CN114685675A - Bispecific antibodies and their use in the treatment of cancer - Google Patents

Abstract

The invention provides a bispecific antibody and application thereof in treating cancer, comprising a first antigen binding domain specifically bound with a first antigen and a second antigen binding domain specifically bound with a second antigen, wherein the first antigen is EGFR, the second antigen is CD3, the first antigen binding domain targeting EGFR can be combined with a target antigen with high affinity, the targeting property of tumor cell recognition is improved, and the second antigen binding domain targeting CD3 selects a single-domain antibody structure with medium and strong affinity, so that the bispecific antibody not only can effectively mediate T cell immune response, but also can prevent the bispecific antibody from gathering in T cell-enriched tissues such as spleen, lymph node and the like in vivo to influence an anti-tumor effect; the introduction of 4-1BB co-stimulation factor into the structure of the bispecific antibody enables effector cells to be activated through CD3 and 4-1BB dual signaling pathways, and a greater anti-tumor effect is exerted; in addition, the bispecific antibody provided by the invention can also reduce the secretion of certain immune factors in the treatment process, and is helpful for reducing toxic and side effects.

Description

Bispecific antibodies and their use in the treatment of cancer

The technical field is as follows:

the invention belongs to the field of tumor immunotherapy, and particularly provides a bispecific antibody and application thereof in cancer treatment.

Background art:

cancer is the third leading cause of death in humans following cardiovascular disease and infectious disease, with one-fourth of deaths in developed countries being caused by cancer. Although the intense research by researchers over decades has greatly facilitated a profound understanding of cancer biology, genomics, proteomics, and prospective therapies, with many positive outcomes, even though modern cancers remain fatal, 1700 thousands of new cases and 950 thousands of deaths in 2020, and new cancer cases are expected to rise to the staggering 2750 thousands worldwide by 2040, highlighting the urgency and necessity to develop new cancer diagnostic and therapeutic approaches. Current cancer treatments are dominated by surgery, radiation therapy and chemotherapy, but traditional treatment modalities are limited for the following reasons: (1) failure to cross biological barriers, (2) non-specific side effects, (3) minimal impact on metastatic tumors, and (4) lack of effective diagnostic/therapeutic screening procedures. Cancers can be classified into hematological tumors and solid tumors according to their occurrence sites and physiological characteristics, and immunotherapy represented by chimeric antigen receptor T-cells (CAR-T cells) has achieved great success in hematological tumors, but unlike hematological tumors, a large amount of fibrous matrix and immunosuppressive cells are present in the solid tumor microenvironment, and at the same time, tumor tissues are protected by physical barriers and immune barriers, and attack by immune cells is resisted. In addition to the barrier, the peripheral of the solid tumor is often accompanied with vascular malformation and fibrous connective tissue hyperplasia to form a microenvironment with hypoxia, acidity and lack of essential amino acids (arginine, tryptophan and the like), and infiltration of T cells in the microenvironment is difficult to survive and activate, so that an ideal tumor killing effect is difficult to achieve.

The bispecific antibody (BsAb, double antibody for short) means that one antibody molecule can be combined with two different antigens or two different epitopes of the same antigen, the concept of double antibody is proposed in the last 60 th century, but due to the limitation of bioengineering technology and genetic technology, breakthrough progress is not achieved until the last decade, and the European medical administration approved double antibody cataxomab targeting EpCAM and CD3 for malignant ascites treatment in 2009 becomes the first approved double antibody drug; in 2014, the U.S. food and drug administration approved a dual-resistant blinatumomab targeting CD19 and CD3 for the treatment of the most common philadelphia chromosome negative relapsing or refractory precursor B cell acute lymphoblastic leukemia (BCP-ALL) of acute lympholeukemias (ALL), followed by further expansion to include philadelphia chromosome positive relapsing or refractory BCP-ALL. It is worth noting that besides the traditional antibodies with light chain and heavy chain structures, bispecific nanobodies (BsNb) have been developed, which have the characteristics of stronger specificity, targeting property and lower off-target toxicity, and have enhanced binding force with target antigen and prolonged serum half-life, so that it has become a research hotspot in diagnosis and treatment in the fields of infection, tumor and immunity.

