CN115029318A

CN115029318A - Mesenchymal stem cell carrying multi-specific antibody gene and pharmaceutical application thereof

Info

Publication number: CN115029318A
Application number: CN202210518596.9A
Authority: CN
Inventors: 余亚杰; 窦欣童
Original assignee: Guangzhou Mingzheng Biotechnology Co ltd
Current assignee: Chengdu Fuzhi Future Technology Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-09-09
Anticipated expiration: 2042-05-12
Also published as: CN115029318B

Abstract

The application provides a mesenchymal stem cell carrying a multi-specificity antibody gene and pharmaceutical application thereof, the multi-specificity antibody adopts a single-domain antibody structure, the molecular mass of the antibody can be reduced, the drug delivery and the in vivo absorption are facilitated, and the construction and expression difficulty of a vector can be reduced; the antibody structure domain targeting EGFR and the antibody structure domain targeting CD3 are arranged in a ratio of 2:1, so that the targeting recognition capability aiming at the EGFR antigen on the surface of tumor cells is improved, and the antibody can be prevented from being gathered at a non-specific part; a costimulatory factor structural domain is arranged in the antibody, and double-way activation of T cells can be realized through CD3 and costimulatory factors, so that the anti-tumor curative effect is improved; MSC is taken as a carrying carrier, so that the blood brain barrier can be effectively broken through in vivo, the MSC reaches a focus part and exerts an anti-tumor effect, and the MSC also carries PEDF gene, so that the anti-tumor effect is further strengthened; can also promote IFN-gamma expression, and the IL-6 expression level is not obviously changed, thereby inhibiting the over secretion of immune factors and providing a foundation for the development of nervous system tumor medicaments.

Description

Mesenchymal stem cell carrying multi-specific antibody gene and pharmaceutical application thereof

The technical field is as follows:

the invention belongs to the field of tumor immunotherapy, and particularly provides mesenchymal stem cells carrying a multi-specific antibody gene and pharmaceutical application thereof.

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.

Among the numerous tumors, glioma is the most common aggressive primary central nervous system tumor in adults, which poses a serious economic burden worldwide. The standard treatment for malignant gliomas is surgical resection and post-operative radiotherapy, followed by adjuvant chemotherapy in combination with temozolomide, which rarely achieves complete resection due to the aggressive growth characteristics of malignant gliomas. Current approaches to treat glioblastoma have been developed, for example, the use of fluorescence guided techniques such as 5-aminolevulinic acid (5-ALA) during surgery, which can increase resection rates but not overall survival of patients with glioblastoma; since the 1950 s, physicians have begun radiation therapy for intracranial tumor patients, but the technology has progressed slowly; temozolomide is the most common and most effective chemotherapeutic drug for patients with glioblastoma, but the results depend on the promoter methylation status of 6-methylguanine methyltransferase (MGMT). In addition, the complex characteristics of malignant gliomas lead to poor prognosis, with median survival rates of the worst-case glioblastoma of only 14.6 months(see

RP, Zong H. Inflation and formalism, biological communication at early and late stages of tumor progression. current Pathiobiol Rep.2013; 1(1):19-28.). In recent years, with the deep understanding of glioma biological characteristics, more and more precise targeted therapies have been generated, including monoclonal antibodies, multispecific antibodies, siRNA, chimeric antigen receptor T cells, and the like.

