CN117801113A

CN117801113A - Bispecific antibody and pharmaceutical composition composed of bispecific antibody and NK cells

Info

Publication number: CN117801113A
Application number: CN202211095906.7A
Authority: CN
Inventors: 姚凯庆; 刘宏宇; 郭天
Original assignee: Jinan Linyun Haida Biotechnology Co ltd
Current assignee: Jinan Linyun Haida Biotechnology Co ltd
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2024-04-02

Abstract

The invention provides a bispecific antibody and a pharmaceutical composition formed by the bispecific antibody and NK cells, wherein the bispecific antibody comprises a targeted CD16 antigen binding domain and a targeted CD33 antigen binding domain, so that the bispecific antibody can be used for targeted recognition of tumor cells, can also be used for effectively activating and recruiting NK cells and strengthening tumor immunity; further, the bispecific antibody adopts an enhanced structure, comprises two 2 targeted CD16 antigen binding domains and 2 targeted CD33 antigen binding domains, wherein the targeted CD16 antigen binding domains and the targeted CD33 antigen binding domains are respectively positioned at two sides of the Fc fragment, and the same antigen binding domains are positioned at the same side, so that the mutual interference between different types of antigen binding domains can be prevented, and the affinity with a target antigen can be improved and maintained; furthermore, the bispecific antibody and the NK cells from the variant source form a pharmaceutical composition, so that the interference of the tumor microenvironment in a tumor patient on autoimmune cells can be avoided, and a strong tumor inhibition effect is exerted. The bispecific antibody and the pharmaceutical composition thereof can effectively inhibit proliferation of leukemia, lymphoma and myeloma cells, regulate expression of immune factors, strengthen immune response of organisms and resist tumor invasion.

Description

Bispecific antibody and pharmaceutical composition composed of bispecific antibody and NK cells

Technical field:

the invention belongs to the field of medicine research, and particularly provides a bispecific antibody, a medicine composition composed of the bispecific antibody and NK cells, and application of the bispecific antibody in preparation of antitumor medicines.

The background technology is as follows:

tumors are diseases that seriously affect human health, and although therapeutic means such as surgery, chemical drugs, radiotherapy, etc. are becoming mature, many clinical difficulties are faced, such as high recurrence rate, difficult further improvement of 5-year survival rate, occurrence of unpleasant serious adverse reactions affecting patient quality of life, etc. In recent years, with the continuous deep research and understanding of the autoimmune regulation mechanism of human body, tumor immunotherapy is gradually in the corner of the head, and is more and more paid attention to, new generation treatment technologies represented by immune checkpoint inhibitors and chimeric antigen receptors acquire surprising treatment effects in clinical application of tumors, and a plurality of star-like drugs are induced, which suggests that tumor immunotherapy is an important development direction of novel anti-tumor drugs.

To date, most immunomodulatory treatments have focused on enhancing T cell responses, such as Chimeric Antigen Receptor (CAR) T cells. CAR-T cells are engineered to recognize specific Tumor Antigens (TAs) and to achieve unprecedented clinical effects in several hematological malignancies, such as non-hodgkin's lymphoma, acute Lymphoblastic Leukemia (ALL), and chronic lymphoblastic leukemia. However, CAR-T cell therapies may produce toxic effects, most commonly systemic cytokine response syndrome and CAR-T cell related encephalopathy syndrome. Meanwhile, bispecific T cell engagers have also been proposed, such as BiTE, to redirect endogenous immune effector T cells near tumors and to aggregate CD3T cell receptor (TCR) complexes within induced immune synapses, triggering T cell signaling. As with CAR-T cell therapies, biTE therapies are also limited in toxicity. In addition to cytotoxicity, T cell-based immunotherapy is also challenged, for example, research has found that although the immune system can prevent the development of tumors, many cancer cells can evade the monitoring and clearance of the immune system of the body through a process called "immune escape", T cells are greatly affected by immune escape, and tumor cells can evade the killing effect of T cells by modulating or mutating the cell antigen receptor T cell receptor or expressing immune checkpoint surface antigens, which is one of the main problems faced in the course of T cell therapy of tumors. For these reasons, researchers have been focusing on immune cells other than T cells in order to develop more effective and convenient anti-tumor therapies.

