and relevant structural units; designing the light chain primers and the fusion gene, introducing the Kozak sequence, the signal peptide, and the corresponding restriction enzyme cleavage sites, and performing the synthesis of Taihe Biotechnology (Beijing) Co., Ltd.; using the plasmids PTSE-JY016L-TetBiAb and PTSE-JY016H-TetBiAb as a template, PCR amplification was performed to obtain the corresponding bands, and homologous recombination was performed with the vector (plasmid profiles are shown in Fig. 2B).

Specific embodiment 2: expression and purification of a bispecific antibody molecule.

a) Expression of a tetravalent antibody

Maxi-preparation of plasmids is carried out by using an endotoxin-free maxi kit (CWBio, CW2104) and the specific work steps are carried out as described in the kit.

Human embryonic kidney cells (HEK293ES suspension cells) are cultivated in FreeStyle 293 expression medium (Gibco, 12338-026), cells are subcultivated once every day or every two days, the initial density of subcultivated cells is maintained at 0.2-0.6x106/ml, and the cell culture volume should be 15-35% of the shake flask volume; the cell culture flask is placed on a shaker (shaker speed: 135 rpm, temperature: 37° C., CO ₂ : 5%); carry out subculturing of HEK293ES cells, which are in a logarithmic phase and in good growth condition, to obtain a flask with a cell density of 0.5x10 ⁶ /ml one day before transfection, culture is carried out on a shaker overnight (135 rpm, 37°C, 5% CO ₂ ) and transfection the next day.

Cultivate the resulting cell suspension with a density of 1x10 ⁶ /ml on a shaker (135 rpm, 39°C, 5% CO ₂ ) for 2 h before transfection, during transfection sequentially add PTSEantiEGFR-H-TetBiAb (having a final concentration of 0 .5 μg/ml), PTSE-antiEGFR-L (having a final concentration of 0.5 μg/ml) and polyethyleneimine (Sigma) (having a final concentration of 2 μg/ml), are uniformly mixed, co-transfected into HEK293ES suspension cells and cultured on a shaker (135 rpm, 39°C, 5% CO ₂ ) for 40 min; continuously cultivate transfected cells on a shaker (135 rpm./min, 37°C, 5% CO ₂ ) and express tetravalent antibody against EGFR and against CD3; then transfected for 96 h and then centrifuged to obtain the expression supernatant.

b) Purification of a tetravalent bispecific antibody

Filter the expression supernatant using a 0.22 μM filter membrane and obtain an Fc domain antibody from the expression supernatant using an affinity chromatography column; respectively, take 50 mm Tris-HCl, 0.15 M NaCl, pH 7.0 and 0.1 M citric acid-sodium citrate, pH 3.0, as a balancing buffer and elution buffer; conduct cation exchange chromatography to obtain the target bispecific antibodies, here the cation exchange column is HiTrap SP FF; equilibrate the SP chromatographic column with equilibration buffer - 20 mM PB (phosphate buffer), pH 6.3, elute with elution buffer - 20 mM MBP plus 1 M NaCl (pH 6.3), and finally change the concentration of the solution using PBS buffer ( phosphate buffered saline solution). Purified by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing conditions, the bispecific molecule is shown in FIG. 3. Here, the sequence of the antibody against EGFR - Bespecifically-1 is the sequence of nimotuzumab; anti-EGFR antibody sequence - 5 042736

Bispecific-2 is cetuximab and the anti-EGFR antibody sequence Bispecific-3 is panitumumab.

Specific Embodiment 3: Binding of Bispecific Antibody Molecules to CD3 Molecules and

EGFR.

The extracellular region of EGFR or the dimer of CD3E and CD3G subunits of the extracellular region is coated with carbonate buffer pH 9.6 overnight under the following conditions: 100 ng/well/100 μl at 4°C; washing with 300 µl/well of 0.1% PBS buffer (PBS-T) 5 times and adding 1% BSA (bovine serum albumin)-PBS to block at room temperature for 2 h, add different dilutions of bispecific antibodies or corresponding antibodies. The highest concentration of each bispecific antibody (or antibody) is 1 μM; carry out a dilution of 3 times, form 10 gradients, add only diluents - PBS to the last well to serve as a negative control, and incubate at 37°C for 1 hour; then washed with 300 μl/well of PBS-T solution 5 times and add secondary anti-human Fc antibody conjugated to HRP (horseradish peroxidase) diluted 1:40000 with 1% BSA-PBS, incubated at 37°C for 1 hour; and developed with a TMB (tetramethylbenzidine) developing kit, 100 μl/well, for 8 min at room temperature and then development was stopped with 2 M H ₂ SO ₄ , 50 μl/well and read at 450 nm/630 nm. The experimental results are shown in FIG. 4. The ability of all bispecific antibodies to bind to EGFR is similar to the ability of the corresponding monoclonal antibodies, the ability of the bispecific antibody constructed from nimotuzumab to bind to EGFR is similar to that of nimotuzumab (Fig. 4A); the ability of the bispecific antibody constructed from cetuximab to bind to EGFR is similar to that of cetuximab (FIG. 4B); and the ability of the bispecific antibody constructed from panitumumab to bind to EGFR is similar to that of panitumumab (FIG. 4B)]; while the binding abilities of different bispecific antibodies to CD3E and CD3G dimers are similar (FIG. 4D). The results show that the form of bispecific antibodies adopted in the invention, almost completely retains the original ability of antibodies to bind to antigens; and, in addition, the ability of anti-CD3 ScFv to bind to CD3 is not relevant for different antibodies associated with it (Fig. 4D).