Leukocyte differentiation cluster 3 (CD 3) on the surface of T cells can mediate T cell activation and recruit T cells to the periphery of tumor target cells, making CD3 bispecific antibodies (CD3-BsAbs) an emerging therapeutic modality in the field of cancer immunotherapy. CD3-BsAbs act by binding both Tumor Associated Antigen (TAA) expressed on tumor cells and CD3 on T cells, CD3-BsAbs cross-linking these two cell types allowing the formation of immunological synapses, similar to native T Cell Receptor (TCR)/peptide-Major Histocompatibility Complex (MHC) complexes, which are capable of both specifically binding to tumor target cells and efficiently inducing T cell activation leading to the secretion of inflammatory cytokines and cytolytic molecules, which are capable of killing tumor cells in the process, thereby exerting multiple anti-tumor effects. CD3-BsAb therapy is a passive form of immunotherapy with similar affinity to adoptive cell therapy of T cells expressing chimeric antigen receptor transgenes, and CARs consist of a TAA binding domain directly linked to the intracellular CD3 zeta chain and from a costimulatory receptor (e.g., 4-1BB) to activate T cells upon antigen recognition. CD3-BsAbs and CAR T cells are similar in many respects: both are directed against surface TAAs, both exploit T cell effector functions, and both are successfully used in the clinical treatment of hematological malignancies and exhibit similar types of toxicity profiles. However, some of the disadvantages of the current clinically approved CAR-T cells compared to CD3-BsAbs are: (1) patients need to undergo lymphoscavenging prior to infusion of CAR-T cells, (2) CAR-T cells must be produced individually for each patient, while CD3-BsAbs can serve as a ready-to-use, large-scale therapeutic, (3) CAR-T cells remain in the patient after tumor clearance, resulting in continued B cell depletion in the presence of CAR-T cells targeted to CD19, while CD3-BsAbs clear from the blood over time. Therefore, the CD3-BsAb has more clinical use advantages compared with CAR-T cells.

The Epidermal Growth Factor Receptor (EGFR), also known as HER1 or ErbB1, is an ErbB family member consisting of HER2(ErbB2), HER3(ErbB3) and HER4(ErbB 4). EFGR is a 170kDa transmembrane receptor comprising 3 domains, including the extracellular, transmembrane and intracellular domains, where the extracellular domain can recognize and bind the corresponding ligand, the intracellular domain has tyrosine kinase activity, once activated EGFR forms homo-or heterodimers with other ErbB family members, then phosphorylates tyrosine kinase and activates downstream signaling pathways, such as RAS-RAF-MEK-ERK, JAK-and STAT PI3K-AKT-mTOR, which ultimately lead to tumor development and progression. Therefore, EGFR is a promising target for tumor therapy, and researchers have developed a variety of EGFR-targeting antibody drugs for the treatment of solid tumors, including: (1) cetuximab, approved by the FDA for the treatment of EGFR-positive advanced colon cancer in 2004, was the first FDA-approved drug for EGFR mab, a human/murine chimeric IgG1 monoclonal antibody that competes with endogenous ligands for binding to the EGFR extracellular domain with high affinity and also induces ADCC to kill tumor cells; (2) panitumumab, the first fully humanized IgG2 monoclonal antibody, has a mechanism of action similar to cetuximab, and is approved by FDA for treatment of metastatic colorectal cancer after chemotherapy failure in 9 months in 2006, and approved by EMEA for treatment of K-ras wild-type colorectal cancer in 12 months in 2007; (3) nimotuzumab is the first EGFR monoclonal antibody drug approved and introduced in China for treating malignant tumors, and in 2008, CFDA is approved for treating EGFR expression positive stage III/IV nasopharyngeal carcinoma by combining radiotherapy or chemotherapy; (4) gaxistabu, a second generation recombinant human IgG1 EGFR antibody, was FDA approved for first-line treatment of metastatic squamous non-small cell lung cancer with gemcitabine, cisplatin in 2015. Although bispecific antibodies targeting EGFR and CD3 have been reported, such as WO2021104430a1, CN111848806A, CN113874396A, etc., they still face the problems of insufficient targeting to solid tumors, and to be improved in tumor killing activity.