In this context, NK cells, i.e. natural killer cells (Natural Killer Cell, NK), are increasingly gaining importance, and the role of NK cells in tumor monitoring is well recognized, especially in controlling blood cancers and tumor metastasis, e.g. the expression levels of NK cell activating receptors NKp30 and NKG2D in metastatic lymph nodes of tumor patients are inversely related to metastasis levels; NK cells from patients with longer overall survival and castration resistance exhibit high expression levels of activating receptors and high cytotoxicity in metastatic prostate cancer patients; in the mouse metastasis model, depletion of NK cells and genetic defects of IFN-gamma or perforin lead to elevated metastasis levels in mice.

In addition to its role in direct tumor monitoring, NK cells also contribute to T cell anti-tumor immunity. NK cells promote T-bet in mouse model ⁺ CD4 ⁺ Accumulation of T cells in tumor areas, infiltration of CD8 by tumors ⁺ T cells promote the production of effector molecules TNF-alpha and IFN-gamma, inhibiting these CD 8' s ⁺ Expression of the failure marker PD-1 in T cells and promotes induction of tumor-specific T cell memory. In vitro data indicate that NK cells might promote differentiation of antitumor Th1 cells by producing IFN- γ in a NKG 2D-dependent manner. Furthermore, NK cells are necessary for accumulation of conventional type I dendritic cells (dcs 1) in tumors in the mouse model, as NK cells produce CCL5 and XCL1 chemoattractants, the recruitment of which dcs 1 is critical for T cell anti-tumor immunity. The above evidence suggests that NK cells act as "helpers" in the formation of potent anti-tumor T cell responses.

NK cells have multiple immunomodulatory effects, and their sources are also quite broad, and NK cells that have been or are proposed to be used clinically at present include: (1) Allogeneic NK cells are inoculated into a patient after in vitro culture and domestication of NK cells from healthy people, and because the immunogenicity of the NK cells is not strong, worrying Graft Versus Host Disease (GVHD) can be avoided, and the method has been used for treating acute leukemia patients by Hematopoietic Stem Cell Transplantation (HSCT), so that the GVHD can be effectively inhibited, and the therapeutic effect of leukemia can be consolidated and enhanced; (2) Autologous NK cells are isolated from peripheral blood of a patient and expanded in vitro, and then are returned to the patient, GVHD can be completely avoided theoretically, but the functional state and expansion capacity of the autologous NK cells are often poor, and the autologous NK cells of the patient can be interfered by tumor microenvironment so that the clinical efficacy is poor, and people try to solve the problem of culturing the autologous NK cells by activating different combinations of cytokines (IL-2, IL-12, IL-15, IL-18 and the like) and using the feeder cells during the ex vivo expansion period, so far under study; (3) Mature NK cell lines, in view of the difficulty in obtaining large amounts of cytotoxic NK cells from peripheral blood, researchers have developed subculturable NK cell lines, of which NK92 cells are widely used clinically, and some clinical trials have demonstrated the safety of NK92 cells as a cancer therapeutic agent, but since it is an immortalized cell line, the potential tumorigenicity has not yet been thoroughly addressed; (4) NK cells are induced, and can be differentiated from stem cells, including Induced Pluripotent Stem Cells (iPSCs) and stem cells obtained from umbilical cord blood, and recently NK cells have been extracted from iPSCs produced by peripheral blood cells, and exhibit low KIR expression and have cytotoxic effects on cancer cell lines in vitro.

Despite the strong immunomodulatory effects and relatively abundant cell sources of NK cells, NK cell therapies still face difficulties, one of which is how to effectively activate NK cells and enrich them in the vicinity of tumor cells. Recently, the generation of bispecific killer cell cement (bispecific killer cell engagers, biKE) has provided new promise for solving this dilemma. Bikes are composed of two scFvs with different specificities via a flexible linkerTogether, one scFv targets a tumor antigen (e.g., CD19, CD20, CD 33), while the other is specific for NK cell receptors. This effectively brings together cancer cells and NK cells, promotes the formation of immune synapses, and allows NK cells to perform their cytolytic function specifically and effectively. The primary target of BiKEs is CD16, as it effectively induces NK activation without additional co-stimulation, CD16 BiKEs have been effectively used to target tumor cells expressing CD19, CD20, CD33, CD133 and EpCAM. NK cells from patients with myelodysplastic syndrome (MDS) can be effectively activated by CD16-CD33 BiKE, and the drug is not only aimed at CD33 ⁺ MDS cells, and also directed against immunosuppressive CD33 ⁺ A population of MDSCs. In these studies, the BiKE is able to redirect autologous NK cells to tumor cells, overcoming the immunosuppression that is prevalent in the tumor microenvironment.

