CN117209611A - Bispecific antibody binding to human CD33 and CD3 - Google Patents

Abstract

The invention discloses a bispecific antibody combining human CD33 and CD3, which relates to the technical field of biological medicine, and the bispecific antibody combining human CD33 and CD3 provided by the invention kills CD33 positive cells by utilizing T cells of a human body so as to achieve the purpose of treating myeloid leukemia, and a novel bispecific antibody molecule is designed on the basis of K193, and the bispecific antibody with a molecular structure is a CD3 epsilon-ScFv single-chain antibody with low affinity connected with the C end of a light chain of a standard IgG1 molecular structure through a hydrophilic linker so as to achieve the purpose of preferentially combining with a CD33 target, and simultaneously, the specificity enables EK333F to be a highly directional therapeutic drug for guiding the T cells to attack leukemia cells, and simultaneously reduces the unnecessary influence on normal cells.

Description

Bispecific antibody binding to human CD33 and CD3

Technical Field

The invention relates to the technical field of biological medicine, in particular to a bispecific antibody combining human CD33 and CD 3.

Background

Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of leukemia cells. Despite a deeper understanding of the biology of acute myeloid leukemia and recent approval of new drugs for the treatment of acute myeloid leukemia, the 5-year overall survival rate of standard cytotoxic chemotherapy is only 30%, thus a new therapy is urgently needed.

In recent years, T cell-based immunotherapy has received a great deal of attention as a promising immunotherapy approach for the treatment of various malignant tumors. AML cells are very sensitive to the cytotoxic effects of functional immune cells, and dual-specific T cell engagers provide an effective means for the treatment of AML.

The CD33 molecule is a sialic acid binding Ig-like lectin (Siglec) transmembrane glycoprotein, has a molecular weight of 67KD, is composed of 364 amino acids, is specifically expressed on the cell surface of a hematopoietic system, and is located on the 19 th chromosome of a human body. It is a member of the immunoglobulin superfamily, having two immunoglobulin-like extracellular domains, and also a fourth member of the sialoadhesive immunoglobulin-like lectin family, which together with sialoadhesive enzymes, CD22 and myelin binding glycoproteins constitute the adhesin family. Studies have shown that CD33 is a specific leukemia antigen of myeloid cells, absent from hematopoietic stem cells and expressed in hematopoietic cell subsets.

CD33 is expressed in 90% of Acute Myeloid Leukemia (AML) cells, but not in normal hematopoietic stem cells, and it has been demonstrated that targeted clearance of CD33 positive cells followed by culture can restore hematopoietic function. Therefore, CD33 becomes an ideal target point for AML specific immunotherapy, and then screening and utilization of CD33 monoclonal antibody or double antibody become hot spots for medical research.

CD33 monoclonal antibodies exert antitumor effects by mediating cytotoxicity through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and the like. Early in the development of immunotherapy, studies using murine anti-CD 33 mab M195, P67.7, HIM3-4, etc., have found that they activate complement-mediated cytotoxicity, induce endocytosis and macrophage chemotaxis, etc., but their repeated use is limited due to the immunogenicity of murine antibodies. Along with the development of antibody humanization technology and antibody engineering, humanized antibodies can be successfully constructed, the defect of immunogenicity is overcome, and good clinical treatment effect is achieved.

In recent years, antibodies bind to toxins and drugs to form antibody-drug conjugates (ADCs), which enhance the cytotoxic effects of antibodies. Currently, a variety of anti-CD 33 antibody drugs are on the market or in clinical trials.

GO (Gemtuzumabozogamicin) is a conjugate of a humanized anti-CD 33 monoclonal antibody and a potent anti-tumor antibiotic, acetylspinosyn, wherein the monoclonal antibody comprises about 98% of the human amino acid sequence and the remainder is derived from a murine animal. The spinetoram is combined with CD33 antigen on the surface of leukemia cells, and released after entering the cells, has obvious effect of killing leukemia cells, thereby treating leukemia alone or in combination with other chemotherapeutics.

GO was officially approved by the united states Food and Drug Administration (FDA) as a single drug for the treatment of cd33+ AML patients who first relapsed older than 60 years old at 5 months 2000. These patients do not meet the criteria of standard chemotherapy, but voluntarily leave the market due to lack of clinical benefit and increased adverse events, the validated clinical trial after approval is terminated prematurely. However, GO was re-approved in 2017, when further studies indicate that if divided doses of drug were used, it would be beneficial to add GO to standard chemotherapy.

CD3 is a marker present on the surface of all T lymphocytes. CD3 alias T3 or Leu-4. There are 3 subtypes, cd3δ, cd3ε, and cd3γ, respectively. The molecular weight of CD3 delta and CD3 epsilon is 20kD, the molecular weight of CD3 gamma is 26kD, and the CD3 gamma is expressed on the surfaces of T lymphocytes, thymus cells and NK cell membranes. 61-85% expression on normal peripheral blood lymphocytes and 60-85% expression on thymus cells. It belongs to the immunoglobulin superfamily. CD3 is a component of the T lymphocyte receptor (TCR) complex, forming a complex with the α/β and γ/δ T lymphocyte receptors (TCRs), the major membrane antigen that conducts TCR signals in combination with peptide/MHC. TCRs are essential in cell surface expression, antigen recognition and signal transduction.

CD3 is a T lymphocyte surface-specific molecule by which T lymphocytes with killing effects can be recruited. Monoclonal antibodies to CD3 may induce or prevent T lymphocyte activation. The anti-CD 3 antibody induces apoptosis of T lymphocytes in the presence of the anti-CD 28 antibody or IL-2. CD3 is one of the best markers (markers) of mature T lymphocytes in peripheral blood, and measurement of CD3+ T lymphocytes is of great importance for evaluation of diagnosis of immunodeficiency disease (T lymphocyte deficiency), leukemia, and lymphoma (T lymphocyte type). The anti-CD 3 monoclonal antibody can be used for immunosuppressive treatment during organ transplantation or bone marrow transplantation, and can also be used for immunomodulatory treatment of severe autoimmune diseases to remove T lymphocytes. U.S. Pat. No. 4,361,549 describes a murine hybrid cell line for the production of monoclonal antibody OKT3 against antigens found in normal human T cells and cutaneous T lymphoma cells, and in U.S. Pat. No. 5,885,573, a humanized monoclonal antibody constructed by transferring murine OKT3 into a human antibody framework in order to reduce its immunogenicity in human applications and to reduce the probability of occurrence of human anti-mouse antibody (HAMA) responses. OKT3 was the first murine monoclonal drug approved by the U.S. FDA in 1986 for the treatment of organ transplant acute phase rejection and was also the first monoclonal antibody drug approved for use by government drug administration worldwide. The main drawbacks of murine OKT3 mab treatment are T cell activation due to cytokine release caused by cross-linking between T cells and fcγr bearing cells and HAMA response, OKT3 has been replaced by humanized antibodies and new small molecule immunosuppressants after more than 10 years of market use. On the other hand, OKT3 or other anti-CD 3 antibodies may be used as immunopotentiators for stimulating T cell activation and proliferation, anti-CD 3 mab in combination with anti-CD 28 antibodies or interleukin-2 in vitro cell culture to induce T cell proliferation. OKT3 is further used alone or as a component of bispecific antibodies for targeting cytotoxic T cells to tumor cells and virus infected cells. To date, the use of antibodies as a means of recruiting T cell agents has been hampered by several findings. First, natural or engineered antibodies with high affinity for T cells often do not activate the T cells to which they bind; second, natural or altered antibodies with low affinity for T cells are often poorly or ineffectively effective in eliciting T cell-mediated cell lysis, and therefore it is important to select an anti-CD 3 mab with the appropriate affinity.