The application provides a novel bispecific antibody targeting EGFR and CD3, which can efficiently identify tumor cells, effectively recruit T cells and play a strong role in killing tumors; the bispecific antibody adopts a form of combining a traditional antibody structure and a nano antibody structure, can improve the quality of effector cells, possibly reduce the risk of an immune factor storm, and improve the safety and the effectiveness of treatment; the bispecific antibody has a certain broad spectrum in the aspect of treating solid tumors, has a killing effect on various solid tumor cells, and provides a new way for developing corresponding antitumor drugs.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a bispecific antibody comprising: a first antigen-binding domain that specifically binds a first antigen and a second antigen-binding domain that specifically binds a second antigen; the first antigen is EGFR, the first antigen binding domain comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2, HCDR3 shown as SEQ ID NO. 3, LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5, and LCDR3 shown as SEQ ID NO. 6; the second antigen is CD3, and the second antigen binding domain is a single-domain antigen binding domain and comprises a CDR1 shown as SEQ ID NO. 7, a CDR2 shown as SEQ ID NO. 8 and a CDR3 shown as SEQ ID NO. 9; the second antigen binding domain is linked to a co-stimulatory factor selected from at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D, and B7-H3.

In the invention, an EGFR target is selected to be matched with a CD3 target, the antibody has a brand new CDR (complementary deoxyribonucleic acid) region, wherein a first antigen binding domain targeting EGFR can be combined with a target antigen with high affinity to improve the targeting property of tumor cell recognition, and a second antigen binding domain targeting CD3 selects a single-domain antibody structure with high and high affinity, so that the T cell immune response can be effectively mediated, and the bispecific antibody can be prevented from being accumulated in tissues enriched with T cells such as spleen, lymph nodes and the like in vivo to influence the anti-tumor effect; the 4-1BB co-stimulation factor is introduced into the structure of the bispecific antibody, so that effector cells can be activated through a CD3 and 4-1BB dual signaling pathway to exert a greater anti-tumor effect; in addition, the bispecific antibody provided by the invention can also reduce the secretion of immune factors in the treatment process, and is beneficial to reducing toxic and side effects.

Further, the first antigen binding domain comprises the heavy chain variable region as set forth in SEQ ID NO 10.

Further, the first antigen binding domain comprises the light chain variable region as set forth in SEQ ID NO. 11.

Further, the second antigen binding domain includes a single domain variable region as shown in SEQ ID NO 12.

Further wherein the bispecific antibody is an IgG1 antibody.

Further, the costimulatory factor is 4-1BB, and the amino acid sequence of the costimulatory factor is shown as SEQ ID NO. 13.

A nucleotide encoding the bispecific antibody is provided.

A pharmaceutical composition comprising the bispecific antibody is provided.

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

Further, the tumor is selected from liver cancer, lung cancer or colorectal cancer.

EGFR is a target which is widely expressed in malignant tumors and widely expressed in various solid tumors, so that the bispecific antibody provided by the invention can be used for liver cancer, lung cancer, non-small cell lung cancer, breast cancer, lymph cancer, colon cancer, kidney cancer, urothelial cancer, prostate cancer, pharyngeal cancer, rectal cancer, renal cell cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma on skin or eyes, uterine cancer, ovarian cancer, colorectal cancer, cancer of anal region, peritoneal cancer, stomach cancer, esophageal cancer, salivary gland cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, malignant glioma, neuroblastoma, cervical cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, cancer of endocrine system, cancer of liver, kidney cancer, colon cancer, kidney cancer, colon cancer, kidney cancer, colon cancer, lung cancer, colon cancer, lung, Treatment of solid tumors such as thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system tumor (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid carcinoma, squamous cell carcinoma; preferably used for liver cancer, lung cancer or colorectal cancer.

Advantageous effects

The invention provides a novel bispecific antibody targeting EGFR and CD3, which has a brand-new CDR region, wherein a first antigen binding domain targeting EGFR can be combined with a target antigen with high affinity, so that the targeting property of tumor cell recognition is improved, and a second antigen binding domain targeting CD3 selects a single-domain antibody structure with high and high affinity, so that the novel bispecific antibody not only can effectively mediate T cell immune response, but also can prevent the bispecific antibody from being gathered in tissues enriched with T cells such as spleen, lymph nodes and the like in vivo to influence the anti-tumor effect; the introduction of 4-1BB co-stimulation factor into the structure of the bispecific antibody enables effector cells to be activated through CD3 and 4-1BB dual signaling pathways, and a greater anti-tumor effect is exerted; in addition, the bispecific antibody provided by the invention can reduce excessive secretion of immune factors in the treatment process, and is helpful for reducing toxic and side effects.