In order to improve tumor immunotherapy means based on NK cells, the invention provides a bispecific antibody, which comprises an antigen binding domain targeting CD16 and CD33, can be combined with a target antigen with high affinity, so that the NK cells can effectively target the tumor cells and play an anti-tumor role; the composition comprising the bispecific antibody and NK cells is constructed on the basis of allogeneic NK cells, so that the interference of tumor microenvironment in a patient on autologous NK cells can be eliminated, and a stronger anti-tumor effect is exerted.

Disclosure of Invention

In order to solve the technical problems, the invention provides a bispecific antibody, which is characterized in that the bispecific antibody comprises a targeting CD16 antigen binding domain and a targeting CD33 antigen binding domain, wherein the heavy chain variable region of the targeting CD16 antigen binding domain comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2 and HCDR3 shown as SEQ ID NO. 3, and the light chain variable region comprises LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6; the heavy chain variable region of the targeting CD33 antigen binding domain comprises HCDR1 shown as SEQ ID NO. 7, HCDR2 shown as SEQ ID NO. 8 and HCDR3 shown as SEQ ID NO. 9, and the light chain variable region comprises LCDR1 shown as SEQ ID NO. 10, LCDR2 shown as SEQ ID NO. 11 and LCDR3 shown as SEQ ID NO. 12.

In the invention, for effectively activating tumor immune response based on NK cells, a bispecific antibody targeting CD16 and CD33 is provided, wherein an antigen binding domain targeting CD16 can be combined with NK cells with high efficiency, so that the effective recruitment of NK cells near tumor cells is realized, and the targeting and the effectiveness of immunotherapy are improved; the antigen binding domain of the target CD33 can effectively bind to CD33 positive tumor cells, such as high-level expression of CD33 in blood tumors of leukemia, lymphoma, myeloma and the like, and experiments prove that the bispecific antibody provided by the invention can effectively kill the blood tumor cells.

Furthermore, the amino acid sequence of the heavy chain variable region of the targeted CD16 antigen binding domain is shown as SEQ ID NO. 13; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 14.

Furthermore, the amino acid sequence of the heavy chain variable region of the targeting CD33 antigen binding domain is shown as SEQ ID NO. 15; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16.

Further, comprising 2 targeted CD16 antigen binding domains and 2 targeted CD33 antigen binding domains and an Fc fragment, the targeted CD16 antigen binding domains and the targeted CD33 antigen binding domains are located on both sides of the Fc fragment, respectively, and the same antigen binding domains are located on the same side.

Further, the Fc fragment is a human IgG Fc fragment, and the amino acid sequence of the Fc fragment is shown as SEQ ID NO. 17.

The structure of the bispecific antibody is complex and flexible, various types of bispecific antibody structures exist in the prior art, and the bispecific antibody provided by the invention has the following characteristics: (1) The human IgG Fc fragment is particularly used, is the most abundant and common Fc fragment in human body, can reduce unnecessary immune rejection reaction, can effectively prolong the half-life of the antibody and improves the bioavailability; (2) The antigen-binding domain contains two 2 targeted CD16 antigen-binding domains and 2 targeted CD33 antigen-binding domains, belongs to an enhanced bispecific antibody, and can improve the binding capacity of the antigen-binding domains and target antigens, thereby efficiently recognizing targets; (3) The targeting CD16 antigen binding domain and the targeting CD33 antigen binding domain are respectively positioned at two sides of the Fc fragment, and the same antigen binding domain is positioned at the same side, so that mutual interference between different types of antigen binding domains can be prevented, and the affinity with a target antigen can be improved and maintained.

A pharmaceutical composition is provided comprising the bispecific antibody and NK cells.

Further, the preparation method of the NK cells comprises the following steps: collecting peripheral blood of a healthy person, and obtaining peripheral blood mononuclear cells by adopting Ficoll density gradient centrifugal separation; autologous plasma was obtained using centrifugation; 100mL of basic serum-free culture medium is added with IFN-gamma with the final concentration of 20 mug/mL, IL-2 with the final concentration of 10 mug/mL, IL-21 with the final concentration of 10 mug/mL, IL-15 with the final concentration of 50 mug/mL and autologous plasma with the final concentration of 5mL, the basic serum-free culture medium is RPMI-1640 liquid culture medium, and the liquid culture medium is added with CO with the concentration of 5.0% at 37 DEG C ₂ Culturing in a constant temperature incubator with saturated humidity, and changing fresh culture medium according to the growth condition of cells; after about 2 weeks of culture, NK cells were obtained by flow cytometry screening.