Several enterprises at home and abroad have studied and developed CD33-CD3 epsilon bispecific antibodies, and 4 bispecific antibodies aiming at CD33 are currently evaluated in phase 1 clinical trials.

AMG330 (research and development institution: amgen) is a short acting BiTE molecule that requires continuous Intravenous (IV) infusion for 2-4 weeks. Preclinical studies have shown that the BiTE-mode anti-CD 33x anti-CD 3 structure is cytotoxic even at low CD33 antigen densities on target cells, making it a candidate for targeting a broad range of cd33+ leukemias, including AML.

By month 11 of 2022, clinical studies have been conducted in groups of 96 people, and clinical trial data have shown that 55 relapsed or refractory (r/r) AML patients have been included in the phase 1 trial (NCT 02520427) to evaluate the safety of AMG330 and determine its maximum tolerated dose. Of 42 patients, 8 (19%) were assessed as responsive to AMG330, 3 of them were fully relieved (CR), 4 were fully relieved with incomplete recovery of blood (CRi), and 1 were non-leukemic status (MLFS). Phase 1 trial results indicate that AMG330 doses up to 720 μg/day can provide early evidence of acceptable safety, drug tolerance and anti-leukemia activity, and support further dose escalation studies to provide a trial basis.

AMG673 (research and development institution: amgen) is a half-life extended BiTE structure, combining the binding specificities of CD33 and CD3, fused to the N-terminus of a single IgGFc region. The expected terminal half-life of AMG673 in humans is expected to increase to 21 days. AMG673 was infused intravenously 2 times on day 1 and day 5 with 14 days as a cycle. By month 3 of 2022, 30 patients were enrolled in phase 1 trial (NCT 03224819) of AMG673, 11 out of 27 evaluable patients (41%) had a maternal cell reduction, with a maternal cell reduction rate of over 50% for 5 patients. 27 (4%) of the patients were assessed for CR in 1. The test result in the phase 1 shows that: preliminary data for doses of AMG673 up to 72 μg provided early evidence that the molecule had acceptable safety, drug tolerance and anti-leukemia activity. AMV564 (research and development institution: amphenaTherapeutics, inc.) is a tetravalent anti-CD 33x anti-CD 3 tandem diabody (TandAb) structure with continuous intravenous infusion at 14 day intervals. Because AMV564 is tetravalent, there are two CD3 binding sites and two CD33 binding sites, the affinity of this drug for both targets is increased. AMV564 has a higher molecular weight than the monovalent bispecific antibody and therefore a longer half-life. Preclinical in vitro and in vivo studies have demonstrated that AMV564 is capable of inducing potent cytotoxicity of cd33+ AML cell lines in a dose-dependent manner.

AMV564 is currently being evaluated in phase 1 trial (NCT 03144245), and by the year 2021, 05, 36 patients have participated in the study. Of 35 patients, 3 (9%) had objective responses, 1 had CR,1 had CRi, and 1 had Partial Remission (PR) were assessed. 17 (49%) of 35 patients were assessed for maternal cell depletion.

JNJ-67561244 (research and development institution: janssen) is a fully human IgG4-PAA bispecific antibody capable of binding to the C2 domain of CD33 and CD3 inducing T cell recruitment and tumor cytotoxicity. JNJ-67571244 specifically binds CD33 expressing cells and mediates specific in vitro T cell dependent cytotoxicity against AML cell lines and primary patient samples.

JNJ-67571244 is well tolerated in cynomolgus monkeys, up to 30mg/kg. JNJ-67571244 mediated potent cytotoxicity of cell lines and primary samples, regardless of their SNP genotype status, indicating potential therapeutic benefit compared to other V-binding antibodies. JNJ-67571244 is currently undergoing a phase 1 clinical trial (NCT 03915379) in patients with relapsed/refractory AML and high-risk myelodysplastic syndromes, and no clinical data has been reported for JNJ-67561244.

However, the specificity of conventional bispecific antibodies is not high, they may have an unwanted effect on normal cells while attacking leukemia cells, a certain probability may trigger nonspecific immune responses during treatment, they may attack normal cells due to lack of high specificity, not just leukemia cells, which results in so-called "nonspecific toxicity", i.e. normal tissues are damaged, resulting in poor therapeutic effects, higher doses are required to achieve therapeutic effects, thus increasing discomfort and risk for patients, while the selection of conventional bispecific antibodies on specific affinities or linking effectors is deficient, possibly resulting in overactivation of T cells or other adverse reactions, if the affinity of bispecific antibodies is too high or the linking effector is too strong, they may trigger strong immune responses, including cytokine release syndromes, reducing the safety of the treatment, and there is therefore a need for a bispecific antibody that can provide highly targeted therapy while binding human CD33 and CD3 in a low affinity manner.

Disclosure of Invention

(one) solving the technical problems

Aiming at the defects of the prior art, the invention provides a bispecific antibody for combining human CD33 and CD3, which solves the problems that the specificity of the bispecific antibody in the prior art is not high, the bispecific antibody can generate unnecessary influence on normal cells, and the selection on specific affinity or a connection effector is defective.

(II) technical scheme

To achieve the above object, the present invention provides a bispecific antibody that binds human CD33 and CD3, comprising:

1) IgG antibodies that specifically bind the cell membrane antigen CD 33;

2) A single chain antibody that binds to a T cell surface antigen CD3 molecule;

the single-chain antibody for recognizing the CD3 molecule is connected with the C end of the CD33 antibody light chain through a hydrophilic connecting peptide;

the bispecific antibody structure is:

and, in addition, the processing unit,

the Linker1: GSTSGSGKPGSGEGSTKGGS;

Linker2：GGGGSGGGGSGGGGS；

wherein the amino acid sequence of the VH-CH 1-finger Region-CH2-CH3 is a sequence 5;

the VL-Cκ -Linker1-VH (CD 3) -Linker2-VL (CD 3) amino acid sequence is shown in sequence 6.