Drawings

FIG. 1: schematic representation of bispecific antibody structure;

FIG. 2: killing effect of the bispecific antibody on Huh-7 cells;

FIG. 3: killing effect of bispecific antibody on A549 cell;

FIG. 4: killing of HCT-15 cells by bispecific antibodies;

FIG. 5: the effect of bispecific antibodies on effector cell perforin secretion;

FIG. 6: changes in tumor volume in animals;

FIG. 7 is a schematic view of: a graph of changes in the expression level of TNF- α;

FIG. 8: IL-6 expression level change profile.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagent biomaterials, test kits, if not specifically indicated, are commercially available.

Example 1CD3 Single Domain antibody preparation

Selecting healthy adult alpaca, uniformly mixing a recombinant human CD3 antigen (stored in a laboratory) and Freund's complete adjuvant according to the proportion of 1:1, injecting immune alpaca to the back of the patient at multiple points subcutaneously for four times, wherein the immune interval is 2 weeks. After the immunization is successful, 10mL of alpaca peripheral blood is collected and used for constructing a phage display library.

Separating lymphocytes from the collected alpaca peripheral blood by using a lymphocyte separation kit (Sigma-Aldrich); take 1X 10⁷Extracting total RNA from each cell by using a Trizol method, wherein the method comprises the following steps: to an EP tube containing lymphocytes, 1ml of trizol (purchased from Sigma) was added, repeatedly whipped, and left on ice for 5 minutes; adding 250 mu L of chloroform, swirling for 30 seconds, and then placing on ice for 5 minutes; centrifuging at 4 ℃ and 12000g for 15 minutes, sucking the water phase and transferring into a new EP tube; adding equal amount of isopropanol, and standing on ice for 10 min; centrifuging at 12000g for 10min at 4 ℃, and removing supernatant; washing with 1mL of precooled 70% ethanol, centrifuging at 4 ℃ and 7500g for 5 minutes, discarding the supernatant and drying for 5 minutes; adding 30 μ L RNase-free water to dissolve the precipitate to obtain total RNA. The total RNA was used as a template, and was converted into cDNA using a reverse transcription kit (purchased from Roche Co.), followed by amplification and enrichment by two-round PCR reactionsThe cDNA is collected, and Pst I and Not I enzyme cutting sites are introduced at two ends of the product. The target nucleic acid molecule is connected to a pMECS vector through enzyme digestion and connection reaction, the vector carrying the target nucleic acid is transformed into escherichia coli DH5 alpha competent cells through an electric transformation method, and positive clones are screened through a PCR method and stored in a refrigerator at the temperature of 20 ℃ below zero.

Collecting the frozen competent cells, recovering, inoculating to YT-AG culture medium, culturing to logarithmic growth phase, adding VCSM13 bacteriophage, standing at 37 deg.C, and infecting for 60 min; centrifuging at room temperature 4000rpm for 10min, discarding supernatant, resuspending thallus with YT-AK culture medium containing ampicillin and kanamycin, and culturing at 37 deg.C 200rpm overnight; centrifuging, placing the supernatant in a 50mL centrifuge tube, adding PEG/NaCl (20%/2.5M) solution, mixing thoroughly, standing at room temperature for 5min, centrifuging at 4000rpm, discarding the supernatant, washing the precipitate with 4 deg.C precooled PBS, and centrifuging; the phage titer is tested, and the requirements of further experiments are met.

Screening positive clones by an ELISA method; electrically transforming the screened positive clone into Escherichia coli HB2151, coating on LB culture plate containing ampicillin and glucose, and culturing at 37 deg.C overnight; and (2) selecting a monoclonal antibody, inoculating the monoclonal antibody into a liquid LB culture solution containing ampicillin, carrying out shake culture at 37 ℃ overnight until the OD600nm value reaches above 0.6, adding 1M IPTG, carrying out shake culture at 28 ℃ for 12H, centrifuging to collect escherichia coli, carrying out ultrasonic method to break thalli, purifying the antibody by a nickel column affinity chromatography method, and carrying out co-screening to obtain 6 required targeting CD3 single-domain antibodies such as 1A2, 1B7, 2C5, 3D6, 3E3 and 5H 9.

