Hydrophilic monoclonal antibodies targeting EphA2 with improved properties, as well as multispecific chimeric antigen receptors and engineered immune cells containing these monoclonal antibodies.

Abstract

This invention relates to a monobody-based chimeric antigen receptor. This monobody-based chimeric antigen receptor includes an extracellular ligand-binding domain comprising a monobody. Furthermore, this invention relates to an immune cell that expresses the monobody-based chimeric antigen receptor on its cell surface membrane. These monobody-based chimeric antigen receptor (CAR) immune cells 1) exhibit excellent efficacy in preventing or treating cancer by targeting receptors expressed on the surface of cancer cells; 2) unlike animal-derived scFv-based CAR immune cells, using human FN3-based monobody antibodies as extracellular ligand-binding domains, they exhibit low immunogenicity when administered to humans; 3) are smaller than scFv, thus possessing high tissue penetration; 4) can minimize the side effects of existing antibody therapies for cancer treatment (e.g., autoimmune diseases caused by nonspecific binding); 5) various monobody-based CAR immune cells can be prepared using pre-constructed monobody libraries targeting various cancer cell antigens, thus enabling their use in the prevention or treatment of refractory solid cancers with various antigens.

Description

Technical Field

[0001] This invention relates to a hydrophilic monobody that replaces a portion of the fibronectin (FN3) sequence that conventionally targets liver ligand receptor A2 (EphA2) with hydrophilic amino acids, and a chimeric antigen receptor containing said hydrophilic monobody. More particularly, it relates to a dual chimeric antigen receptor that simultaneously targets both EphA2 and programmed death protein ligand 1 (PD-L1). The invention also relates to an isolated immune cell containing said dual chimeric antigen receptor and its use in cancer therapy. Background Technology

[0002] In treating solid tumors, surgical resection alone is often insufficient to completely eliminate cancer stem cells, residual cancer cells, or metastatic cancer cells. Therefore, a combination therapy is employed, combining surgical resection with radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In particular, immunotherapy utilizes the body's immune system, thus minimizing the side effects seen with radiotherapy or chemotherapy.

[0003] T cells are representative examples of immune cells, mediating adaptive immune responses and comprising 75% of all lymphocytes. Most T cells exist in an inactive state in the blood, but upon stimulation by antigens, they transform into an active form, capable of eliminating cells carrying specific antigens. The functions of T cells can be activated through antigen-recognizing receptors (T cell receptors (TCRs)), co-stimulatory molecules, and cytokines.

[0004] Immune cells, including T cells, bind to the major histocompatibility complex (MHC) molecule, which is bound to tumor-specific antigens, to kill cancer cells. Cancer cells, in turn, kill cancer cells by inhibiting MHC expression or by altering the function of immune checkpoints expressed on the surface of immune cells, such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4), programmed cell death protein-1 (PD-1), lymphocyte-activation gene-3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA), or by expressing immune checkpoint ligands (programmed cell death protein ligand 1). The mechanism of ligand-1 is to inhibit the function of immune cells and facilitate immune evasion. Furthermore, it alters the tumor microenvironment, favoring cancer cell growth and intravascular migration. In particular, one of the greatest challenges in treating solid tumors is that even within the same type of cancer, there can be differentiation into different cancer cells, resulting in multiple antigens that make it difficult to target a specific cancer, unlike hematologic malignancies. Therefore, for the treatment of solid tumors, the development of at least two targeted therapies has begun. Examples include catutoxumab (peritoneal cancer), first patented as a dual antibody in 2009 (discontinued in 2017), blinatumomab (hematologic malignancies), the second to be licensed in 2014, and tefazizumab (Kimmtrak) and mutuzumab (Lunsumio), which were recently licensed in the US and Europe in 2022.

[0005] It was discovered that cancer cells can utilize immune checkpoints to fight immune cells and survive, leading to active development of drugs that interfere with the binding of immune checkpoint proteins to ligands on cancer cells. Ultimately, a monoclonal antibody was developed that specifically binds to PD-1 / CTLA-4 proteins, which are immune checkpoint proteins. The PD-1 antibody or CTLA-4 antibody binds to PD-1 and CTLA-4 proteins, respectively, located on the membranes of immune cells, thereby preventing these immune checkpoint proteins from binding to ligands present on cancer cells, thus maintaining the function of immune cells in killing cancer cells. Examples of this antibody-based immunotherapeutic agent include atezolizumab, avelumab, and ipilimumab.