The invention is further arranged to: the structure of the single-chain antibody recognizing the CD3 molecule adopts the form of ScFv, is aimed at human CD3 epsilon, and can be derived from various currently known monoclonal antibody variable region gene sequences, including but not limited to CD3 specific antibodies of OKT3, X35-3, WT31, WT32, SPv-T3b, TR-66, 11D8, 12F6, M-T301, SMC2 and F101.01;

The invention is further arranged to: the variable region gene sequences of various monoclonal antibodies which are known at present can be sourced, or the sequences of the variable regions of the anti-CD 33 monoclonal heavy chain and the anti-human CD33 monoclonal light chain which are built by the company are used;

the invention is further arranged to: the nucleotide sequence and the amino acid sequence of the heavy chain containing the leader peptide are shown as a sequence 1 and a sequence 2;

the nucleotide sequence and the amino acid sequence of the light chain containing the leader peptide are shown as sequence 3 and sequence 4;

the amino acid sequence of the heavy chain without the leader peptide is shown in sequence 5;

amino acids not contained in the leader peptide-free light chain are shown in sequence 6;

the invention also provides a preparation method of the bispecific antibody, which adopts a gene recombination technology to prepare, uses different types of mammalian cell expression vectors, uses the expression in CHO cells, adopts chemical components to limit a culture medium for the culture of the CHO cells, and does not add hormone and various animal-derived proteins or hydrolysates thereof in the culture process;

the invention is further arranged to: carrying out enzyme digestion on a single plasmid vector containing the bispecific antibody genes by adopting a single endonuclease to carry out linearization;

After transfection of CHO cells, a positive clone strain is obtained, then a bioreactor culture is carried out, and the product is secreted into the supernatant of the culture solution;

purifying by ion exchange chromatography medium or affinity chromatography combined with ion exchange chromatography to obtain bispecific antibody capable of specifically binding to human CD33 and CD 3;

the invention is further arranged to: the bispecific antibody is used for treating recurrent and refractory myelogenous leukemia, and is an immune therapeutic drug for killing myelogenous leukemia cells in a T cell dependent manner;

the invention also provides a pharmaceutical composition of any of the bispecific antibodies described above;

the invention is further arranged to: the pharmaceutical composition can be prepared into liquid preparations, freeze-dried preparations, sustained delivery by using a sustained infusion pump, timing delivery by using a pulse type infusion pump, intravenous delivery by recommendation and subcutaneous injection by using a subcutaneous injection;

the invention is further arranged to: the liquid preparation and the freeze-dried preparation comprise the following components in percentage by weight:

acetic acid: 0.2 to 0.4g/L

L-methionine: 1.0-20 g/L

L-histidine: 1.0-2.0 g/L

Tween-20: 0.2-1.0 g/L

Sucrose: 50-150 g/L.

(III) beneficial effects

The invention provides a real-time calculation method for the tide level of an intelligent channel design. The beneficial effects are as follows:

the EK333F is a bispecific antibody targeting CD33 and CD3, and can kill CD33 positive cells by utilizing T cells of a human body, so as to achieve the aim of treating myeloid leukemia.

The EK333F bispecific antibody of the invention is a bispecific antibody targeting CD33 and CD3, and after the EK333F is used for 7-10 days of low-dose slow induction treatment, the EK333F antibody is used for treatment, and an alternative treatment method is provided for patients with recurrent/refractory myelogenous leukemia.

The molecular structure of EK333F is essentially different from that of other bispecific antibodies which have entered clinical research stage at present, and has the advantage of better targeting, and meanwhile, the low-affinity CD3 epsilon-ScFv of the invention is adopted as a weak binding molecule for connecting effector-T cells, so that even if each EK333F molecule contains two CD3 binding sites, T cells cannot be activated like OKT 3.

In the research and development of EK333F, the molecular structure of K193 of the company is also referred to (patent number 201711131955.0) initially, but the K193 aims at a CD19 target, the B cells express higher B7 and easily activate T cells, the CD33 positive myelogenous leukemia cells express lower B7, and the early NSG mouse animal experiments show that the treatment effect is poor, so that a novel bispecific antibody molecule is designed on the basis of the K193, and the structure is shown as figure 1. The bispecific antibody with the molecular structure is a CD3 epsilon-ScFv single-chain antibody with low affinity, which is connected with the C end of a light chain with a standard IgG1 molecular structure through a hydrophilic linker, so as to achieve the aim of preferentially binding with a CD33 target.

In summary, the EK333F provided by the present application is a bispecific antibody targeting CD33 and CD3, and recognizes CD33 positive myeloid leukemia cells and T cells of the human body itself, and this specificity makes EK333F a highly targeted therapeutic drug capable of directing T cells to attack leukemia cells while reducing unnecessary effects on normal cells.

Meanwhile, the treatment method provided by the application comprises the steps of slow induction treatment at a low dose for 7-10 days, and then treatment by using an EK333F antibody at a proper dose, wherein the treatment method combines the use of a bispecific antibody and specific treatment time and dosage, and provides a new treatment option for patients with recurrent/refractory myelogenous leukemia.

In molecular structure, the molecular structure of EK333F adopts CD3 epsilon-ScFv with low affinity as a connecting effector, and has weaker affinity to be combined with T cells. Helps to prevent overactivation of T cells, thereby reducing adverse effects, while ensuring preferential binding to CD33 targets.

Solves the problems that the specificity of the bispecific antibody in the prior art is not high, the bispecific antibody can generate unnecessary influence on normal cells, and the selection on specific affinity or a connection effector is defective.

Drawings

FIG. 1 is a block diagram of the EK333F of the present invention;

FIG. 2 is a map of plasmid DGV-EK333F-Line linearized agarose electrophoresis;

FIG. 3 is a purification profile of an EK333F bispecific antibody by affinity chromatography;

FIG. 4 is a HPLC-SEC chromatogram after purification by EK333F bispecific antibody affinity chromatography;

FIG. 5 is an EK333F bispecific antibody anion exchange chromatography purification profile;

FIG. 6 is a HPLC-SEC chromatogram after purification by EK333F bispecific antibody ion exchange chromatography;

FIG. 7 is a HPLC-SEC chromatogram after EK333F bispecific antibody ultrafiltration;

FIG. 8 shows the binding activity of EK333F bispecific antibodies to CD33 positive cells;

FIG. 9 shows the binding activity of EK333F bispecific antibodies to T cells;

FIG. 10 is the ability of EK333F bispecific antibodies to efficiently activate T cells in the presence of CD33 positive cells;

FIG. 11 is a graph showing the change in the intensity of an in vivo animal imaging luminescence signal as a function of EK333F dose;

fig. 12 is a photograph of an animal living body image showing the therapeutic effect of EK 333F.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Examples

Referring to FIGS. 1-12, the present invention provides a bispecific antibody that binds human CD33 and CD3, namely EK333F, by which the purpose of killing CD33 positive cells by human T cells can be achieved, thereby achieving the purpose of treating myeloid leukemia.