The affinity of the above-mentioned single-domain antibody to human CD3 was measured using a Fortebio biomacromolecule interactor (obtained from eisi bio, usa), and the results are shown in table 1:

TABLE 1 affinity detection of Individual Single Domain antibodies to target antigens

Single domain antibodies	Affinity (nM)
		1A2	3.17E-07
1B7	6.67E-06
		2C5	5.82E-07
3D6	3.72E-08
		3E3	3.55E-09
5H9	4.19E-09

It has been reported that in the CD3-BsAb double antibody, the affinity of the antigen binding domain targeting CD3 and the Target antigen CD3 should not be too high, otherwise, CD3-BsAb would be excessively enriched in T Cell-rich tissues such as spleen and lymph node, and the binding of CD3-BsAb to Tumor cells would be affected (see Mandiian, D.et al., relative Target affinity of T-Cell-Dependent Bispecific Antibodies derivative biological distribution a Solid Tumor model. mol. cancer Ther.2018,17, 776785). In the previous development of bispecific antibodies against blood tumors based on targets such as CD19, CD20 and the like, 2C5 single-domain antibodies with moderate affinity are selected, and good anti-tumor effects are achieved in vivo and in vitro experiments. However, solid tumors differ greatly from the immune environment in blood and the tumor microenvironment, on the one hand, the availability of effector cells in solid tumors is low, and for hematological malignancies, cancer cells in blood are surrounded by T cells, so that CD3-BsAb can be extracted from an endless pool of effector cells, while solid tumors require T cell infiltration for therapeutic effect, which results in the possible occurrence of "immune deserts" (Lanitis, e.e. al. mechanisms regulating T-cell infiltration and activity in soluble tumors. ang. oncol.2017,28), the reduction or lack of sufficient effector cells at the tumor site, resulting in very few tumor-specific triggering T cells being attributed to the tumor, and the possible occurrence of "immune rejection" (see Kuczek, d.e. al. genes differentiation, tumor-specific targeting of tumor, tumor-invasion T-cell matrix j.68, cancer-infiltration T-cell j.2019, cancer-infiltration t.2017, rather than tumor nests, i.e., effector cells present at unintended sites; on the other hand, the quality of effector cells is also affected, causing immune Cell Dysfunction, impaired ability to proliferate and produce cytolytic molecules, including granzymes and perforins, to exhibit some tumor resistance (see Thommen, D.S.; Schumacher, T.N.T. Cell Dysfunction in cancer. cancer Cell 2018,33, 547-. In order to cope with more complex tumor microenvironment of solid tumors and weakened immune environment, the invention selects the antigen binding domain of the 3D6 antibody with higher affinity strength to form the bispecific antibody in the medium affinity antibody, which can ensure effective recruitment and activation of beneficial cells such as T cells, maintain relatively medium affinity, prevent the bispecific antibody from converging in non-targeting parts such as spleen, lymph and the like, and improve the anti-tumor effect.

The sequencing results of the 3D6 clone strain were analyzed for antibody light and heavy chain genes using the sequence alignment software Vector NTI to determine the Framework Regions (FRs) and Complementarity Determining Regions (CDRs) of the variable Regions. Through identification, the CDR regions of the 3D6 clone strain are shown as CDR1 in SEQ ID NO. 7, CDR2 in SEQ ID NO. 8, CDR3 in SEQ ID NO. 9, and the variable region is shown as SEQ ID NO. 12.

Example 2 design and preparation of bispecific antibodies

2.1 design of bispecific antibodies

Conventional structural antibodies targeting EGFR and CD3 were stored by the laboratory and their nucleic acid sequences were cloned into PTT5 vector, where the EGFR targeting antibody has high affinity for the target antigen with HCDR1 shown in SEQ ID No. 1, HCDR2 shown in SEQ ID No. 2, HCDR3 shown in SEQ ID No. 3, LCDR1 shown in SEQ ID No. 4, LCDR2 shown in SEQ ID No. 5, and LCDR3 shown in SEQ ID No. 6; and has a heavy chain variable region as shown in SEQ ID NO. 10 and a light chain variable region as shown in SEQ ID NO. 11.

Bispecific antibodies were constructed in this example using a traditional antibody targeting CD3 and a single domain antibody, respectively, as shown in figure 1, in which EGFR-CD3VH/VL BsAb has a traditional antibody structure (as shown in figure 1A), binding to EGFR and CD3 targets, respectively; EGFR-CD3VHH BsAb (shown in figure 1B) has a traditional antibody structure targeting EGFR and a single domain antibody structure targeting CD3, and the single domain antibody has medium and strong affinity to the target antigen; further, in order to improve the quality of the benefit cells, by using the method of introducing the costimulatory factor into the CART cells, the 4-1BB costimulatory factor is introduced into the EGFR-CD3VHH BsAb so as to improve the activation degree of the T cells in the vicinity of the solid tumor, and the amino acid sequence of the 4-1BB costimulatory factor is shown as SEQ ID NO. 9.