[0006] Although it is a representative cancer antigen, no antibody therapeutic has yet been developed for the liver ligand receptor (Ephrin receptor). It is a membrane receptor tyrosine kinase (RTK) with a size of 108 kDa. It consists of three parts: an outer membrane region containing a ligand-binding domain, a cystein-rich region, and two fibronectin III repeats; a transmembrane region in the middle; and a cytoplasmic region containing a kinase-active domain (Labrador et al., 1997; Pasquale, 1997). Hepatin receptors are proteins that bind to their ligands, hepatinin, and transmit extracellular signals into cells via phosphorylation. They participate in cell proliferation, cell migration, differentiation, synaptic transmission in nerve cells, tissue remodeling, osteoblast differentiation, and angiogenesis (Pasquale EB. Cell. 133:38-52, 2008; Barquilla A et al., Annu. Rev. Pharmacol. Toxicol. 55:465-487, 2015). Based on the structure of hepatic ligand receptors and the ligand-receptor binding specificity, they are divided into nine types: group A (EphA1-8 and EphA10) and five types: group B (EphB1-4 and EphB6) (Ieguchi K. Endocrine, Metabolic and Immune Disord Drug Targets. 15:119-128, 2015; Eph Nomenclature Committee. Cell. 90:403-404, 1997; Lindberg RA et al., Molecular Cell Biology. 10(12):6316-6324, 1990; Davis S et al., Science. 266(5186):816-819, 1994).Ephrin receptor A2 (EphA2) is expressed at extremely low levels in normal cells, but it is non-specifically overexpressed in most cancer cells, including liver cancer, prostate cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, lung cancer, cervical cancer, and ovarian cancer. It promotes cancer progression, metastasis, angiogenesis, and resistance to tumors. EphA2 shares approximately 65% ​​protein homology with EphA1 and is found in almost the same location in the mouse lung. It is overexpressed along with EphA1 and its ligand Ephrin-A1 in a variety of malignant tumors, including non-small cell lung cancer, and participates in carcinogenesis and malignant progression through its interaction with EphA2 / A1-Ephrin-A1 (Naj AC et al., Nat Genet. 43:436-441, 2011; Finney AC et al., Circulation. 136:566-582, 2017).

[0007] Since 2017, immune cell-based immunotherapies have been developed. Unlike existing immunotherapies, these agents specifically bind to cancer cells to eliminate them within the body through immune cells. These are cancer therapeutic agents that enable immune cells to specifically attack cancer cells. They include either CAR T cells (which express chimeric antigen receptors, which can directly recognize tumor-associated antigens) or T cells that express specific antibody sequences of immune checkpoint receptors on cancer cells.

[0008] Currently developed chimeric antigen receptors are structured into a single-chain variable fragment (scFv) portion that recognizes the antigen, a transmembrane domain, and a signaling domain that transmits the signal into the cell. Pharmaceutical companies such as Novartis, Gilead, Celgene, AbClon, and Janssen, along with clinicians, have developed CAR-T therapy using scFv that specifically binds to the CD19 antigen of hematological malignancies or nanobodies that specifically bind to the BCMA antigen of multiple myeloma, as a treatment for hematological cancers.

[0009] Solid tumors, which account for a large proportion of all cancers, absolutely require CAR-based immunotherapy that integrates various genetic engineering technologies. However, solid tumors are constrained by many factors, such as the heterogeneity of different antigens expressed by individual patients, the complex characteristics of the tumor microenvironment (hypoxia), and the migration and activation of T cells. Recent clinical trial results have shown that using dual-targeting to overcome heterogeneity, compared to targeting a single antigen, results in a stronger immune response, leading to serious side effects including cytokine storms and neurotoxicity. Furthermore, reports indicate that directly using the antibodies used in antibody drugs—that is, using the antigen recognition site directly as a CAR immunotherapy agent without considering the characteristics of the antibody drug, i.e., its strong binding properties to the antigen—can lead to serious side effects such as on-target / off-tumor detumescence.

[0010] Existing anticancer immunotherapy is a blood cancer therapy that relies on murine antibody molecules, specifically antibody fragments (scFv) to recognize and bind to human cancer antigens, and then eliminate cancer cells through the secretion of various cytokines or the attack of surrounding immune cells. However, due to the side effects of animal-derived antibodies, the low tissue permeability caused by the size of scFv, and the heterogeneity of different antigens expressed by different patients in solid cancer, there is a problem that it is necessary to recognize at least two antigens at the same time.