The EK333F bispecific antibody of the invention is a bispecific antibody targeting CD33 and CD3, and after the EK333F is used for 7-10 days of low-dose slow induction treatment, the EK333F antibody is used for treatment, and an alternative treatment method is provided for patients with recurrent/refractory myelogenous leukemia.

The molecular structure of EK333F is essentially different from that of other bispecific antibodies which have entered clinical research stage at present, and has the advantage of better targeting, and meanwhile, the low-affinity CD3 epsilon-ScFv of the invention is adopted as a weak binding molecule for connecting effector-T cells, so that even if each EK333F molecule contains two CD3 binding sites, T cells cannot be activated like OKT 3.

In the research and development of EK333F, the molecular structure of K193 of the company is also referred to (patent number 201711131955.0) initially, but the K193 aims at a CD19 target, the B cells express higher B7 and easily activate T cells, the CD33 positive myelogenous leukemia cells express lower B7, and the early NSG mouse animal experiments show that the treatment effect is poor, so that a novel bispecific antibody molecule is designed on the basis of the K193, and the structure is shown as figure 1.

The bispecific antibody with the molecular structure is a CD3 epsilon-ScFv single-chain antibody with low affinity, which is connected with the C end of a light chain with a standard IgG1 molecular structure through a hydrophilic linker, so as to achieve the aim of preferentially binding with a CD33 target.

The EK333F bispecific antibody was expressed using a CHO cell expression system and the specific antibody yield per unit was up to 1.5 g/L or more after optimization of the chemical composition defining medium.

The invention provides a bispecific antibody combining human CD33 and CD3, which consists of a CD33 molecule and a human CD3 molecule which specifically identify the surface of human myeloid leukemia cells, wherein the heavy chain variable region gene of the anti-human CD33 molecule is connected with the genes of a human IgG CH1 and Fc region, wherein the genes containing the light chain (containing kappa chain Ckappa region) of the anti-human CD33 monoclonal antibody are connected with the heavy chain gene of the anti-human CD3 single chain antibody through a hydrophilic connecting peptide Linker1 and then connected with the light chain gene of the anti-human CD3 single chain antibody through a hydrophilic connecting peptide Linker 2;

the bispecific antibody structure is as follows:

Linker1：GSTSGSGKPGSGEGSTKGGS；

Linker2：GGGGSGGGGSGGGGS；

the amino acid sequence of the VH-CH 1-finger Region-CH2-CH3 is shown in a sequence 5;

the amino acid sequence of VL-Cκ -linker1-VH (CD 3) -linker2-VL (CD 3) is shown in sequence 6;

wherein the nucleotide sequence contained in the heavy chain containing the gene sequence encoding the leader peptide is shown in sequence 1, the position of the 1 st to 70 th nucleotides relative to the structural formula;

The CD33 heavy chain variable region sequence, the human IgG1 CH1, and the Fc sequence relative to nucleotides 71-1408;

the amino acid sequence of the heavy chain containing the leader peptide sequence is shown in sequence 2;

the leader peptide sequence is relative to amino acids 1-19. The 20 th to 135 th amino acids are VH (CD 33), the 136 th to 233 th amino acids are human IgG1 CH1, and the 237 th to 465 th amino acids are IgG1 Fc;

the nucleotide sequence contained in the light chain containing the gene sequence of the code guide peptide is shown in the sequence 3, and the position of the nucleotide sequence is relative to the 1 st to 73 rd nucleotides;

the position of the CD33 light chain variable region relative to nucleotides 74-406;

the position of human IgG1 C.kappa.relative to nucleotides 407-727;

the position of the connecting peptide relative to nucleotides 728-787, 1163-1207;

VH (CD 3) position relative to nucleotides 788-1162; VL (CD 3) relative to positions 1208-1534.

The amino acid sequence of the light chain containing the leader peptide sequence is shown as a sequence 4, and the leader peptide sequence is relative to the 1 st to 20 th amino acids;

amino acids 21 to 238 are VH (CD 33) +IgG 1C kappa, amino acids 239 to 258 are connecting peptide GSTSGSGKPGSGEGSTKGGS, amino acids 384 to 398 are connecting peptide (G4S) 3, amino acids 259 to 383 are VH (CD 3), and amino acids 399 to 507 are VL (CD 3).

The sequence without the amino acids contained in the leader peptide heavy chain is shown in sequence 5.

The amino acid sequence not contained in the leader peptide-free light chain is shown in sequence 6.

The Fab structural fragment for specifically recognizing the human CD33 antigen can be derived from the sequences of the light chain and heavy chain variable regions of various known mouse anti-human CD33 monoclonal antibodies, or the sequences of the variable regions of other known human anti-human CD33 monoclonal antibodies, or the sequences of the heavy chain and light chain variable regions of anti-CD 33 monoclonal antibodies which are self-constructed by the company.

Wherein the single chain antibody recognizing CD3 molecule adopts the form of ScFv, is aimed at human CD3 epsilon, and can be derived from various monoclonal antibody variable region gene sequences known at present, including but not limited to the CD3 specific antibodies of OKT3, X35-3, WT31, WT32, SPv-T3b, TR-66, 11D8, 12F6, M-T301, SMC2 and F101.01.

The present invention further provides a method for preparing the bispecific antibody of the present invention, which uses genes

The recombinant preparation can be expressed by using various forms of mammalian cells, preferably CHO cells, and the culture of the CHO cells adopts a chemical composition limiting culture medium, and hormone and proteins or hydrolysates thereof from various toxicants are not added in the culture process.

The preparation method comprises the steps of carrying out linearization on a single plasmid vector containing a bispecific antibody gene by adopting single endonuclease digestion, obtaining a positive clone strain after transfection of CHO cells, culturing in a bioreactor, secreting the product into a culture solution supernatant, and purifying by utilizing ion exchange chromatography media or affinity chromatography combined ion exchange chromatography to obtain the bispecific antibody capable of specifically combining human CD33 and CD 3.