The pharmaceutical composition provided by the invention comprises the bispecific antibody and the NK cells from the variant source, and has evidence that the immunogenicity of the variant NK cells is in an acceptable range, severe rejection reaction can not be caused, and the NK cells from the healthy human source are not interfered by tumor immune microenvironment, so that the killing capacity to tumors is stronger, and the composition is more beneficial to exerting the tumor immunotherapy effect.

Provides the application of the bispecific antibody or the pharmaceutical composition in preparing antitumor drugs.

Further, the tumor is selected from at least one of bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, a tumor of the central nervous system, lymphoma, leukemia, and myeloma.

Further, the tumor is selected from the group consisting of lymphoma, leukemia, and myeloma.

CD33 is highly expressed in a variety of tumor cells including, but not limited to, bladder, breast, uterine, cervical, ovarian, prostate, testicular, esophageal, pancreatic, colorectal, colon, renal, head and neck, lung, gastric, germ cell, bone, liver, thyroid, skin, central nervous system tumors, lymphomas, leukemias, myelomas, and the like; particularly in hematological tumors such as lymphoma, leukemia and myeloma, CD33 has become a recognized effective therapeutic target, various antibodies and immunocyte medicaments have been approved or are in clinical test stage, and the bispecific antibody and the pharmaceutical composition can effectively inhibit the proliferation of tumor cells of lymphoma, leukemia and myeloma and play a role in killing tumors.

Advantageous effects

The application provides a bispecific antibody and a pharmaceutical composition formed by the bispecific antibody and NK cells, which have the following advantages:

(1) The bispecific antibody can be combined with target antigens CD16 and CD33 with high affinity, so that tumor cells can be identified in a targeted manner, NK cells can be effectively activated and recruited, and the tumor immune function is enhanced;

(2) The bispecific antibody adopts an enhanced structure, contains two 2 targeted CD16 antigen binding domains and 2 targeted CD33 antigen binding domains, belongs to the enhanced bispecific antibody, and can improve the binding capacity of the antigen binding domains and target antigens, thereby efficiently recognizing targets; the targeting CD16 antigen binding domain and the targeting CD33 antigen binding domain are respectively positioned at two sides of the Fc fragment, and the same antigen binding domain is positioned at the same side, so that the mutual interference between different types of antigen binding domains can be prevented, and the affinity with a target antigen can be improved and maintained;

(3) The bispecific antibody contains an Fc fragment, and specifically uses a human IgG Fc fragment which is the most abundant and common Fc fragment in human body, so that unnecessary immune rejection reaction can be reduced, the half life of the antibody can be effectively prolonged, and the bioavailability can be improved;

(4) The bispecific antibody and the NK cells from the variant source form a pharmaceutical composition, so that the interference of the tumor microenvironment in a tumor patient on autoimmune cells can be avoided, and a strong tumor inhibition effect is exerted.

Drawings

Fig. 1: schematic of the general structure of bispecific antibodies;

fig. 2: schematic of the general structure of an enhanced bispecific antibody;

fig. 3: tumor cell inhibition rate;

fig. 4: tumor volume change in animal model;

fig. 5: serum IL-6 expression levels;

fig. 6: serum IFN-gamma expression levels.

Detailed Description

The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.

The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.

Example 1 design and preparation of bispecific antibodies

1.1 design of bispecific antibodies

The antigen binding domains targeted to CD16 and CD33 are obtained by screening phage display libraries in the early experiments of the invention, wherein the heavy chain variable region of the targeted CD16 antigen binding domain comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2 and HCDR3 shown as SEQ ID NO. 3, and the light chain variable region comprises LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6; the heavy chain variable region of the targeting CD33 antigen binding domain comprises HCDR1 shown as SEQ ID NO. 7, HCDR2 shown as SEQ ID NO. 8 and HCDR3 shown as SEQ ID NO. 9, and the light chain variable region comprises LCDR1 shown as SEQ ID NO. 10, LCDR2 shown as SEQ ID NO. 11 and LCDR3 shown as SEQ ID NO. 12. Furthermore, the amino acid sequence of the heavy chain variable region of the targeted CD16 antigen binding domain is shown as SEQ ID NO. 13; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 14. Furthermore, the amino acid sequence of the heavy chain variable region of the targeting CD33 antigen binding domain is shown as SEQ ID NO. 15; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16.