The difficulties encountered in the treatment of malignant glioma mainly come from two aspects, one is that malignant glioma is mostly generated in the nervous system and can be divided into astrocytoma, medulloblastoma, glioblastoma multiforme, ependymoma, oligodendroblastoma and the like according to the pathology, and due to the existence of blood brain barrier, the conventional medicine is difficult to reach the focus part, so that the curative effect is poor; secondly, the immune microenvironment of glioma is complex, and glioma releases soluble factors which can inhibit immunity and support tumor into the microenvironment, thus leading to the accelerated proliferation, invasion and immune escape of cancer.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that have all the commonalities of stem cells, including self-renewal capacity and multipotentiality. The mesenchymal stem cells are clinically applied to the aspects of solving various blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, repair of partial resection injury of knee joint meniscus, autoimmune diseases and the like. Up to now, the us FDA has approved nearly 60 clinical trials with MSCs, mainly including: (1) hematopoietic stem cell transplantation, which enhances the hematopoietic function, promotes the transplantation of the hematopoietic stem cell graft and treats graft-versus-host disease; (2) repair of tissue damage, such as bone, cartilage, joint damage, heart damage, liver damage, spinal cord damage, and neurological diseases; (3) autoimmune diseases, such as systemic lupus erythematosus, scleroderma, inflammatory enteritis, etc.; (4) as a vector for gene therapy. Mesenchymal stem cells are widely considered to be of low immunogenicity, enabling them to cross Major Histocompatibility Complex (MHC) barriers. Although MSC is not immune-privileged, it is considered to be immune-evasive and largely undetected by the immune system, MSC has the innate ability to migrate remotely, cross the blood-brain barrier and communicate with surrounding immune cells, all making them ideal "trojan horses" for brain cancer treatment. In this regard, a number of research results have been reported, for example, MSC carries different effector genes such as an immune factor gene, an oncolytic virus, a suicide gene, etc. to treat brain tumors.

Pigment Epithelium Derived Factor (PEDF) is one of the effective anti-tumor effector factors, is glycoprotein secreted by 50kDa, can activate a Fas/FasL pathway to induce endothelial cell death and regulate the balance between an inducer and an angiogenesis inhibitor, and plays an important role in angiogenesis and tumorigenesis of glioma. However, the molecular mechanisms by which PEDF causes glioma cell apoptosis and antiangiogenesis are not fully understood, and it has been found by researchers that conditioned media from phosphatase and tensin homolog mRNA engineered MSCs induce U251 cell death in vitro via the PI3K-AKT-mTOR pathway.

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) in acute lympholeukemia (ALL), which was subsequently further expanded to include philadelphia chromosome-positive relapsing or refractory BCP-ALL. It should be noted that besides the traditional antibodies with light chain and heavy chain structures, bispecific nanobodies (BsNb) have been developed, and such diabodies have the characteristics of stronger specificity, targeting property, lower off-target toxicity, enhanced binding force with target antigen and prolonged serum half-life, so that they have been the research focus in diagnosis and treatment in the fields of infection, tumor and immunity. In order to further improve the therapeutic effect of antibodies, researchers have proposed the concept of multispecific antibodies (msabs) based on bispecific antibodies, which means that one antibody molecule can bind to more than two different antigens or two different epitopes of the same antigen, and thus the targeting property is enhanced.

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, and CD3-BsAbs cross-link these two cell types allowing the formation of immunological synapses, similar to the native T Cell Receptor (TCR)/peptide-Major Histocompatibility Complex (MHC) complex, 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 that can kill tumor cells in the process, thus 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 to 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 CAR-T cell infusion, (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 targeting 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 homodimers 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 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 the tumor killing activity needs to be improved, especially in the treatment of brain tumors, how to effectively break through the inhibition of blood brain barrier and tumor immune environment is becoming a hot issue in the research of brain tumors.

The application provides a mesenchymal stem cell carrying multi-specific antibody genes of targeting EGFR and CD3, the multi-specific antibody adopts a single-domain antibody structure, the molecular mass of the antibody can be reduced, the drug delivery and the in vivo absorption are facilitated, and the construction and expression difficulty of a vector can be reduced; the antibody structure domain targeting EGFR and the antibody structure domain targeting CD3 are arranged in a ratio of 2:1, so that the targeting recognition capability aiming at the EGFR antigen on the surface of tumor cells is improved, and the antibody can be prevented from being gathered at a non-specific part; a costimulatory factor structural domain is arranged in the antibody, and double-way activation of T cells can be realized through CD3 and costimulatory factors, so that the anti-tumor curative effect is improved; MSC is taken as a carrier, can effectively break through blood brain barrier in vivo, reaches a focus part, and plays an anti-tumor effect, and the MSC also carries PEDF gene, which is beneficial to further strengthening the anti-tumor effect, thereby laying a foundation for the development of novel anti-tumor drugs.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a mesenchymal stem cell characterized by carrying a multispecific antibody gene, the multispecific antibody comprising a first antigen-binding domain specifically binding to a first antigen, a second antigen-binding domain specifically binding to a second antigen, and a third antigen-binding domain specifically binding to a third antigen, the antigen-binding domain having a single domain antibody structure; the first antigen and the third antigen are both EGFR, the first antigen binding domain and the third antigen binding domain respectively comprise CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3; the second antigen is CD3, comprising CDR4 shown in SEQ ID NO. 4, CDR5 shown in SEQ ID NO. 5, and CDR6 shown in SEQ ID NO. 6; 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.