The bispecific antibody provided by the invention is a therapeutic drug for treating recurrent/refractory myelogenous leukemia, and is an immune therapeutic drug for killing myelogenous leukemia cells in a T cell dependent manner.

The invention further provides pharmaceutical compositions comprising the bispecific antibodies of the invention. The pharmaceutical composition can be prepared into liquid preparations, freeze-dried preparations, sustained delivery by using a sustained infusion pump, timing delivery by using a pulse type infusion pump, intravenous delivery by using a recommendation and subcutaneous injection.

The bifunctional antibody constructed by the technology successfully realizes high-level stable expression in CHO cells, obtains high-purity bifunctional antibody by purification methods such as liquid chromatography and the like, and develops various liquid preparations aiming at the antibody; by adopting the liquid preparation formula provided by the invention, the bispecific antibody has stable quality under the condition of light-shielding storage at 2-8 ℃ within the concentration range of 0.1-30 mg/ml.

The invention provides a tumor targeting molecule antibody capable of generating high affinity and a CD3 antibody with relatively weak binding capacity, which are connected through hydrophilic peptide chains with proper lengths, so that a sufficient free stretching space is provided for an antibody specific binding part.

The 4 bispecific antibodies against CD33 of the prior art are compared with the molecular structure diagram of the present invention, see table 1:

table 1: comparison of the molecular Structure of the present invention with the 4 existing CD33 bispecific antibodies

Example 1

Construction of plasmid expression vectors

Construction of plasmids SGV-K33H (pKC2+CD33VH+hIgG 1 CH 1+finger region+hIgG1 Fc) and SGV-EK333FL (pKC1+CD33Vk+hIgG 1 Cκ+anti-Human-CD 3-ScFv).

After the plasmids SGV-K3331H (containing Anti-Humanized CD33VH sequence), pKC2 (vector sequence) and IgGCT-1 (hIgG 1 CH 1+finger region+hIgG1 Fc sequence) were treated with the corresponding restriction enzymes, the cleavage products CD33VH-HindIII/SalI, igGCT-SalI/EcoRI and pKC2-HindIII/EcoRI were obtained, the three cleavage products were ligated, DH 5. Alpha. Was transformed and the positive clone SGV-K33H (pKC2+CD33VH+hIgG 1 CH 1+finger region+hIgG1 Fc) was obtained by screening.

The primers VSNF1 and CKL3R and L3M3F and M3VR were used to amplify the fragment CD33VL+hIgG1 Cκ and introduce KOZAK sequence and light chain signal peptide sequence from the humanized Anti-CD 33 monoclonal antibody light chain containing plasmid SGV-K3331L and to amplify the fragment Anti-Human-CD3-ScFv (containing Linker2 ((G4S) 3) and introduce Linker1 (GSTSGSGKPGSGEGSTKGGS), plasmid pKC1 (vector sequence) was treated with restriction enzymes to obtain the cleavage products pKC1-HindIII/EcoRI, the two fragments were bridged and then homologous recombined with pKC1-HindIII/EcoRI to obtain plasmid SGV-EK333FL. Different clones were sequenced and selected for the next correct cloning test.

Table 2:

construction of DGV-EK 333F:

the SGV-K33H and SGV-EK333FL are treated by using restriction enzymes NotI and PvuI to obtain cleavage products K33H-N/P and EK333FL-N/P, the cleavage products are connected by a ligase, DH5 alpha is transformed and screened to obtain a positive clone DGV-EK333F, the plasmid DGV-EK333F is extracted in a large quantity, linearization treatment and phenol extraction purification are carried out by using restriction enzymes PvuI to obtain a linearization plasmid DGV-EK333F-Line, and a linearization plasmid agarose electrophoresis photograph is shown in figure 3.

Example 2

Establishment and screening of stable clones

Setting the perforation voltage of a gene pulse generator Xcell (Bio-Rad) to 300V, 900 mu F and exponential pulse in a sterile laminar flow workbench, taking out a disposable electric shock cup with a gap of 4mm, adding 40 mu g of linear plasmid DNA (100 mu l) and 0.7ml of CHO K1 cell (GSKO) suspension, setting the resistance of an electroporation instrument to infinity, directly transfecting the linearized plasmid 193 HVkP-into CHOK1 cells by an electrotransfection method, transferring the cells in the electric shock cup into a triangular flask, adding a CD CHO culture solution, culturing on a 5% CO2 shaker at 36-37 ℃, centrifuging at a low speed for 135 revolutions per minute, collecting cells after 24 hours, replacing the cells with the CD CHO culture solution (without glutamine) containing 50 mu M MSX, obtaining monoclonal cell strains by a limited dilution method, screening out clone with higher expression quantity by an ELISA method (mouse anti-human kappa chain monoclonal antibody+expression product K33+goat anti-human-HRP), culturing to obtain the clone with the highest expression quantity, and culturing the clone with the highest expression quantity according to the highest expression quantity, and carrying out the final clone growth condition of the clone with the highest expression quantity according to the obtained expression quantity; and obtaining a monoclonal cell strain by a limiting dilution method, screening out a clone strain with higher expression level by adopting the same ELISA method, subculturing, finally obtaining 5 clone strains, and selecting the clone strain (named EK 333F) with highest expression level for expansion culture.

Example 3

EK333F fermentation and purification

The obtained EK333F clone strain is inoculated into a 2L or 3L triangular flask containing 500ml of CD CHO culture solution, and is cultured on a 5% CO2 shaking table at 36-37 ℃ for 130-140 revolutions per minute, and after 3-6 days, the strain is cultured in a transfer bioreactor.

Reactor culture conditions: initial seeding density of the reactor: 50-200 ten thousand, the culture temperature is 32-37 ℃, the pH is 6.5-7.5, the stirring revolution is 50-120 r/min, the dissolved oxygen is 50% +/-20%, the intermittent feeding mode is adopted for feeding (feeding, such as sugar, a culture medium or alkali liquor, and the like) during the culture, the culture time is 12-18 days, and when the density of living cells is reduced to 60-70%, the living cells are harvested.

Centrifuging the obtained culture solution at 10000 r/min for 30 min at high speed, removing cells and cell fragments, collecting cell culture supernatant, purifying by affinity chromatography gel (such as Protein At Bead LX), performing predissociation and dissociation process on affinity chromatography, wherein the concentration of dissociation solution is 0.1mol/L citric acid-sodium citrate (pH2.0-4.5), the protein purity after affinity chromatography reaches above 96%, the affinity chromatography dissociation diagram is shown in figure 3, and the purity diagram is shown in figure 4; purifying with anion exchange chromatography gel such as DEAE Sepharose Fast Flow (shown in figure 5), wherein the purity can reach more than 99%, and the purity map is shown in figure 6; the purified liquid is concentrated by ultrafiltration and fully replaced by a stabilizer solution to form a stock solution.