Bispecific antibodies have been reported to have a relatively flexible structure, as shown in fig. 1, and various specific structures have been reported in the prior art, and an Fc fragment can be included in these structures, which is advantageous for increasing the biological half-life of the antibody in vivo and improving the bioavailability; on the other hand, in order to simplify the structure of antibodies while preventing the antibodies from being overdriven to cause a strong immune response, attempts have been made to accelerate the in vivo clearance of bispecific antibodies by employing a simplified structure that omits the Fc fragment (as in fig. 1C).

In the present invention, in order to enhance the binding capacity to the target antigen, an "enhanced" bispecific antibody structure is used, i.e. in the case that the bispecific antibody comprises 2 targeted CD16 antigen binding domains and 2 targeted CD33 antigen binding domains, the selectable structure is as shown in fig. 2, in fig. 2A, the targeted CD16 antigen binding domain and the targeted CD33 antigen binding domain are arranged in sequence on the same side of the Fc fragment; in fig. 2B, the targeting CD16 antigen binding domain and the targeting CD33 antigen binding domain are flanking the Fc fragment, respectively linked to the Fc fragment by a flexible linker GGGGS; in fig. 2C, the Fc fragment is omitted and the targeting CD16 antigen binding domain and the targeting CD33 antigen binding domain are directly linked by a flexible linker.

In preliminary experiments, the bispecific antibodies of the structures shown in fig. 2A and 2B have longer half-lives in experimental animals, which are advantageous for prolonging the antibody action time, and severe immune rejection and immune factor storm are not observed, so that the use of bispecific antibody structures with Fc fragments is preferred. The Fc fragment is a human IgG Fc fragment, and the amino acid sequence of the Fc fragment is shown as SEQ ID NO. 17.

1.2 preparation of bispecific antibodies

Ligating the polynucleotide sequences encoding the targeted CD16 antigen binding domain and the targeted CD33 antigen binding domain to a polynucleotide sequence encoding a human IgG1 Fc fragment in the order shown in fig. 2A, 2B; the polynucleotide sequence is introduced into an expression vector PCDNA3.1, and after the sequencing verification is correct, the vector is introduced into escherichia coli to prepare the plasmid on a large scale.

The target plasmid is introduced into CHO cells by electroporation, continuous fermentation culture is carried out by using a 5L micro-fermenter, and when the cell density reaches 90%, the supernatant is centrifugated to purify the protein. Purifying by using Protein A column to obtain bispecific antibody BiKE-A with structure shown in figure 2A and bispecific antibody BiKE-B with structure shown in figure 2B, respectively, and determining that the purity of the obtained antibodies reaches more than 95%, which meets the requirement of subsequent experiments.

1.3 bispecific antibody affinity assay

The affinity of the antibodies obtained by the molecular interaction analysis platform Biacore detection and screening with target proteins human CD16 and CD33 is shown in Table 1, and the result is shown in Table 1, the affinity of BiKE-A with target antigens is weaker than that of BiKE-B, and the target CD16 antigen binding domain and the target CD33 antigen binding domain in the BiKE-A are possibly affected by steric hindrance, so that a certain interference is presumed, and the subsequent experiments of the invention are carried out by using the BiKE-B antibodies.

Table 1 bispecific antibodies and target antigen affinity assays

Remarks: anti-CD 16 is a targeting CD16 monoclonal antibody screened in the earlier stage; anti-CD 33 is a targeting CD33 monoclonal antibody screened in advance.