Many CD3-BsAbs are proposed in the prior art, but most of them are used for treating blood tumors such as leukemia, lymphoma, etc., and have satisfactory curative effect, but as mentioned above, the treatment of solid tumors is difficult, especially in the treatment of brain tumors, the treatment is affected by blood brain barrier and complex tumor microenvironment, and the curative effect of common drugs is greatly reduced. According to 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, 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 medium and strong affinity, so that not only can T cell immune response be effectively mediated, but also the bispecific antibody can be prevented 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; 4-1BB co-stimulation factor is introduced into a multi-specific antibody structure, so that effector cells can be activated through a CD3 and 4-1BB dual-signaling pathway, and a greater anti-tumor effect is exerted; the antibody structure domain of the targeting EGFR and the antibody structure domain of the targeting CD3 are arranged in a ratio of 2:1, so that the targeting recognition capability of the targeting EGFR antigen on the surface of tumor cells is improved; mesenchymal stem cells are used as a drug delivery carrier, so that the blood brain barrier can be smoothly broken through and enriched in tumor focus positions, and a PEDF factor is introduced, so that the anti-tumor effect is further enhanced; in addition, the cells of the invention can prevent the over-secretion of immune factors in the treatment process, and help to reduce toxic and side effects.

Further, the first antigen binding domain includes a single domain variable region as shown in SEQ ID NO. 7.

Further, the first antigen binding domain includes a single domain variable region as shown in SEQ ID NO 8.

Further, the costimulatory factor is 4-1BB, and the amino acid sequence of the costimulatory factor is shown as SEQ ID NO. 9.

Furthermore, the nucleotide sequence of the multispecific antibody is shown as SEQ ID NO. 10.

Further, the mesenchymal stem cell also carries PEDF gene, and the nucleotide sequence of the PEDF gene is shown as SEQ ID NO. 11

Further, the mesenchymal stem cells are umbilical cord Wharton jelly mesenchymal stem cells.

The mesenchymal stem cells comprise various sources, such as umbilical cords, umbilical cord blood, bone marrow and the like, wherein the Wharton jelly mesenchymal stem cells are MSC source types newly proposed in recent years, and have remarkable advantages in cell differentiation capacity, cytokine expression capacity and metabolism regulation capacity, so that the Wharton jelly mesenchymal stem cells are selected as the basis to carry out gene modification and modification so as to have higher activity of drug delivery capacity and drug safety.

A pharmaceutical composition is provided, which comprises the mesenchymal stem cell provided by the invention.

Provides an application of the mesenchymal stem cells or the pharmaceutical composition in preparing antitumor drugs.

Further, the tumor is selected from glioma and brain cancer.

EGFR is a target widely expressed in malignant tumors and widely expressed in various solid tumors, so that the mesenchymal stem cells 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, esophagus 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 the anal region, peritoneal cancer, stomach cancer, esophagus cancer, salivary gland cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, penile cancer, malignant glioma, neuroblastoma, cervical cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, esophagus cancer, small intestine cancer, cancer of endocrine system, endocrine system cancer, colon cancer, Treatment of solid tumors such as thyroid, parathyroid, adrenal, soft tissue sarcoma, urinary tract, penile, pediatric solid tumors, bladder, renal or ureteral cancer, renal pelvis, Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, neuroendocrine tumors (including carcinoid, gastrinoma, and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid carcinoma, squamous cell carcinoma; preferably used for glioma and brain cancer.