2.2 preparation of bispecific antibodies

Introducing DNA sequences targeting EGFR and CD3 antibodies into a pcDNA3.3 expression vector, wherein the expression vector contains a constant region of human IgG1, and constructing an EGFR-CD3VH/VL BsAb expression vector; the EGFR targeting antibody DNA sequence, CD3 targeting single domain antibody DNA sequence and 4-1BB co-stimulatory factor DNA sequence are introduced into pcDNA3.3 expression vector, and flexible joints (GGGGS) are adopted between different parts₃And connecting the expression vector containing a constant region of human IgG1 to construct an EGFR-CD3VHH BsAb expression vector. The expression vector was electrically transformed into E.coli competent cell DH 5. alpha. and spread on LB plate containing antibiotic after transformation, and cultured overnight at 37 ℃ to select positive clones. Plasmids were extracted and sequenced to show that the bispecific antibody sequences were correct.

The expression plasmids were co-transfected into Expi293 cells using the Expi293 expression system kit (purchased from thermo fisher), 5 days after transfection, the supernatants were collected and the antibodies were purified by nickel column affinity chromatography to obtain EGFR-CD3VH/VL BsAb and EGFR-CD3VHH BsAb bispecific antibodies, respectively. Antibody purity was checked by HPLC-SEC with 94.5% for EGFR-CD3VH/VL BsAb and 95.6% for EGFR-CD3VHH BsAb.

Example 3 bispecific antibody in vitro anti-tumor experiments

3.1 efficient cell isolation and culture

Human PBMC cells were isolated using Ficoll density gradient centrifugation: 10mL of fresh human peripheral blood is extracted and mixed with 10mL of serum-free RPMI1640 medium, and the mixture is slowly added to the upper layer of 10mL of density gradient centrifugation liquid Ficoll; centrifuging at 12000rpm for 10min at room temperature; taking out the centrifuge tube, discarding the upper plasma layer, carefully sucking white layer PBMC between the plasma and the Ficoll, and placing the white layer PBMC in a 50mL centrifuge tube; adding 15mL serum-free RPMI1640 culture medium, centrifuging at 3000rpm for 10min after resuspension, and repeating the operation for 2 times; 15mL of RPMI1640 medium containing 10% serum was added thereto at 37 ℃ with 5% CO₂Culturing under the condition.

3.2 tumor cell culture

In the invention, a liver cancer cell line Huh-7, a lung cancer cell line A549 and a colorectal cancer cell line HCT-15 are selected as experimental objects, and the anti-tumor effect of the bispecific antibody is investigated. Recovering the above cells, inoculating in complete culture medium containing 10% serum, wherein the A549 cells are RPMI1640 medium, the Huh-7 and HCT-15 cells are DMEM medium, and culturing at 37 deg.C with 5% CO₂Culturing the cells under conditions to logarithmic growth phase; the tumor cells are transfected by a lentiviral vector carrying a Green Fluorescent Protein (GFP) gene, so that subsequent observation and experiments are facilitated.

3.3 in vitro antitumor experiments

Inoculating effector cells and target cells into a 96-well plate according to a ratio of 5:1, respectively adding 10ng/mL of EGFR-CD3VH/VL BsAb and EGFR-CD3VHH BsAb bispecific antibodies, taking a sterile culture medium as a control, culturing at 37 ℃ for 24h, adding 2 mu l of 7-AAD into each well, incubating at 37 ℃ for 1h, photographing by using a Perkin Elmer Opetta high content imager, and detecting the number of living cells of each group of cells; percent cell killing was calculated, concentration percent killing ═ number of viable cells in blank-number of viable cells in each concentration antibody group)/number of viable cells in blank.

As shown in fig. 2-4, the bispecific antibody provided in the present invention can effectively kill tumor cells in vitro, and the killing effect of the EGFR-CD3VHH BsAb antibody is generally better than that of the EGFR-CD3VH/VL BsAb antibody, which may be related to the addition of 4-1BB domain to the EGFR-CD3VHH BsAb antibody, which can promote the activation of effector cells, and can also be derived from the use of single domain antibody domain, which can reduce the molecular weight of the antibody, thus facilitating drug delivery and target binding. Among the three tumor cells, the EGFR-CD3VHH BsAb has more advantages on the liver cancer cell line Huh-7, and the tumor killing rate is obviously higher than that of the EGFR-CD3VH/VL BsAb. The results show that the bispecific antibody provided by the invention can effectively kill tumor cells of liver cancer, lung cancer, colorectal cancer and the like, and provides support for development of related clinical drugs.