[0011] In order to overcome the shortcomings of this scFv and the hydrophobic monobody, the inventors conducted research and confirmed that using a hydrophobic monobody based on PD-L1 scFv and hydrophilic human fibronectin FN3 as the extracellular ligand binding site of the chimeric antigen receptor has excellent preventive and therapeutic effects on solid cancer, thus completing the present invention.

[0012] (Existing technical documents)

[0013] (Patent Documents)

[0014] (Patent Document 1) Korean Patent Registration No. 10-2533540

[0015] (Patent Document 2) Korean Patent Registration No. 10-2048477

[0016] (Patent Document 3) Korean Patent Registration No. 10-2157197 Summary of the Invention

[0017] Technical issues

[0018] The present invention aims to provide a hydrophilic monobody that specifically binds to EphA2.

[0019] The present invention aims to provide a chimeric antigen receptor comprising the monobody.

[0020] The present invention aims to provide a vector comprising a polynucleotide containing a nucleic acid sequence encoding the Monobody or chimeric antigen receptor.

[0021] The present invention aims to provide the aforementioned polynucleotide.

[0022] The present invention aims to provide an immune cell that expresses an extracellular ligand-binding domain containing the Monobody or the chimeric antigen receptor on its cell membrane surface.

[0023] The present invention aims to provide a pharmaceutical composition comprising the immune cells for the prevention or treatment of cancer.

[0024] Technical solutions

[0025] This invention provides a hydrophilic monobody comprising a monobody with one or more polar nonionic amino acids replaced by hydrophobic amino acids, wherein the monobody is composed of an amino acid sequence numbered 46. The monobody may include the amino acid sequence numbered 8.

[0026] The monobody can be linked to the PD-L1 scFv directly or via a linker peptide. The PD-L1 scFv may include the amino acid sequence at sequence number 27.

[0027] The present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor includes the monobody as an extracellular ligand binding domain, and further includes one or more of a group consisting of a transmembrane domain and an intracellular signal transduction domain.

[0028] The transmembrane domain may be selected from the following groups: T cell receptor α chain, T cell receptor β chain, T cell receptor ζ chain, CD8 α chain, CD8 β chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a portion thereof, and any combination thereof.

[0029] The intracellular signal transduction domain can be selected from any of the following groups: TCR ζ, FcR γ, FcR β, FcRε, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD5, CD22, CD79a, CD79b and CD66d and any combination thereof.

[0030] The intracellular signal transduction domain may be selected from one or more of the following groups: OX40 domain, CD2 domain, CD27 domain, CD28 domain, CDS domain, ICAM-1 domain, LFA-1 domain, and 4-1BB domain, and any combination thereof.

[0031] The chimeric antigen receptor may also include a hinge region.

[0032] The present invention provides a polynucleotide comprising a nucleic acid sequence encoding the Monobody or the chimeric antigen receptor.

[0033] The present invention provides a vector containing the polynucleotide.

[0034] The present invention provides an immune cell that expresses an extracellular ligand-binding domain containing the Monobody or the chimeric antigen receptor on its cell surface membrane.

[0035] The immune cells may be selected from a group consisting of free leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, and dendritic cells.

[0036] The present invention provides a pharmaceutical composition comprising the immune cells for the prevention or treatment of cancer.

[0037] The cancers mentioned can be selected from the group consisting of melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colorectal cancer, hematopoietic system tumors and their metastatic cancers.

[0038] The cancer mentioned can be pancreatic cancer, prostate cancer, or ovarian cancer.

[0039] Invention Effects

[0040] The chimeric antigen receptor based on hydrophilic monobody according to the present invention, immune cells expressing the receptor on cell surface membranes, and compositions using the present invention for the prevention or treatment of cancer can exhibit the following effects.

[0041] 1) It can reduce the side effects (e.g., hair loss and digestive disorders) caused by inhibiting normal cell growth in existing methods such as radiotherapy or chemotherapy with deoxyribonucleic acid (DNA) nucleotide analogs (cisplatin series and 5-fluorouracil (5-FU) series) that only inhibit the rapid proliferation of cells.