Advantageous effects

The present application provides mesenchymal stem cells carrying genes for multispecific antibodies targeting EGFR and CD3, having technical advantages in several respects, including:

(1) the multi-specificity antibody adopts a single-domain antibody structure, so that the molecular mass of the antibody can be reduced, drug delivery and in-vivo absorption are facilitated, and the difficulty of vector construction and gene expression can be reduced;

(2) the antibody structure domain of the target EGFR and the antibody structure domain of the target CD3 are arranged in a ratio of 2:1, so that the target recognition capability of the EGFR antigen on the surface of tumor cells is improved, and the antibody can be prevented from being gathered at a non-specific part;

(3) a costimulatory factor structural domain is arranged in the antibody, and double-way activation of T cells can be realized through CD3 and costimulatory factors, so that the anti-tumor curative effect is improved;

(4) MSC is used as a drug delivery carrier, can effectively break through a blood brain barrier to reach a focus part in vivo, can also utilize the physiological characteristics of MSC stem cells to enable the MSC stem cells to gather and reach a tumor part to play a targeted anti-tumor effect, and the MSC also carries PEDF genes, so that the anti-tumor effect is further enhanced;

(5) can regulate the secretion of immune factors, so that the expression of IFN-gamma is increased, the expression level of IL-6 is not obviously changed, and the over secretion of the immune factors is inhibited.

Drawings

FIG. 1: a schematic diagram of a multispecific antibody structure;

FIG. 2: killing effect of glioma cell in vitro;

FIG. 3: mean survival of in vivo glioma animal models;

FIG. 4: a profile of IFN- γ expression levels;

FIG. 5 is a schematic view of: 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 1 design and acquisition of multispecific antibodies

In previous studies, the inventors developed and obtained multispecific antibodies targeting EGFR and CD 3. The conventional bispecific antibody targeting the CD3 target at present usually only comprises an antigen binding domain targeting tumor cells and an antigen binding domain targeting CD3 of T cells, i.e. a structure of formula 1+1, which easily causes weak affinity to solid tumors, generates nonspecific binding to cause side effects, and may also cause a reduction in the amount of EGFR surface antigen due to tumor cell variation, thereby being difficult to exert a predictable anti-tumor effect. Compared with the conventional bispecific antibody comprising a CD3 target, the invention provides the multispecific antibody of the formula of '2 + 1', wherein the multispecific antibody comprises 2 antigen binding domains targeting tumor cell surface antigens and 1 CD3 antigen binding domain targeting T cells, and the antigen binding domains are single-domain antibody structures, so that the recognition capability of solid tumor cells can be improved, the operation difficulty and the carrier loading difficulty in molecular biology are reduced, and the effective delivery of a medicament is facilitated. In addition, in the method for selecting the affinity of the antigen binding domain, the antigen binding domain targeting EGFR selects the binding domain with high affinity, and the antigen binding domain targeting CD3 selects the binding domain with medium and high affinity, so that the multispecific antibody can effectively identify target cells, and can avoid excessive aggregation at non-tumor focus parts.

As shown in fig. 1, the multispecific antibody EGFR-CD3-EGFR MsAb comprising a first antigen-binding domain that specifically binds a first antigen, a second antigen-binding domain that specifically binds a second antigen, and a third antigen-binding domain that specifically binds a third antigen, the antigen-binding domains having a single domain antibody structure; the first antigen and the third antigen are both EGFR, the first antigen binding domain and the third antigen binding domain respectively comprise CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3; the second antigen is CD3, including the CDR4 shown in SEQ ID NO. 4, the CDR5 shown in SEQ ID NO. 5, and the CDR6 shown in SEQ ID NO. 6. The first antigen-binding domain and the third antigen-binding domain comprise a single domain variable region as set forth in SEQ ID NO. 7 and the second antigen-binding domain comprises a single domain variable region as set forth in SEQ ID NO. 8. Furthermore, in order to improve the quality of the benefit cells, by using the method of introducing costimulatory factors into CART cells, 4-1BB costimulatory factors are introduced into a multispecific antibody so as to improve the activation degree of T cells in solid tumors, wherein the amino acid sequence of the 4-1BB costimulatory factors is shown as SEQ ID NO. 9. The nucleotide sequence of the multispecific antibody is shown as SEQ ID NO. 10.