EXAMPLE 2NK cell preparation

Collecting peripheral blood of healthy volunteers, pouring the peripheral blood into a 50mL centrifuge tube, adding ice-precooled lymphocyte separation liquid (purchased from Beijing Soy Bao technology Co., ltd.) at 4 ℃, and separating by Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells; autologous plasma was obtained using centrifugation; 100mL of basic serum-free culture medium is added with IFN-gamma with the final concentration of 20 mug/mL, IL-2 with the final concentration of 10 mug/mL, IL-21 with the final concentration of 10 mug/mL, IL-15 with the final concentration of 50 mug/mL and autologous plasma with the final concentration of 5mL, wherein the basic serum-free culture medium is RPMI-1640 liquid culture medium, and is put into CO with the concentration of 5.0% at 37 DEG C ₂ Constant saturation humidityCulturing in a warm incubator, and changing fresh culture medium according to the growth condition of cells; after about 2 weeks of incubation, the flow cytometer detects CD3-CD56 ⁺ Cell proportion, NK cells were obtained by screening.

Example 3 in vitro anti-tumor experiment

3.1 selection and cultivation of target cells

In order to verify the killing effect of the bispecific antibody and NK cells on tumors, leukemia cell lines HL-60 and K562, a multiple myeloma cell line RPMI-8226 and a lymphoma cell line Raji are selected as experimental objects. Resuscitates the cells, inoculates them in RPMI 1640 medium containing 10% FBS, 37 ℃ and 5.0% CO ₂ Culturing at constant temperature and saturated humidity to logarithmic phase for subsequent experiment.

3.2 cell killing experiments

Tumor cells in section 3.1 were treated at 5X 10 ⁴ Inoculating the culture medium into 96-well plate, and placing 96-well culture plate at 37deg.C with 5% CO ₂ The incubation was continued overnight until the cells adhered to the wall and the growth state was good. Positive wells were added with 10. Mu.g/mL of BiKE-B antibody and 5X 10, respectively ⁵ NK cells per well, cell culture medium was used as negative control. Continuously placing at 37deg.C with 5% CO ₂ After 48h incubation in the cell incubator, the 96-well plates were removed, the medium in the plates was discarded, and medium containing 10% cck8 reagent was added at a volume of 100 μl per well and returned to the 37 ℃ cell incubator for waiting to develop color. After the color development is completed for the required time, the 96-well ELISA plate is placed in an ELISA reader to read the OD450 value, and the reading is recorded and the cell inhibition rate is calculated.

As shown in FIG. 3, both BiKE-B antibody and NK cell have obvious inhibition effect on the tumor cells, especially on HL-60 cells, the inhibition rate can reach more than 90%, and the inhibition rate on K562 and RPMI-8226 cells is maintained at a high level, but the tumor killing ability in Raji cells is reduced. This shows that the BiKE-B antibody and NK cells provided by the invention can effectively inhibit proliferation of various liquid tumor cells including leukemia, myeloma and lymphoma, and further play an anti-tumor role.

Example 4 in vivo anti-tumor experiment

4.1 animal model preparation and treatment

Taking HL-60 cells in logarithmic growth phase, inoculating under the right armpit skin of 30 nude mice under aseptic condition, and inoculating 5×10 cells ⁶ And/or just. The diameter of the transplanted tumor is measured by a vernier caliper, and the tumor grows to 80mm ³ Selecting 40 tumor-bearing nude mice with good growth state and uniform tumor size, randomly dividing the nude mice into 4 groups, wherein 10 nude mice in each group are BiKE-B groups, and injecting 100mg/kg BiKE-B antibody into tail veins every two days; NK cell group, tail intravenous injection 10 every two days ⁶ individual/NK cells; combination group, 100mg/kg BiKE-B antibody and 10 were injected intravenously every two days ⁶ individual/NK cells; in the control group, equal volumes of physiological saline were injected into the tail vein every two days.

4.2 tumor volume measurement

Tumor volumes were measured and calculated using vernier calipers every 3 days for a total of 21 days of observation. As shown in fig. 4, the bispecific antibody or NK cell provided by the present invention can inhibit the tumor growth rate in vivo, wherein the inhibition effect of the combination group is strongest, the BiKE-B antibody is secondary, and the NK cell effect is secondary, which may be due to the fact that the BiKE-B antibody is used alone, although the dual effects of targeting tumor cells and recruiting NK cells are achieved, the tumor model animal is affected by tumor microenvironment, and the activation and enrichment of NK cells may be inhibited to some extent, thereby affecting the overall anti-tumor effect; after the injection of the fresh NK cells, the NK cell sources are supplemented, so that the anti-tumor effect can be exerted in vivo.