[0042] 2) Because of the use of small monobody-based human FN3, the side effects of autoimmune diseases and cancer cell metastasis caused by anticancer drug resistance that occur in targeted antibody therapy used as animal-derived antibody anticancer therapy and chimeric T-cell therapy using scFv obtained from mouse monoclonal antibody library can be minimized.

[0043] 3) It can overcome the limitations of monobody antibodies, which have no ability to kill cancer cells, are unstable, have a short lifespan, and were previously only used for diagnosis. It can effectively inhibit or eliminate solid cancers that express cancer antigens (e.g., EphA2), thereby enabling the simultaneous diagnosis and treatment of cancer.

[0044] 4) Compared with traditional T-cell therapy for solid cancer, monobody-based CAR immune cells can exhibit superior anti-cancer effects and can be used as immunotherapeutic agents for the treatment or prevention of solid cancers (such as pancreatic cancer, prostate cancer, and ovarian cancer that express EphA2 as an antigen).

[0045] 5) FN3 monobodies have the advantage of being able to prepare various types of monobodies targeting specific antigens. Therefore, CAR immune cells based on specific monobodies can be prepared for various antigens, thereby treating cancer more effectively.

[0046] 6) Monobody-based CAR immune cells are smaller than other CAR immune cells (e.g., the size of a monobody is about one-third the size of an scFv). Therefore, they penetrate cancer tissue more easily than scFv-based CARs. When creating multivalent CARs in viral vectors, considering viral packing, up to four monobody sequences recognizing cancer antigens can be loaded into the vector, thus overcoming the heterogeneity of cancer antigens, a characteristic of solid tumors. Furthermore, due to their sufficient selectivity and specificity for cancer antigens, they can block the escape of cancer immune cells, effectively preventing or treating cancer.

[0047] 7) Although existing research reports have shown that the existing hydrophilic fibronectin FN3 domains enhance their function as biomarkers by strongly binding to targets and being absorbed into intracellular endosomes (Sirois AR, Deny DA, Baierl SR, George KS, Moore SJ (2018). Modified Fn3 proteins that recognize the tumor biomarker mesothelin internalize upon binding. PLoS ONE 13(5): e0197029. https: / / doi.org / 10.1371 / journal.pone.0197029), they have not yet been used for therapeutic purposes, especially as cell gene therapy agents. It has been confirmed that the expression rate of hpEphA2 monobody-based CAR immune cells (hpVECs) using the hydrophilic fibronectin FN3 domain, which is currently patent-pending, is more than twice that of existing EphA2 monobody CAR immune cells (VECs), meaning that they have a much higher probability of recognizing and targeting cancer cells in vivo. Immunogenicity can be further reduced during insillico immunogenicity prediction analysis.

[0048] The two cancer antigen-specific chimeric antigen receptors PD-L1 and EphA2 of the present invention, and the immune cells containing them, not only have excellent PD-L1 and EphA2 specific targeting efficiency, but also exhibit excellent anti-cancer effects on cancer cells that only express PD-L1 or EphA2 single antigens, even if cancer cells do not express PD-L1 or EphA2 to escape the immune system or undergo self-clearance. They can be used to prevent or treat tumor growth and metastasis. Attached Figure Description

[0049] Figure 1 This is a schematic diagram illustrating the structure of a vector expressing a chimeric antigen receptor based on a hydrophilic monobody.

[0050] Figure 2a A schematic diagram comparing the amino acid sequences of the non-hydrophilic monobody (E1) and the hydrophilic monobody (hpE1).

[0051] Figure 2bThis is a schematic diagram showing the structural similarity of a chimeric antigen receptor by superimposing the three-dimensional structures of a non-hydrophilic monobody (E1) and a hydrophilic monobody (hpE1).

[0052] Figure 2c A graph showing the protein binding ability of the non-hydrophilic monobody (E1) and the hydrophilic monobody (hpE1) to the human antigen EphA2.

[0053] Figure 3 A graph showing the transduction rates between T immune cells containing chimeric antigen receptors based on hydrophilic monobody antibodies (hpVEC-1) and T immune cells containing chimeric antigen receptors based on non-hydrophilic monobody antibodies (VEC-1).

[0054] Figure 4 A graph showing the activation markers between pVEC-1 or VEC-1.

[0055] Figure 5a and Figure 5b A graph showing the inhibition markers between hpVEC-1 or VEC-1.