The DNA sequence encoding EGFR-CD3-EGFR MsAb was introduced into pcDNA3.3 expression vector, the expression vector was electrically transformed into E.coli competent cell DH5 α, and after transformation, spread on LB plate containing antibiotic, 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 plasmid was co-transfected into Expi293 cells using an Expi293 expression system kit (purchased from thermo fisher), and 5 days after transfection, the supernatant was collected and the antibody was purified by nickel column affinity chromatography to obtain EGFR-CD3-EGFR MsAb antibody, respectively. The purity of the EGFR-CD3-EGFR MsAb antibody was 95.8% as measured by HPLC-SEC.

Example 2 preparation of umbilical cord mesenchymal stem cells

Taking human umbilical cord tissue, cleaning with sterile normal saline for 3 times,removing attached blood and impurities, separating and removing arteriovenous vessels, and shearing umbilical cord tissue into small pieces of 5cm multiplied by 2cm by using sterile surgical scissors; tearing open the amniotic membrane on the umbilical cord surface, taking out all Wharton's jelly, cleaning with normal saline for 3 times, cutting Wharton's jelly into small pieces of tissue with size of about 1cm × 1cm, placing in DMEM complete culture medium (containing 10% FBS and 10ng/mL bFGF), 37 deg.C, and 5% CO ₂ Culturing; changing the culture medium every day, observing the growth condition of the cells, and carrying out subculture after the cell fusion degree reaches about 80%; cells were harvested and stored in a-80 ℃ freezer.

Example 3 genetic modification of umbilical cord mesenchymal Stem cells

According to the bioinformatics search result, the nucleotide sequence of the PEDF gene is obtained and is shown as SEQ ID NO. 11. The nucleotide sequence was inserted into a pLent-EF1 alpha expression vector (purchased from Vigene Co.) to construct a lentiviral vector carrying the PEDF gene. The MSC cells prepared in example 2 were revived to 5 × 10 ⁵ The cells/well were seeded in 6-well plates at 37 ℃ with 5% CO ₂ After overnight incubation, 5X 10 was added ⁶ Lentiviral of cfu/well, 5% CO at 37 ℃ ₂ After 48h of incubation, the cells are harvested, and the transfected PEDF-MSC cells are identified to be capable of stably expressing the PEDF factor by ELISA.

Based on a similar method, a lentivirus vector carrying EGFR-CD3-EGFR MsAb antibody nucleotide is constructed, then PEDF-MSC cells are transfected to obtain MsAb-PEDF-MSC cells capable of expressing the multispecific antibody, and the cells are identified to be capable of stably expressing EGFR-CD3-EGFR MsAb antibody.

Example 4 in vitro antitumor assay

4.1 efficient cell isolation and culture

Separating human PBMC cells by 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 medium, and centrifuging at 3000rpm for 10mi after resuspensionn, repeating the operation for 2 times; 15mL of RPMI1640 medium containing 10% serum was added at 37 ℃ with 5% CO ₂ Culturing under the condition.

4.2 tumor cell culture

In the invention, a human glioma cell line U87 is selected as an experimental object, and the anti-tumor effect on a cell level is examined. Recovering U87 cells, inoculating in DMEM complete medium containing 10% serum, and culturing at 37 deg.C under 5% CO ₂ Culturing 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.