Example 4

The EK333F stock solution stabilizer formula:

acetic acid: 0.2 to 0.4g/L

L-methionine: 1.0-20 g/L

L-histidine: 1.0-2.0 g/L

Tween-20: 0.2-1.0 g/L

Sucrose: 50-150 g/L

The purified liquid is the EK333F bispecific antibody. Bispecific antibody was analyzed on Shimadzu LC-20AT HPLC TSK 3000SW xl (7.8. Times.300 mM) column using 40mM PBS (containing 0.5M Na2SO4,pH6.5) as mobile phase, and the results showed that the purity was not less than 95% and the polymer content was less than 5%, and no visible impurity peaks were found, as shown in FIG. 7.

Example 5

Binding Activity assay of CD33-CD3 bispecific antibodies against CD 33-positive cells

The cell surface of U937 expresses CD33 antigen, and the antibody recognizing CD33 can be specifically combined with the CD33 antigen on the cell surface. The CD33-CD3 bispecific antibody sample EK333F was tested for site-specific binding to U937 cells CD33 using a flow cytometer. The experiment selects K333 antibody (Fab-ScFv) solution and CD33 antibody K33 solution as control samples, wherein EK333F molecule contains CD33 and CD3 double binding site, K333 only contains CD33 and CD3 single binding site, and K33 is human CD33 monoclonal antibody. The EK333F antibody was tested for site-specific binding activity to U937 cells CD33 using a flow cytometer (NovoCyte 3130, elsen biology (hangzhou)).

EK333F antibody was diluted to an initial protein concentration of 3mg/ml with 0.02mol/L PBS (pH 7.4, 1% BSA), 1 piece of 96 well U-plate was taken, and 50. Mu.l of 0.02mol/L PBS (pH 7.4, 1% BSA) was added to row B1-3 wells as a blank well. 50 μl of 0.02mol/L PBS (pH 7.4, containing 1% BSA) is added to each of 1-11 rows of C, D, E and 1-3 rows of F-H, 75 μl of EK333F antibody with 3mg/ml antibody content is added to each of C12, D12 and E12 rows, 25 μl of antibody solution in each of C12, D12 and E12 rows is sucked by a pipette, the mixture is sequentially diluted from right to left to columns of C1, D1 and E1 rows according to a gradient of 3 times, the mixture is uniformly mixed and then transferred to F3, G3 and H3 rows, gradient dilution is continued until F1, G1 and H1 rows are continued, 25 μl of antibody solution is sucked out after uniform mixing, and 50 μl of antibody per each row is reserved. The dilution range is 3mg/ml to 0.627ng/ml, and the total dilution is 15. Cell suspensions with a cell density of 5.0X106 cells/ml were prepared, 100. Mu.l of U937 cell suspension was added to each of the above-mentioned sample wells, and after mixing and allowing to react at room temperature for 60 minutes, the supernatant was carefully aspirated after centrifugation and discarded.

Except for the blank B2 wells, 50. Mu.l of 0.02mol/L PBS (pH 7.4, 1% BSA) was added, 50. Mu.l of murine anti-human IgG-kappa chain mab diluted to 2. Mu.g/ml was added to each well, and after mixing and allowing to react at room temperature for 60 minutes, the supernatant was carefully aspirated by centrifugation and discarded. 50 μl of 0.02mol/L PBS (pH 7.4, 1% BSA) was added to the blank B1 well, 50 μl of diluted FITC-labeled goat anti-mouse IgG (1:500) was added to the sample well, and after mixing and standing at room temperature for 30 minutes in the dark, the supernatant was carefully aspirated off by centrifugation. To each well, 170. Mu.l of 0.02mol/L PBS (pH 7.4) was added, and the cells in the wells were resuspended and mixed well. 10000 sample loading amounts in a flow cytometer door are set, the sample flow rate is high, the cell fluorescence value is measured, and the fluorescence value is counted (blank control fluorescence value is subtracted).

EC50 values of EK333F antibody were 2.406 ×10-9 mol/L calculated by using GraphPad prism 5.0 software, EC50 values of control sample K333 bifunctional antibody, CD33 antibody K33 were 1.074×10-8 mol/L and 8.220E-10mol/L, respectively, and binding activity of EK333F to U937 cells was superior to that of K333 protein. The binding activity of K33 mab to CD33 was superior to EK333F (see FIG. 8).

Example 6

Flow cytometry to determine the binding activity of EK333F antibodies to T cells

To test the ability of EK333F bispecific antibodies to bind to T cell surface molecule CD3, we performed flow cytometry analysis (FACS) on the bispecific antibodies obtained.

Jurkat cells are T lymphocytes, and the cell surface of the Jurkat cells is provided with a CD3 antigen, so that the Jurkat cells can be specifically combined with the CD3 antigen on the cell surface. The bispecific antibody sample EK333F was tested for site-specific binding to Jurkat cells CD3 using a flow cytometer. In the experiment, a K333 antibody solution and an OKT3 antibody solution are selected as control samples, wherein EK333F is a tetramer, K333F is a dimer, the molecular weight of EK333F is 2.7 times of that of K333, and OKT3 is CD3 locus monoclonal antibody. The EK333F antibody was tested for site-specific binding activity to Jurkat cell CD3 using a flow cytometer (NoveCyte 3130, elsen biology (hangzhou)).

EK333F antibody was diluted to an initial protein concentration of 300. Mu.g/ml with 0.02mol/L PBS (pH 7.4, 1% BSA), 1 piece of 96 well U-plate was taken, and 50. Mu.l of 0.02mol/L PBS (pH 7.4, 1% BSA) was added to row B1-3 wells as a blank well. 50. Mu.l of 0.02mol/L PBS (pH 7.4, 1% BSA) was added to each of lines 1 to 11 of C, D, E, 75. Mu.l of antibody solution diluted to 300. Mu.g/ml was then added to each of the C12, D12 and E12 wells, 25. Mu.l of antibody solution in each of the C12, D12 and E12 wells was pipetted, diluted sequentially from right to left to the columns C1, D1 and E1 wells in a 3-fold gradient, and 25. Mu.l was aspirated after mixing, and discarded, and the volume of each well was kept at 50. Mu.l. The dilution range is 300 mu g/ml-1.694 ng/ml, and the total dilution is 12. Cell suspensions having a cell density of 2.0X106 cells/ml were prepared, 100. Mu.l of Jurkat cell suspension was added to each of the above-mentioned sample wells, and after mixing and allowing to react at 37℃for 60 minutes, the supernatant was carefully aspirated after centrifugation and discarded.