[0056] Figure 6 The graph shows the cytotoxicity of hpVEC-1 and VEC-1 against HEK293 kidney cell lines overexpressing the human cancer antigen EphA2.

[0057] Figure 7a and Figure 7b This chart compares the killing ability of HPVEC-1 and VEC-1 against cancer cells (ovarian cancer, gastric cancer, glioblastoma, and pancreatic cancer) in vitro using real-time cell analysis (RTCA) technology.

[0058] Figure 8 A graph showing the results of fluorescence flow cytometry (FACS) analysis of the characteristics of hpVEC-1 and immune cells prepared from VEC-1 24 days later, as well as the expression levels of activating factors CD62L, CD25, CD69 and repressing factors CTLA-4, TIGIT, TIM-3, LAG-3, and PD-1.

[0059] Figure 9 A chart showing the cancer cell killing ability of hpVEC-1 and VEC-1 immune cells using RTCA analysis.

[0060] Figure 10 The graphs show the LDH efflux of dead cancer cells in the 3D spheroid co-culture method and culture medium, as determined by an enzyme-linked immunosorbent assay (ELISA) analyzer, and the killing ability of hpVEC-1 and VEC-1 against ovarian and gastric cancer cells over time.

[0061] Figure 11 A graph showing changes in tumor size and mouse weight recovery in a mouse model of ovarian cancer xenograft.

[0062] Figure 12 This is a schematic diagram illustrating the structure of a dual chimeric antigen receptor vector that simultaneously recognizes both PD-L1 and EphA2 antigens.

[0063] Figure 13 A chart comparing activators and inhibitors of T lymphocytes as immune cells containing heterologous chimeric antigen receptors.

[0064] Figure 14 The graphs show the cytotoxic effects of T lymphocyte immune cells that recognize chimeric antigen receptors using RTCA analysis, specifically using HEK293 cell lines that simultaneously overexpress PD-L1, EphA2, and both antigens.

[0065] Figure 15 A graph showing the killing effect of T lymphocyte immune cells containing PD-L1 and EphA2 dual antigens recognizing chimeric antigen receptors on two subpopulations within the OVCAR-5 ovarian cancer cell line derived from gastric cancer, using RTCA analysis.

[0066] Figure 16 A graph showing changes in tumor size and mouse weight recovery in a mouse model of ovarian cancer xenograft.

[0067] Figure 17 A graph showing the survival curves of mice in an ovarian cancer xenograft mouse model, plotted using the Kaplan-Meier formula. Detailed Implementation

[0068] Throughout the specification, when a part is described as "including" a certain component, unless otherwise stated, it does not mean that other components are excluded, but rather that other components may be included.

[0069] Unless otherwise defined in detail herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of gene therapy, biochemistry, genetics and molecular biology.

[0070] Unless otherwise specified, the present invention may be carried out using conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA and immunology, which may fall within the scope of the relevant fields.

[0071] The terms “prevention” as used in this invention refer to all actions that inhibit or delay the onset of a disease by administering the composition; “treatment” refers to all actions that improve or benignly change the symptoms of an individual suspected of having or already having the disease by administering the composition; and “improvement” refers to all actions that at least reduce disease-related parameters, such as the severity of symptoms, by administering the composition.

[0072] PD-L1 scFv and chimeric antigen receptors based on hydrophilic monobody antibodies

[0073] This invention relates to PD-L1 scFv and chimeric antigen receptors (CARs) based on hydrophilic monobody antibodies.

[0074] The PD-L1 scFv and the chimeric antigen receptor based on hydrophilic monobody antibodies of the present invention may include an extracellular ligand binding domain.

[0075] The term "extracellular ligand-binding domain" as used in this invention can refer to an oligopeptide or polypeptide capable of binding ligands. Preferably, the domain can interact with cell surface molecules. For example, an extracellular ligand-binding domain can be selected to recognize ligands that function as cell surface markers on target cells associated with any disease.

[0076] The term "linker" used in this invention refers to an amino acid that connects protein domains. The "linker" of this invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence of sequence number 19.

[0077] The term "hinge region" used in this invention can refer to a naturally occurring hinge region or a synthetic amino acid sequence, preferably a part of the CD8 α chain.

[0078] The PD-L1 scFv sequence number of the present invention can have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of sequence number 18.