4.3 in vitro antitumor experiments

Inoculating effector cells and target cells into a 96-well plate according to a ratio of 5:1, respectively adding 50ng/mL of EGFR-CD3-EGFR MsAb antibody, PEDF-MSC cells with the ratio of 1:1 to the target cells, MsAb-PEDF-MSC cells with the ratio of 1:1 to the target cells and sterile physiological saline (a control group), taking a sterile 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 Operetta 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, the multispecific antibody or MSC cell provided by the present invention can effectively kill tumor cells in vitro, wherein the killing efficiency of the EGFR-CD3-EGFR MsAb antibody is highest, and the cell killing rate can exceed 80%, compared with the low killing ability of the MSC cell, although the MsAb-PEDF-MSC cell carries the EGFR-CD3-EGFR MsAb antibody gene, the MSC cell does not show the expected strong anti-tumor activity in vivo. In addition, the antigen binding domain targeting EGFR and CD3 in the multispecific antibody adopts the characteristic design of 2:1 and is added with 4-1BB co-stimulation factor, so that the in vitro anti-tumor activity is consolidated and strengthened; the MSC cells are influenced by the expression efficiency of the anti-tumor genes or other intracellular regulation mechanisms, and the anti-tumor effect is slightly low.

Example 5 in vivo antitumor assay

5.1 nude mouse brain tumor model

Selecting female BALB/c nude mice, breeding for 1 week to adapt to the environment in an experiment at the room temperature of 26-28 ℃ and the air humidity of 40% -60%. Culturing U87 glioma cells to logarithmic growth phase (as in section 4.2), adjusting cell density to 2X 10 using sterile PBS ⁵ mu.L of single cells, nude mice were anesthetized and fixed on a console, and 5. mu.L of U87 cells (cell number 1X 10) were injected intracerebrally with a microinjector ⁶ One), after one week of molding, the mold is successfully molded through detection of a living body imager.

Randomly dividing the experimental animals into 4 groups and a BsAb antibody group, and injecting EGFR-CD3-EGFR MsAb antibody at a ratio of 5mg/kg in tail vein every 3 days; PEDF-MSC group, 1X 10 injections weekly ⁶ PEDF-MSC cells; MsAb-PEDF-MSC group, 1X 10 injections weekly ⁶ A plurality of MsAb-PEDF-MSC cells; control group, 0.5mL sterile saline was injected every 3 days in tail vein.

5.2 mean Life time Observation

Observing the survival condition of experimental animals every day, recording and calculating the average survival time of each group of model animals, wherein the result is shown in figure 3, the EGFR-CD3-EGFR MsAb multispecific antibody has poor anti-tumor effect in vivo experiments, is different from the antibody which can strongly inhibit the growth of glioma in vitro experiments, and hardly shows the expected anti-tumor effect in vivo experiments, which shows that the antibody is related to the fact that the antibody is difficult to effectively pass through a blood brain barrier to reach the tumor focus part in vivo; on the contrary, the MSC shows stronger tumor inhibition effect in vivo experiments, the survival time of the model animals in related treatment groups is prolonged, particularly in the MsAb-PEDF-MSC group, compared with that of a control group, the survival time of the model animals is prolonged by nearly 1 time, which shows that the MSC can reach tumor focus positions through a blood brain barrier, and the combined anti-tumor effect is realized by expressing PEDF factors and the multispecific antibody provided by the invention, and the humoral immunity mechanism and the cellular immunity mechanism are mobilized to play a synergistic anti-tumor effect.

5.3 detection of the concentration of immune factors in serum

After 3 weeks of administration, nude mouse plasma was centrifuged at 3000r/min for 15min, and the supernatant was collected and measured for IFN-. gamma.and IL-6 content in serum using ELISA kit (purchased from Dr. Bioengineering Co., Ltd., Wuhan doctor) according to the procedures described in the specification.

As shown in figure 4, the expression of IFN-gamma in the model animal body is obviously improved after MSC treatment, and the IFN-gamma is considered to be an anti-tumor factor with wide function, and can promote the activity of NKL cells, the antigen presentation and the activity of macrophage lysosome by activating immune factors such as TNF-alpha, IL-2 and the like, so as to play an anti-tumor role.