Specific embodiment 4: binding of bispecific antibody molecules to cells overexpressing EGFR or CD3.

a) Binding of bispecific antibodies and A431 cells

In the present invention, a tumor cell line (A431) overexpressing EGFR is taken to detect binding of various bispecific antibodies and EGFR on the cell surface, the corresponding antibody is used as a positive control, and human IgG (hIgG) is used as an isotype control. Digest with 0.25% trypsin and centrifuge to collect A431 cells; while diluting different antibodies, controlling the highest concentration - 1 μm and performing a gradient dilution 3 times; wash the selected cells with PBS plus 1% BSA three times, then add PBS plus 1% BSA to the resuspended cells, spread the cells into a 96-well plate at a dose of 1x105 cells per well, add 100 µl of diluted bispecific antibodies and incubate at room temperature for 1 h, centrifuge to remove supernatant, wash cells with PBS three times, then resuspend cells using diluted anti-human IgG-Fc antibody labeled with Alexa488, incubate in the dark at room temperature for 1 h, wash with PBS three times, resuspend with 100 µl PBS and detect fluorescence intensities by flow cytometry. The results are analyzed using Graphpad Prism. The results show that (FIG. 5) the binding capacity of each bispecific antibody and A431 is comparable to that of the corresponding monoclonal antibody, while the binding of hIgG and A431 is very weak. The bispecific antibody has been shown to well support the activity of the parent antibody, which specifically binds to EGFR on the cell surface.

b) Binding of bispecific antibodies and Jurkat cells

CD3 overexpressing Jurkat cells are used to detect bispecific antibody and CD3 cell surface binding. The experimental method for binding bispecific antibodies to Jurkat cells is similar to that of embodiment 4a, with the difference being that Jurkat cells are suspension cells while A431 cells are adherent cells. Jurkat cells are selected by centrifugation, different antibodies are added, and the rest of the operations are the same as those in the A431 experiment. The results show that (FIG. 6) the ability of the isotype control to bind to Jurkat is very weak; and different bispecific antibodies are able to bind well to cells, and the binding abilities of the three bispecific antibodies to Jurkat cells are consistent, indicating that the ability of the anti-CD3 ScFv in the bispecific antibody in FIG. 1 to binding to cell surface antigens is not affected by any antibody associated with it.

- 6 042736

Specific Embodiment 5: Effect of death mediated by a bispecific antibody molecule

PBMC on effector cells.

a) PKH26 labeling in A431 or H520 cells

A431 is a tumor cell strain capable of overexpressing EGFR, while H520 does not express EGFR. In this experiment, A431 serves as the test cell strain, while H520 serves as the negative control.

Add 2x106 cells to a 1.5 ml centrifuge tube at 1500 rpm for 5 min, remove complete medium and purify cells accordingly twice with serum-free medium; resuspend cells with diluent C solution in PKH26 kit, add 2xPKH26 staining solution in equal volume (proportion: dissolve 0.4 µl stock stain solution in 100 µl diluent C), mix evenly and place at room temperature for 1 min; immediately add the same volume of 0.5% BSA-PBS solution as the volume of the solution in the tube, to complete the reaction, add 1 ml of the appropriate complete medium to dilute and resuspend cells, centrifuge at 1500 rpm for 5 min to collect cell pellet and culture cells resuspended in complete medium in cell culture bottles for later use.

b) Separation of RVMS

Add 20 ml of separation medium for mononuclear cells to a 50 ml tube, dilute the collected blood with whole blood diluent according to a 1:1 ratio, then mix evenly, slowly spread the top layer of separation medium at a constant speed along the inner walls of the Corning tubes, and adjust the volume of whole blood to each tube up to 20 ml; place each tube filled with liquid in a centrifuge pre-cooled to 22°C, and carry out horizontal centrifugation at 600 g for 15 min (set acceleration and deceleration 1); take out the centrifuge tubes after centrifugation is completed, carefully pipette the cell layer - mononuclear cells (PBMC) in the distribution in the form of an arc of a circle between the separating medium and serum and place in a new 50 ml tube; add cell washing liquid to the cytoplasmic matrix according to the ratio of 1:5, mix and centrifuge completely and evenly, remove the supernatant, wash once again, collect the cell pellet, resuspend using RPMI-1640 medium, and culture the cells in cell culture bottles for subsequent use .

c) Separation with magnetic beads of CD3 T cells plus PBS (pH 7.2 containing 0.5% BSA, 2 mM EDTA (ethylenediaminetetraacetic acid)), filtered, sterilized, and prevented blistering.