[0079] The term "hydrophilic monobody (hp)" used in this invention can refer to the substitution of polar uncharged amino acids with hydrophobic amino acid sequences in the backbone of EphA2 monobody, allowing it to specifically bind to ligands or antigens present on the surface of target cells in a stable form such as hydrogen bonds between proteins.

[0080] Hydrophilic monobodies are composed of amino acids with uncharged polar sidechains. Therefore, they can carry a charge depending on pH. The serine (S) and threonine (T) side chains contain hydroxyl groups, allowing them to form hydrogen bonds. This is crucial for stabilizing protein structures and other biochemical reactions, particularly in protein-protein interactions or interactions between proteins and other molecules. Asparagine (N) and glutamine (Q), in particular, may be important for targeting and protein recognition.

[0081] Hydrophilic monobodies can be prepared by replacing hydrophobic amino acids such as glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), phenylalanine (F), and tryptophan (W) with hydrophilic amino acids such as serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), aspartic acid (D), and glutamic acid (E); or by adding hydrophilic amino acids. For example, hydrophobic valine (V) can be replaced with hydrophilic amino acids, serine (S), or threonine (T), hydrophobic leucine (L) can be replaced with hydrophilic threonine (T), or hydrophilicity can be increased by adding 1 to 1000 amino acid sequences containing hydrophilic amino acids (E, S).

[0082] Monobody can be selected in a variety of ways depending on the desired target. For example, monobodies can specifically bind to proteins selected from the following groups: CRL1, liver ligand receptor, EphA2, β-galactosidase, RAS, Abl kinase, VEGFR2, MBP, SARS-CoV-2, Bcr-Abl kinase, STAT3, EGFR, VEGFR2, the SH3 domain of human Lyn tyrosine kinase, MLKL, Flu homologues, mitogen-activated protein kinase (MAPK), ERK2, MAPK14, the SH2 domain of kinase, SUMO, AurA, WDR5, the Flu family, GFP, the SARS-CoV-2 receptor-binding domain, the SH3 domain of FYN, the SH2 domain of ABL, SUMO1, Bcr-Abl, GPR56, ECR, PD-L1, glycoprotein 3 (Glypican-3), PCSK9, Gp41, CD4, and IL-23, but are not limited to these.

[0083] In addition, monobodies can be known.

[0084] Monobody antibodies can specifically bind to liver glycoprotein receptors, EphA2, or human EphA2.

[0085] The monobody has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of sequence number 8.

[0086] Monobody-based CARs may also include one or more of a group consisting of hinge regions, transmembrane domains, and intracellular signal transduction domains.

[0087] Furthermore, monobody-based CARs are expressed on the cell surface membrane. Therefore, monobody-based CARs may include transmembrane domains. Distinguishing features of appropriate transmembrane domains include expression on the cell surface, preferably on the surface of the immune cells of the present invention (particularly lymphocytes or natural killer (NK) cells), and the ability to interact to induce a cellular response of the immune cells to predetermined target cells. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-binding protein or transmembrane protein.

[0088] The transmembrane domain can be selected from one or more of the following groups: T cell receptor α chain, T cell receptor β chain, T cell receptor ζ chain, CD8 α chain, CD8 β chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a subset thereof, and combinations thereof.

[0089] The hinge region and transmembrane domain may contain a portion of the human CD8 α chain. The hinge region and transmembrane domain may contain the amino acid sequence of sequence number 15, preferably, may contain an amino acid sequence having at least 70%, more preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of sequence number 22.

[0090] The intracellular signaling domain of a monobody-based CAR is what enables the extracellular ligand-binding domain to bind to immune cells and targets that activate the immune response, thereby triggering intracellular signal transduction. The signaling domain can be the cause of activation of at least one of the normal effector functions of immune cells expressing a monobody-based CAR. For example, the effector functions of T cells can include cytotoxic or helper activities, including cytokine secretion.

[0091] The term "signal transduction domain" used in this invention can refer to a part of a protein that transduces effector signals within immune cells and induces the cell to perform a specific function.

[0092] Intracellular signal transduction domains may include one or more of the following groups: TCR ζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

[0093] Intracellular signal transduction domains may include one or more of the group consisting of the OX40 domain, CD2 domain, CD27 domain, CD28 domain, CDS domain, ICAM-1 domain, LFA-1 domain, and 4-1BB domain.