As shown in figure 5, IL-6 is also an immune factor with a complex regulation mechanism, IL-6 shows different effects at different time of tumor development, and in addition, the IL-6 is also a main effector factor in immune factor storm, if the expression level is too high, severe adverse reactions such as fever, syncope and even death can be caused, the antibody or MSC cell provided by the invention has no obvious influence on the expression of IL-6, and the treatment has a guarantee on the safety aspect and the generation mechanism of the immune factor storm is inhibited.

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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1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Ile Thr Gly Leu Ser Thr Lys Ile

1 5

<210> 6

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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

1 5 10

<210> 7

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Cys Ala Ala Ser Gly Met Ser Thr Gly Met Trp His Arg Pro Thr Pro

20 25 30

Gly Arg Glu Arg Glu Leu Ile Ala Leu Lys Ala Pro Trp Pro Gly Thr

35 40 45

Ala Pro Ser Ser Trp Arg Phe Thr Ile Ser Arg Asp Asn Ala Ser Asn

50 55 60

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

65 70 75 80

Tyr Phe Cys His Ala Gly Gly Leu Leu Glu Trp Glu Leu Leu Val Val

85 90 95

Ala Ala Ser Trp Gly Gln Gly Thr Val Ser Ser

100 105

<210> 8

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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> 9

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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

<210> 10

<211> 1254

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaaagcggcg gcggcctgac ccagccgggc ggcagcctgc gcctgagctg cgcggcgagc 60

ggcatgagca ccggcatgtg gcatcgcccg accccgggcc gcgaacgcga actgattgcg 120

ctgaaagcgc cgtggccggg caccgcgccg agcagctggc gctttaccat tagccgcgat 180

aacgcgagca acaccgtgta tctgcagatg gcgaacctga acaccgaaga taccgcggtg 240

tatttttgcc atgcgggcgg cctgctggaa tgggaactgc tggtggtggc ggcgagctgg 300

ggccagggca ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360

ggcagccagg tgaaactgga agaaagcggc agcgaactgg tgcagccggg cggcccgctg 420

cgcctgagct gcgcggcgag cgcgaacatt tttagccgca ccgaagatga ttggatttgg 480

tatcgccagg cgccgggcaa acagcgcgcg tttgtggcgt ggattaccgg cctgagcacc 540

aaaattggcc gctttaccat tagcgtggat aaaagcaaag gcaccattta tctgcagatg 600

aacgcgctga aaccggaaga taccgcggtg tattatgcga acgcggcgga acatgcgagc 660

aaagtggtgg aagtgagcac cagctggggc cagggcaccg tggtgaccgt gagcagcggc 720

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcaaacgcgg ccgcaaaaaa 780

ctgctgtata tttttaaaca gccgtttatg cgcccggtgc agaccaccca ggaagaagat 840

ggctgcagct gccgctttcc ggaagaagaa gaaggcggct gcgaactggg cggcggcggc 900

agcggcggcg gcggcagcgg cggcggcggc agcgaaagcg gcggcggcct gacccagccg 960

ggcggcagcc tgcgcctgag ctgcgcggcg agcggcatga gcaccggcat gtggcatcgc 1020

ccgaccccgg gccgcgaacg cgaactgatt gcgctgaaag cgccgtggcc gggcaccgcg 1080

ccgagcagct ggcgctttac cattagccgc gataacgcga gcaacaccgt gtatctgcag 1140

atggcgaacc tgaacaccga agataccgcg gtgtattttt gccatgcggg cggcctgctg 1200

gaatgggaac tgctggtggt ggcggcgagc tggggccagg gcaccgtgag cagc 1254

<210> 11

<211> 1254

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgcaggcgc tggtgctgct gctgtgcatt ggcgcgctgc tgggccatag cagctgccag 60