Centrifuge to remove blood platelets (20°C, 200 g, 10-15 min) and remove the cell aggregate using a 30 μm membrane filter; select PBMC cells by centrifugation at 200 g for 10 min and remove the supernatant; resuspend 2x107 cells in 60 µl buffer plus 20 µl FcR-based blocking reagent (Fc receptor); add 20 µl of magnetic beads, mix evenly and incubate at 2-8°C for 15 min; add 1-2 ml of buffer, centrifuge at 300 g for 10 min and remove the supernatant; resuspended by 500 µl; place the sorting column on a sorting rack and wash with 500 µl; add cell suspension and select unbound cells; wash with 500 μl three times after liquid flows from the top of the column; remove the sorting column from the sorting rack and place it in the collection tube, rinse quickly with 1 ml, select the cells, count and note the ratio of viable cells.

d) Identification of the influence and function of a bispecific antibody

Dilute according to the ratio 1:3 from a final concentration of 1 μm and create 10 gradients in the wells in duplicate; install 2 drug-free control wells, top up with medium, then evenly mix labeled A431 or H520 cells (2x104 cells/well/50 µl) and CD3 plus T cells (2x105 cells/well/50 µl) according to the required amount for application , dispense cells into each well of a 96-well V-bottom plate, and at the same time create three flow-through control wells as follows: (1) unlabeled Raji cells (2x 10 ⁴ cells/well), (2) labeled PKH26 cells (2x104 cells/well) and (3) CD3 plus T cells (2x105 cells/well); culture for 18 hours, add TOPRO3 dye according to the ratio of 1:50000 after completion of incubation and incubate in the dark at 37°C for 10 minutes; simultaneously create flow control (4) cells labeled TO-PRO3; and centrifuged at 3000 rpm for 5 min, partially remove the medium supernatant, resuspend with 0.5% BSA-PBS solution and carry out detection with a flow cytometer.

Calculation formula:

Cell death rate % = (1 - number of PKH26+ TOPRO3 cells ^- (drug group)/number of PKH26+ TOPRO3 cells ^- (no effect group)) x 100 e) Analysis of results

- 7 042736

As shown in FIG. 7, PBMC mediated by a bispecific antibody molecule has an excellent killing effect on A431 effector cells overexpressing EGFR; while the corresponding antibody and its hIgG have a weak killing effect. In the meantime, all bispecific antibodies have a very weak killing effect on H520 cells that do not express EGFR and have a similar effect on the isotype control hIgG (FIG. 8). This indicates that the killing effects exhibited by all engineered bispecific antibodies on target cells are specific; and the ability of three different bispecific antibodies to kill specific target cells are similar, and the cell death rate is about 80%. The above description shows that the bispecific antibodies in FIG. 1 have good biological activity.

The above are only preferred embodiments of the invention and are not intended to limit the invention. For those skilled in the art, the invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, improvements and the like, made within the spirit and principle of this invention, should fall within the protection scope of this invention.

Claims (9)

1. A bispecific antibody containing: (a) a complete monoclonal antibody, (b) a single chain antibody (ScFv) and (c) a linker, where (a) it specifically binds to the EGFR antigen (epidermal growth factor receptor) and consists of two heavy chains of the antibody and two antibody light chains; (b) specifically binds to an immune cell antigen cluster of differentiation 3 (CD3); and (b) refers to two ScFv single chain antibodies; moreover, two single-chain ScFv antibodies (b) are respectively linked to the C-terminus of the two heavy chains of the antibody (a) via a linker (c), where the amino acid sequences of the heavy chain variable region and the light chain variable region of the complete monoclonal antibody (a) are one of three combinations: SEQ ID NO: 1 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 5 or SEQ ID NO: 3 and SEQ ID NO: 6; where both amino acid sequences of the two single chain ScFv antibodies (b) are SEQ ID NO: 7. 2. The bispecific antibody of claim 1, wherein the linker (c) has the amino acid sequence (GGGGX)n, X is Ala or Ser, and n is an integer 1-4. 3. The bispecific antibody of claim 2, wherein X is Ser and n is 3. 4. The bispecific antibody according to claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the single chain ScFv antibody (b) is SEQ ID NO: 8 and the amino acid sequence of the variable region of the light chain of the single chain ScFv antibody (b) is SEQ ID NO: 9. 5. The bispecific antibody of claim 1, wherein the heavy chain constant region of the complete monoclonal antibody (a) is one of the IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions. 6. A recombinant DNA expression vector containing a polynucleotide sequence encoding a bispecific antibody according to any one of claims 1-5. 7. A host cell transfected with a recombinant DNA expression vector according to claim 6, which is a prokaryotic cell, a yeast cell, or a mammalian cell. 8. A pharmaceutical composition containing a bispecific antibody according to any one of claims 1-5. 9. The use of a bispecific antibody according to any one of claims 1 to 5 in the preparation of a medicament for the treatment of neoplastic diseases or other diseases caused by the specific expression of an EGFR antigen.