[0094] Intracellular signal transduction domains may include one or more of the group consisting of CD28 domain, 4-1BB domain and CD3ζ domain, preferably including CD28 domain and CD3ζ domain, or 4-1BB domain and CD3ζ domain.

[0095] The CD28 domain may include the amino acid sequence of sequence number 23 or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with it.

[0096] The 4-1BB domain may include the amino acid sequence of sequence number 11 or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with it.

[0097] The CD3ζ domain may include the amino acid sequence of sequence number 12 or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with it.

[0098] Polynucleotides and carriers

[0099] This invention may relate to a polynucleotide comprising a nucleic acid sequence encoding a hydrophilic monobody or a CAR comprising a hydrophilic monobody and a PD-L1 scFv, or a vector comprising such a nucleic acid sequence or polynucleotide. Here, vector may refer to an expression vector.

[0100] The polynucleotide may include the nucleic acid sequence of sequence number 2. The polynucleotide may include a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the nucleic acid sequence of sequence number 45.

[0101] The nucleic acid sequence encoding a CAR based on a hydrophilic monobody can be codon-optimized for expression in human cells.

[0102] The term "vector" as used in this invention can refer to a construct capable of delivering one or more genes or sequences of interest into a host cell, preferably one that can be expressed within a host cell. Examples include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors polymerized with cationic flocculants, DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells (e.g., producer cells).

[0103] Viral vectors can include: retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus)), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canarypox). Other viruses that can be used as vectors include, for example, noroviruses, capsid viruses, flaviviruses, reoviruses, papillomaviruses, hepatotropic DNA viruses, and hepatitis viruses. Examples of retroviruses can include avian leukosis-sarcoma, mammalian C-viruses, B-viruses, D-viruses, HTLV-BLV groups, lentiviruses, and foamy viruses.

[0104] Engineered immune cells

[0105] This invention may relate to isolated cells or cell lines that can be obtained through the preparation or modification methods of the aforementioned immune cells. The isolated cells herein may include monobody-based CARs. Preferably, the monobody-based CAR may be a CAR according to the present invention comprising a hydrophilic monobody and PD-L1 scFv.

[0106] The isolated cells of the present invention may include a group of CARs comprising extracellular ligand-binding domains that differ from each other. In particular, the isolated cells may include exogenous polynucleotide sequences encoding the CARs.

[0107] The immune cells of this invention can refer to cells derived from hematopoietic organs that function in the initiation and / or execution of innate and / or acquired immune responses. The immune cells of this invention can be derived from stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, non-human stem cells, umbilical cord blood stem cells, precursor cells, bone marrow stem cells, induced pluripotent stem cells, fully differentiated stem cells, or hematopoietic stem cells.

[0108] The immune cells of this invention can be human CD3+ T cells. In one embodiment, the isolated cells can be dendritic cells, cytotoxic dendritic cells, mast cells, NK cells, B cells, or T cells selected from a group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In one embodiment, the cells can be derived from a group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0109] Cells can be obtained from subjects using a variety of non-restrictive methods. Cells can be obtained from a variety of non-restrictive sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infected sites, ascites, pleural effusion, spleen tissue, and tumors.

[0110] Any T cell line known and available to those skilled in the art can be used. The cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with an infectious disease. The cells can be part of a mixed group of cells exhibiting different phenotypic characteristics. Cell lines obtained from T cells engineered according to the above method can be used, and the immune cells according to the invention are resistant to immunosuppressive treatments.

[0111] The immune cells may be leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, dendritic cells, or combinations thereof, but are not limited thereto.

[0112] Treatment using PD-L1 scFv and CAR immune cells based on hydrophilic monobody dual antigen recognition

[0113] PD-L1 scFv and dual-antigen recognition CAR immune cells based on hydrophilic monobody antibodies, engineered immune cells, or populations of these cells can be used as pharmaceutical compositions for the prevention or treatment of cancer.

[0114] This invention relates to a pharmaceutical composition comprising PD-L1 scFv and a dual-antigen recognition CAR immune cell or engineered immune cell based on a hydrophilic monobody, for the prevention or treatment of cancer.

[0115] This invention relates to a method for preventing or treating cancer in a desired subject, the method comprising the steps of administering PD-L1 scFv and CAR immune cells based on hydrophilic monobody dual antigen recognition to the desired subject.