Except for the blank B2 wells, 50. Mu.l of 0.02mol/L PBS (pH 7.4, 1% BSA) was added, 50. Mu.l of murine anti-human IgG-kappa chain mab diluted to 2. Mu.g/ml was added to each well, and after mixing and allowing to react at room temperature for 60 minutes, the supernatant was carefully aspirated by centrifugation and discarded. 50 μl of 0.02mol/L PBS (pH 7.4, 1% BSA) was added to the blank B1 well, 50 μl of diluted FITC-labeled goat anti-mouse IgG (1:100) was added to the sample well, and after mixing and placing in a dark place at 37deg.C for 60 minutes, the supernatant was carefully aspirated off by centrifugation. To each well, 170. Mu.l of 0.02mol/L PBS (pH 7.4) was added, and the cells in the wells were resuspended and mixed well. 10000 sample loading amounts in a flow cytometer door are set, the sample flow rate is high, the cell fluorescence value is measured, and the fluorescence value is counted (blank control fluorescence value is subtracted). EC50 values for EK333F antibodies were calculated using GraphPad prism 5.0 software. EK333F, K333, OKT3

EC50 values were 8.585E-09, 2.216E-09, and 4.379E-10 (mol/L), respectively, and EK333F bispecific antibodies were assayed for binding to Jurkat cells (FIG. 9).

Example 7

Flow cytometry to determine the activity of EK333F bispecific antibody to activate T cells (CD 69)

EK333F antibodies were diluted to serial concentrations (6.4 pg/ml to 500. Mu.g/ml) with 10% FCS 1640 medium and added to 48 well cell culture plates, respectively. Jurkat cells are regulated to 3X 106 cells/ml by 10% FCS 1640 culture solution, U937 cells are regulated to 3X 105 cells/ml, jurkat cells and U937 cells are uniformly mixed in equal volume (the effective target proportion is 10:1), 200 mu l/hole of mixed cell suspension is added into a 48-hole cell culture plate; a control group without B cells was also set, i.e.200. Mu.l/well of each of the serial diluted antibody solution and Jurkat cell solution at a density of 3X 106 cells/ml was added to a 48-well cell culture plate. Then, after culturing in a 10% CO2 incubator at 37℃for 18 hours, 150. Mu.l of the co-culture was collected, centrifuged to remove the supernatant, and then reacted with 6. Mu.l (1:1) of a mixed sample of Anti-Human CD69PE (clone: FN50, LOT:2264967, ebioscience) in the absence of light for 120 minutes, the supernatant was centrifuged to discard, the cells were resuspended in 0.02mol/L PBS (pH 7.4), the average fluorescence intensity of the cell-expressed CD69 was measured by using a flow cytometer, the obtained results were subjected to a fitting treatment by using GraphPad Prism 5.0 software, the measured concentrations were plotted as an abscissa, the average fluorescence intensity of the cells was plotted as an ordinate, the quantitative reaction relationship of the expression of CD69 molecules on the surfaces of Jurkat cells after stimulation with EK333F at the respective concentrations was analyzed, and EC50 values were fitted. The EC50 in the presence of U937 cells was 2.927E-11 (mol/L), and the EC50 value in the absence of U937 cells was 9.742E-09 (mol/L) only. From the above data, it was found that EK333F was 333-fold more capable of activating T cells in the presence of U937 cells.

In the absence of CD33 positive cells, the fluorescence value of CD69 was low, even at EK333F reaching 500ng/ml, approximately 2.7 times the basal value, so EK333F alone did not stimulate T cell activation well.

The test result shows that the surface of Jurkat cells activated by EK333F can express a large amount of CD69 molecules, which is the co-stimulation effect of EK333F antibodies under the existence of co-stimulation of U937 cells, and the two conditions are not indispensable, so that the efficient activation of T cells can not be realized under a single condition. In the presence of CD33 positive cells, activation of T cells (the amount of CD69 molecule produced) and the amount of EK333F were in a logarithmic positive relationship over a range of concentrations. The determination of high efficiency activation of T cells by EK333F in the presence of U937 is shown in figure 10.

EXAMPLE 8,

Anti-tumor efficacy test of bifunctional antibody CD33-CD3 (EK 333F) on tumor-bearing model of human acute promyelocytic leukemia cell HL60 humanized mouse

1 test method

1.1 Preparation of mouse model of HSC-NPG humanized immune system

4-week-old NPG female mice were irradiated with X-ray biological irradiation for 24 hours and then injected with multiple cord blood-derived (mixed donor) CD34+ hematopoietic stem cells by tail vein. After 20 weeks of transplantation, the mice are subjected to orbital blood sampling, EDTA-Na2 anticoagulation tubes are used for collecting blood samples, the contents of humanized CD45 and CD3 positive cells in the peripheral blood of the animals are analyzed through flow cytometry, and the humanized immune reconstruction effect is evaluated. HuHSC-NPG with uniform CD3+ proportion and hCD45 chimeric rate more than 25% is selected for establishing tumor-bearing model.

1.2 Tumor-bearing model preparation of HL60-luc-gfp cell line of HSC-NPG mice

HL60-luc-gfp tumor cells were cultured to log phase and the cell concentration was adjusted to 1×107 cells/mL after cell concentration counting. 40 humanized mouse HSC-NPG mice were weighed, all animals were injected with HL-60-luc-gfp cells via the tail vein, and each mouse was injected with a volume of 0.2mL (number of vaccinated cells 2X 106). After 10 days of transplantation, all animals were subjected to in vivo imaging to detect tumor-bearing conditions, and were grouped according to a hierarchical randomization grouping method based on total photon count, with 8 animals per group, and a total of 5 females.

1.3 Tail intravenous injection of bifunctional antibody EK333F

The five groups are respectively a solvent control group (PBS), an EK333F low-dose administration group (1 mug/only), an EK333F medium-dose administration group (10 mug/only), an EK333F high-dose administration group (30 mug/only) and a ciladaniline control group. The vehicle and EK333F were administered by tail vein injection in a volume of 0.1mL. The administration route of the ciladaniline is intragastric administration, and the administration volume is 5mL/kg of the body weight of the mice.

The frequency of dosing with vehicle and EK333F was 2 times per week for 7 weeks. The frequency of initial dosing of EK333F high dose group D7 was adjusted to 1 time per week. The frequency of administration of the ciladaniline control group was 3 times per week, followed by 7 weeks.