3.4 secretion of anti-tumor factors by Effector cells

Selecting a hepatoma cell line Huh-7 to be co-cultured with effector cells at a ratio of 1:5, adding 10ng/mL EGFR-CD3VH/VL BsAb and EGFR-CD3VHH BsAb bispecific antibody, taking a sterile culture medium as a control, culturing at 37 ℃ for 24h, collecting cell supernatant, detecting the perforin concentration in the cell supernatant by using an ELISA kit (purchased from Abcam company of America), and carrying out the specific steps according to the kit instructions.

As shown in FIG. 5, perforin secretion by effector cells was low, whereas with the bispecific antibody provided by the present invention, perforin levels were elevated, which was more pronounced in EGFR-CD3VHH BsAb, probably due to increased T cell activation and induction of large perforin secretion by dual stimulation with CD3 and 4-1 BB.

Example 4 bispecific antibody in vivo anti-tumor experiments

In order to further verify the in vivo anti-tumor effect of the bispecific antibody provided by the invention, Huh-7 is selected and adopted to construct an animal model in the section and study the in vivo anti-tumor effect.

4.1 animal model preparation and treatment

Adopting C57BL/6 mice, 6-8 weeks old, and breeding experimental animals in SPF-level constant temperature and humidity room for one week; culture of Huh-7 fineAdjusting cell concentration to 1 × 10⁷one/mL, the right flank hairs of the C57BL/6 mice were shaved off, and 100. mu.L of the cell suspension was injected subcutaneously into the right anterior flank of the mice. The growth of the tumors was observed daily, and when the tumor diameter reached between 3mm and 5mm, the experimental animals were randomly divided into three groups, and EGFR-CD3VH/VL BsAb antibody (5mg/kg), EGFR-CD3VHH BsAb antibody (5mg/kg) and an equal volume of physiological saline were injected every 3 days for 4 times.

4.2 tumor volume detection

The first day of administration of the experimental animals was taken as day 0, after which tumor volumes were not measured every 3 days for a total of 30 days. Tumor size, tumor volume (L x W) was measured using a vernier caliper²) The/2 estimate, where L is the length or longest dimension and W is the width of the tumor.

The results are shown in fig. 6, after about 2 weeks of treatment, the bispecific antibody treatment group showed a significant trend of tumor volume reduction, indicating that the bispecific antibody provided by the present invention can significantly inhibit the tumor growth process in vivo; at the later stages of treatment, the therapeutic effect of the EGFR-CD3VHH BsAb antibody was initially significantly better than that of the EGFR-CD3VH/VL BsAb antibody, suggesting that the EGFR-CD3VHH BsAb antibody is more effective in vivo tumor treatment. In previous experiments, the inventor finds that the half-life of the bispecific antibody in plasma can be improved by using a single-domain antibody compared with the traditional antibody, the recruitment degree and quality of immune effector cells can be improved by using the EGFR-CD3VHH BsAb provided by the invention which also carries a 4-1BB domain capable of promoting T cell activation, and cytological experiments prove that the perforin secretion can be promoted by using the corresponding bispecific antibody, and the immunity of the organism is limited by contacting with a tumor microenvironment to a certain extent.

4.3 detection of the concentration of immune factors in serum

After 4 weeks of administration, the orbital veins of the mice were bled, centrifuged at 3000r/min for 15min, and the supernatant was collected and the TNF-. alpha.and IL-6 contents in the serum were measured using an ELISA kit (purchased from Dr. Wuhan, Dride bioengineering, Ltd.) according to the procedures described in the specification.

TNF-alpha and IL-6 are reported to be one of the main cytokines in immune factor storm caused by tumor immunotherapy, and although the action mechanism of TNF-alpha and IL-6 on tumors is still controversial, it is basically determined that if the factors are released in a large amount in a short period, the factors can cause excessive stress of the immune system, damage normal tissues and organs and cause serious adverse reactions such as fever, malignancy, syncope, organ failure and the like, so how to manage and control the immune factor storm occurring in the tumor immunotherapy becomes a problem which must be carefully treated and solved in the tumor therapy. As shown in FIGS. 7 and 8, the expression levels of TNF-alpha and IL-6 are increased to different extents after the bispecific antibody provided by the invention is injected into animals, which is probably related to T cell activation and participating in tumor immune process, and the secretion level of immune factors is increased; the rising trend of TNF-alpha in the EGFR-CD3VHH BsAb treatment group is obviously inhibited, but no obvious difference is found between EGFR-CD3VHH BsAb and EGFR-CD3VH/VL BsAb in IL-6 secretion, which indicates that the interleukin level still needs to be effectively monitored and managed during the treatment process of using the bispecific antibody, and unacceptable adverse reactions are avoided.