aacccggcga gcccgccgga agaaggcagc ccggatccgg atagcaccgg cgcgctggtg 120

gaagaagaag atccgttttt taaagtgccg gtgaacaaac tggcggcggc ggtgagcaac 180

tttggctatg atctgtatcg cgtgcgcagc agcaccagcc cgaccaccaa cgtgctgctg 240

agcccgctga gcgtggcgac cgcgctgagc gcgctgagcc tgggcgcgga acagcgcacc 300

gaaagcatta ttcatcgcgc gctgtattat gatctgatta gcagcccgga tattcatggc 360

acctataaag aactgctgga taccgtgacc gcgccgcaga aaaacctgaa aagcgcgagc 420

cgcattgtgt ttgaaaaaaa actgcgcatt aaaagcagct ttgtggcgcc gctggaaaaa 480

agctatggca cccgcccgcg cgtgctgacc ggcaacccgc gcctggatct gcaggaaatt 540

aacaactggg tgcaggcgca gatgaaaggc aaactggcgc gcagcaccaa agaaattccg 600

gatgaaatta gcattctgct gctgggcgtg gcgcatttta aaggccagtg ggtgaccaaa 660

tttgatagcc gcaaaaccag cctggaagat ttttatctgg atgaagaacg caccgtgcgc 720

gtgccgatga tgagcgatcc gaaagcggtg ctgcgctatg gcctggatag cgatctgagc 780

tgcaaaattg cgcagctgcc gctgaccggc agcatgagca ttattttttt tctgccgctg 840

aaagtgaccc agaacctgac cctgattgaa gaaagcctga ccagcgaatt tattcatgat 900

attgatcgcg aactgaaaac cgtgcaggcg gtgctgaccg tgccgaaact gaaactgagc 960

tatgaaggcg aagtgaccaa aagcctgcag gaaatgaaac tgcagagcct gtttgatagc 1020

ccggatttta gcaaaattac cggcaaaccg attaaactga cccaggtgga acatcgcgcg 1080

ggctttgaat ggaacgaaga tggcgcgggc accaccccga gcccgggcct gcagccggcg 1140

catctgacct ttccgctgga ttatcatctg aaccagccgt ttatttttgt gctgcgcgat 1200

accgataccg gcgcgctgct gtttattggc aaaattctgg atccgcgcgg cccg 1254

Claims

1. A mesenchymal stem cell, characterized in that said mesenchymal stem cell carries a multispecific antibody gene, said multispecific antibody comprising a first antigen-binding domain which specifically binds a first antigen, a second antigen-binding domain which specifically binds a second antigen, and a third antigen-binding domain which specifically binds a third antigen, said antigen-binding domains having a single domain antibody structure; the first antigen and the third antigen are both EGFR, the first antigen binding domain and the third antigen binding domain respectively comprise CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3; the second antigen is CD3, comprising CDR4 shown in SEQ ID NO. 4, CDR5 shown in SEQ ID NO. 5, and CDR6 shown in SEQ ID NO. 6; 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 mesenchymal stem cell of claim 1, wherein the first antigen-binding domain and the third antigen-binding domain comprise a single domain variable region as set forth in SEQ ID No. 7.

3. The mesenchymal stem cell of claim 1, wherein the second antigen-binding domain comprises a single domain variable region as set forth in SEQ ID NO 8.

4. The mesenchymal stem cell of claim 1, wherein the co-stimulatory factor is 4-1BB, the amino acid sequence of which is set forth in SEQ ID NO 9.

5. The mesenchymal stem cell of claim 1, wherein the nucleotide sequence of the multispecific antibody is set forth in SEQ ID NO 10.

6. The mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell further carries a PEDF gene having a nucleotide sequence set forth in SEQ ID No. 11.

7. The mesenchymal stem cell of any one of claims 1-6, which is an umbilical cord Wharton's jelly mesenchymal stem cell.

8. A pharmaceutical composition comprising the mesenchymal stem cell of any of claims 1-7.

9. Use of the mesenchymal stem cell of any one of claims 1 to 7 or the pharmaceutical composition of claim 8 in the preparation of an anti-tumor medicament.

10. The use according to claim 9, wherein the tumor is selected from glioma, brain cancer.