1.4 animal observations

At least 1 observation was made within 30min after the administration, and the response of the animals to the test sample was closely observed within 4h after the administration. Non-dosing time points, observed 1 time per day. The observation index includes clinical symptoms (such as animal appearance, behavior, diet, response to stimulus, secretion, excretion, etc.), death. Recording the starting time, severity and duration of symptoms of the animal in detail; the death status (time of death, number of dead animals, moribund reaction, etc.) of the animals was recorded in detail.

1.5 Living animal imaging detection of tumor

After tumor loading and after drug administration, D7, D14, D21, D28, D35, D42 and D50, mice are injected with fluorescein substrate solution (3 mg/mouse) intraperitoneally, and after 10min, the mice are anesthetized by inhalation of isoflurane gas and transferred into a camera obscura of a living body imager to image all living animals or dead or dying animals, an automatic exposure mode is set to shoot bright field and dark field pictures, and the luminous photon intensity is automatically detected. The total number of photons emitted by light is collected and analyzed through the software of Living Image cube 4.3.1

1.6 statistical analysis

Weight data and in vivo imaging data ± Data were analyzed using Graphpad prism8.0 statistical analysis software. First, normal distribution and variance alignment are checked on the data. Is in accordance with normal distribution (+) > >0.20 Level of variance (+)> >0.10): multiple comparisons were performed using LSD method of one-way anova for comparison between multiple groups> <0.05 is considered statistically significant; non-conforming to normal distribution or variance misalignment: the comparison between sets uses the Kruskal-Wallis H method of non-parametric testing if the Kruskal-Wallis H test results are significant (> <0.05 After the data is subjected to rank conversion, the data are subjected to comparison of groups of data and each other, and the data are subjected to +_f> <The difference was considered statistically significant at 0.05.

Animal survival statistical analysis is carried out to carry out chi-square test, <the difference was considered statistically significant at 0.05.

Normalization (Normalized) treatment of total photon number in living body imaging:

relative level= (post-dose/pre-dose value) ×100%.

Tumor inhibition = (1-total photon number of dosing group/total photon number of control group) = 100%.

2. Results

The results of bioluminescence in vivo imaging showed that the fluorescence signal intensity values of tumors in the control animals were all increased with the time after tumor cell inoculation, indicating that tumor bearing in the model mice was continuously growing (fig. 10). At each imaging time point after treatment, the tumor luminescence signals of animals in the vehicle control group and the ciladaniline control group are obviously stronger than those of animals in 3 treatment groups with different doses of EK 333F; animals in the treatment group (G2-G5) all showed significant differences in tumor site luminescence signal intensity at different imaging time points after treatment compared to vehicle control (fig. 12). The higher the treatment dose of EK333F after 3 weeks of treatment, the better the tumor suppression effect. By homogenizing the total photon number values, tumor inhibition rates were calculated, and the inhibition rates of tumors in the EK333F medium and high dose treatment groups reached more than 90% at D21 post-treatment, with obvious dose-effect relationships, and the tumor inhibition rate was highest in the high dose group (table 3).

Table 3 tumor inhibition rates for each treatment group

Claims (10)

1. A bispecific antibody that binds human CD33 and CD3, comprising:

1) IgG antibodies that specifically bind the cell membrane antigen CD 33;

2) A single chain antibody that binds to a T cell surface antigen CD3 molecule;

the single-chain antibody for recognizing the CD3 molecule is connected with the C end of the CD33 antibody light chain through a hydrophilic connecting peptide;

the bispecific antibody structure is:

and, in addition, the processing unit,

the Linker1: GSTSGSGKPGSGEGSTKGGS;

Linker2：GGGGSGGGGSGGGGS；

wherein the amino acid sequence of the VH-CH 1-finger Region-CH2-CH3 is a sequence 5;

the VL-Cκ -Linker1-VH (CD 3) -Linker2-VL (CD 3) amino acid sequence is shown in sequence 6.

2. A bispecific antibody binding to human CD33 and CD3 according to claim 1, characterized in that the single chain antibody structure recognizing the CD3 molecule takes the form of ScFv, directed against human CD3 epsilon, and can be derived from the variable region gene sequences of various monoclonal antibodies known to date, including but not limited to OKT3, X35-3, WT31, WT32, SPv-T3b, TR-66, 11D8, 12F6, M-T301, SMC2 and F101.01 CD3 specific antibodies.

3. The bispecific antibody of claim 1, wherein the sequence of the heavy chain variable region of the CD33 molecule and the sequence of the light chain anti-human CD33 monoclonal antibody are recognized, and the light chain anti-human CD33 monoclonal antibody comprises kappa region, and the variable region gene sequences of various monoclonal antibodies known at present or the sequences of the heavy chain and light chain variable regions anti-CD 33 monoclonal antibody constructed by the company are used.

4. A bispecific antibody binding to human CD33 and CD3 according to claim 1, characterized in that the heavy chain comprising a leader peptide comprises a nucleotide sequence, an amino acid sequence as shown in sequence 1, sequence 2;

the nucleotide sequence and the amino acid sequence of the light chain containing the leader peptide are shown as sequence 3 and sequence 4;

the amino acid sequence of the heavy chain without the leader peptide is shown in sequence 5;

the amino acids not contained in the leader peptide light chain are shown in sequence 6.

5. A method for preparing bispecific antibody is characterized in that the method adopts gene recombination technology, uses different types of mammalian cell expression vectors, uses CHO cells for expression, adopts chemical composition-limited culture medium for culturing CHO cells, and does not add hormone and various animal-derived proteins or hydrolysates thereof during the culturing process.

6. The method for preparing a bispecific antibody according to claim 5, wherein:

carrying out enzyme digestion on a single plasmid vector containing the bispecific antibody genes by adopting a single endonuclease to carry out linearization;

after transfection of CHO cells, a positive clone strain is obtained, then a bioreactor culture is carried out, and the product is secreted into the supernatant of the culture solution;

Purifying by ion exchange chromatography medium or affinity chromatography combined with ion exchange chromatography to obtain bispecific antibody capable of specifically binding to human CD33 and CD 3.

7. The bispecific antibody of claim 1 which is a therapeutic agent for the treatment of relapsed, refractory myelogenous leukemia, which is a T-cell dependent immunotherapeutic agent for killing myelogenous leukemia cells.

8. A pharmaceutical composition comprising the bispecific antibody of claim 1.

9. The pharmaceutical composition of claim 8, which is used in a method comprising: making into liquid preparation, lyophilizing, and sustained administration with sustained infusion pump.

10. The pharmaceutical composition formulation of claim 8 comprising:

acetic acid: 0.2 to 0.4g/L

L-methionine: 1.0-20 g/L

L-histidine: 1.0-2.0 g/L

Tween-20: 0.2-1.0 g/L

Sucrose: 50-150 g/L.