4.3 determination of the content of immunomodulatory factors

The immune regulating factor plays an important role in tumorigenesis and development and immunotherapy, and researches show that the immune factors such as IL-6, IFN-gamma and the like can not only directly induce apoptosis, but also be beneficial to activating immune cells such as NK cells, T cells, macrophages and the like to play a synergistic anti-tumor role. In this example, after 21 days of treatment, the rat orbit was bled, the serum was collected by centrifugation, and the levels of mouse serum IL-6 and IFN-gamma were measured according to ELISA kit instructions (purchased from Methano Biotechnology Co., ltd.) in Beijing.

The results are shown in FIGS. 5 and 6, and the expression level of IL-6 is obviously up-regulated after tumor modeling, which is related to the self-immune mechanism of the organism, and the high-level secretion of IL-6 is further induced after treatment by using BiKE-B antibody and/or NK cells, and the effect is stronger and more obvious in combined use; IFN-gamma is also an important anti-tumor factor, and human recombinant IFN-gamma is approved for treating tumors by injection at present, and after treatment by using BiKE-B antibodies and/or NK cells, the IFN-gamma level in an animal model body is obviously improved, wherein the expression level of a BiKE-B antibody group is similar to that of a combined group, and the expression level of the BiKE-B antibody group and the combined group are not obviously different, but are higher than that of the condition of using NK cells singly, so that the bispecific antibody provided by the invention has the advantage in the aspect of effectively activating humoral immunity.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A bispecific antibody comprising a CD16 antigen binding domain and a CD33 antigen binding domain, wherein the heavy chain variable region of the CD16 antigen binding domain comprises HCDR1 as shown in SEQ ID No. 1, HCDR2 as shown in SEQ ID No. 2 and HCDR3 as shown in SEQ ID No. 3, and the light chain variable region comprises LCDR1 as shown in SEQ ID No. 4, LCDR2 as shown in SEQ ID No. 5 and LCDR3 as shown in SEQ ID No. 6; the heavy chain variable region of the targeting CD33 antigen binding domain comprises HCDR1 shown as SEQ ID NO. 7, HCDR2 shown as SEQ ID NO. 8 and HCDR3 shown as SEQ ID NO. 9, and the light chain variable region comprises LCDR1 shown as SEQ ID NO. 10, LCDR2 shown as SEQ ID NO. 11 and LCDR3 shown as SEQ ID NO. 12.

2. The bispecific antibody of claim 1, wherein the heavy chain variable region amino acid sequence of the targeted CD16 antigen binding domain is set forth in SEQ ID No. 13; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 14.

3. The bispecific antibody of claim 1, wherein the heavy chain variable region amino acid sequence of the targeting CD33 antigen binding domain is set forth in SEQ ID No. 15; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16.

4. A bispecific antibody according to any one of claims 1-3, characterized in that it comprises 2 targeting CD16 antigen binding domains and 2 targeting CD33 antigen binding domains and an Fc fragment, said targeting CD16 antigen binding domains and targeting CD33 antigen binding domains being located on both sides of the Fc fragment, respectively, and the same antigen binding domains being located on the same side.

5. The bispecific antibody of claim 4, wherein the Fc fragment is a human IgG Fc fragment having the amino acid sequence shown in SEQ ID No. 17.

6. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-5 and NK cells.

7. The pharmaceutical composition of claim 6, wherein the NK cell is prepared by a method comprising: collecting peripheral blood of a healthy person, and obtaining peripheral blood mononuclear cells by adopting Ficoll density gradient centrifugal separation; autologous plasma was obtained using centrifugation; 100mL of basic serum-free culture medium is added with IFN-gamma with the final concentration of 20 mug/mL, IL-2 with the final concentration of 10 mug/mL, IL-21 with the final concentration of 10 mug/mL, IL-15 with the final concentration of 50 mug/mL and autologous plasma with the final concentration of 5mL, wherein the basic serum-free culture medium is RPMI-1640 liquid culture medium which is put into 37 ℃ and 5.0% CO ₂ Culturing in a constant temperature incubator with saturated humidity, and changing fresh culture medium according to the growth condition of cells; after about 2 weeks of culture, NK cells were obtained by flow cytometry screening.

8. Use of a bispecific antibody according to any one of claims 1-5 or a pharmaceutical composition according to claim 7 or 8 for the preparation of an antitumor drug.

9. The use according to claim 8, wherein the tumor is selected from at least one of bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphomas, leukemias, and myelomas.

10. The use according to claim 9, wherein the tumour is selected from lymphoma, leukemia and myeloma.