While this invention has been particularly shown and described with references 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.

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Asn Ser Met Ile Asp

1 5

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Tyr His Thr Ser Val Gly Tyr Trp Asp Leu Ile Asn

1 5 10

<210> 6

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Tyr Leu Cys Gly Asp Val Val Glu Pro Asn Ser Ser Leu Glu Gln

1 5 10 15

Thr

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Arg Thr Glu Asp Asp Trp Ile

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Trp Ile Thr Gly Leu Ser Thr Lys Ile

1 5

<210> 9

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ala Glu His Ala Ser Lys Val Val Glu Val Ser Thr Ser

1 5 10

<210> 10

<211> 129

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Thr Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser

20 25 30

Leu Glu Ser Ile Tyr Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu

35 40 45

Trp Val Ala Thr Thr Ala Asp Glu Ser Cys Pro Asn Lys Glu Glu Tyr

50 55 60

Cys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Val Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Leu Asp Phe Thr Ala Tyr Ile Gln Ile Ser Pro Gly Met

100 105 110

Glu Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Thr

115 120 125

Ser

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Thr Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Asp Asn Ser Met Ile Asp Trp Tyr Gln

20 25 30

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Tyr His Thr

35 40 45

Ser Val Gly Tyr Trp Asp Leu Ile Asn Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Glu Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Tyr Leu Cys Gly Asp Val

85 90 95

Val Glu Pro Asn Ser Ser Leu Glu Gln Thr Phe Gly Gly Gly Thr Lys

100 105 110

Val Glu Ile Lys Ser Ser

115

<210> 12

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Val Lys Leu Glu Glu Ser Gly Ser Glu Leu Val Gln Pro Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Ala Asn Ile Phe Ser Arg Thr

20 25 30

Glu Asp Asp Trp Ile Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Ala

35 40 45

Phe Val Ala Trp Ile Thr Gly Leu Ser Thr Lys Ile Gly Arg Phe Thr

50 55 60

Ile Ser Val Asp Lys Ser Lys Gly Thr Ile Tyr Leu Gln Met Asn Ala

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Ala Asn Ala Ala Glu His

85 90 95

Ala Ser Lys Val Val Glu Val Ser Thr Ser Trp Gly Gln Gly Thr Val

100 105 110

Val Thr Val Ser Ser

115

<210> 13

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

Claims (10)

1. A bispecific antibody comprising: a first antigen-binding domain that specifically binds a first antigen and a second antigen-binding domain that specifically binds a second antigen; the first antigen is EGFR, the first antigen binding domain comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2, HCDR3 shown as SEQ ID NO. 3, LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5, and LCDR3 shown as SEQ ID NO. 6; the second antigen is CD3, and the second antigen binding domain is a single-domain antigen binding domain and comprises a CDR1 shown as SEQ ID NO. 7, a CDR2 shown as SEQ ID NO. 8 and a CDR3 shown as SEQ ID NO. 9; the second antigen binding domain is linked to a co-stimulatory factor selected from at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D, and B7-H3.

2. The bispecific antibody of claim 1, wherein the first antigen-binding domain comprises the heavy chain variable region as set forth in SEQ ID NO 10.

3. The bispecific antibody of claim 1, wherein the first antigen-binding domain comprises the light chain variable region as set forth in SEQ ID NO. 11.

4. The bispecific antibody of claim 1, wherein the second antigen-binding domain comprises a single domain variable region as set forth in SEQ ID NO 12.

5. The bispecific antibody of claim 1, wherein the bispecific antibody is an IgG1 antibody.

6. The bispecific antibody of claim 1, wherein the co-stimulatory factor is 4-1BB, the amino acid sequence of which is shown in SEQ ID NO 13.

7. A nucleotide encoding the bispecific antibody of any one of claims 1-6.

8. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-6.

9. Use of the bispecific antibody of any one of claims 1 to 6 or the nucleotide of claim 7 or the pharmaceutical composition of claim 8 for the preparation of an anti-tumor medicament.

10. The use according to claim 9, wherein the tumor is selected from liver cancer, lung cancer or colorectal cancer.

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