[0116] This invention relates to the use of PD-L1 scFv for manufacturing medicaments for the prevention or treatment of cancer and the use of dual-antigen recognition CAR immune cells based on hydrophilic monobody antibodies.

[0117] This invention can be a PD-L1 scFv for the prevention or treatment of cancer and a dual-antigen recognition CAR immune cell based on a hydrophilic monobody antibody.

[0118] As used in this invention, the term "object" refers to mammals, more preferably humans. Mammals include, but are not limited to, livestock, pets, primates, horses, dogs, cats, mice, and rats.

[0119] In this invention, the treatment may be part of autoimmune therapy or allogeneic immunotherapy. "Autologous" can refer to cells, cell lines, or cell populations used for the treatment subject that are derived from the subject or from a donor compatible with human leukocyte antigen (HLA). "Allogeneic" can refer to cells or cell populations used for the treatment subject that are not derived from the subject but from a donor.

[0120] Cancer can be melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colorectal cancer, hematopoietic system tumors, or their metastatic cancers, but is not limited to these. Furthermore, cancer can be a non-solid tumor or a solid carcinoma.

[0121] Non-solid tumors or non-solid cancers can be myeloma, lymphoma, or leukemia, but are not limited to these.

[0122] Solid cancer can be one or more of the following groups: pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lung cancer, but is not limited to these.

[0123] The embodiments and examples of this application will now be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily implement them. However, this application may take many forms and is not limited to the embodiments and examples described herein.

[0124] [Example 1]

[0125] Preparation of vectors expressing chimeric antigen receptors based on hydrophilic monobody antibodies

[0126] 1-1. Preparation for the preparation of recombinant lentiviral vectors

[0127] Prepare the following strains and vectors to prepare recombinant plasmid vectors expressing chimeric antigen receptors based on hydrophilic monobody antibodies.

[0128] (1) The carrier as the object of copying

[0129] Thermo Fisher Scientific (GeneArt) has synthesized the pMX-hpE1 vector containing the nucleic acid sequence of the hpE1 Monobody (Sequence Number 2) that specifically binds to human EphA2, and is preparing a CAR vector to express the chimeric antigen receptor. Specifically, the expression vector used is the pLenti7.3 / V5-DEST vector from Invitrogen, which includes a nucleic acid sequence for expressing a chimeric antigen receptor between the 5'UTR and 3'UTR of the pLenti7.3 / V5-DEST vector. The chimeric antigen receptor is composed of the CD8α leader sequence of sequence number 1 - the E1 Monobody sequence of sequence number 45 - the CD8 H&TM sequence of sequence number 3 - the CD28 sequence of sequence number 4 - the CD3ζ sequence of sequence number 6; or the CD8α leader sequence of sequence number 1 - the E1 Monobody sequence of sequence number 45 - the CD8 H&TM sequence of sequence number 3 - the 4-1BB sequence of sequence number 5 - the CD3ζ sequence of sequence number 6. Two lentiviral vectors were thus prepared: pLenti7.3::CD8α leader sequence-E1 Monobody sequence-CD8 H&T-CD28-CD3ζ vector and pLenti7.3::CD8α leader sequence-E1 Monobody sequence-CD8 H&T-4-1BB-CD3ζ vector.

[0130] The nucleic acid sequences that constitute the chimeric antigen receptor based on hydrophilic monobody in the recombinant plasmid vector are shown in Table 1 below.

[0131] Table 1

[0132]

[0133]

[0134] The nucleic acid sequences in Table 1 can be expressed in immune cells and can be expressed on the cell surface membrane as chimeric antigen receptors based on Monobody antibodies containing the amino acid sequences in Table 2 below.

[0135] Table 2

[0136]

[0137] (2) Prepare cells for vector replication

[0138] Host cells for the pMX-hpE1 vector: E. coli DH5α(F- endA1 glnV44 thi-1 recA1relA1 gyrA96 deoR nupG purB20 φ80dlacZ△M15 △(lacZYA-argF)U169, hsdR17(rK-mK+), λ-)

[0139] Host cells for p-lentiviral vector: *Escherichia coli* (E. coli) Stbl3(F-mcrB mrrhsdS20(rB-, mB-) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R ) xyl-5 λleumtl-1), Invitrogen™

[0140] The host cells were cultured in LB (Luria-Bertani) solid medium (Difco Laboratories, USA) at 37°C and 200 rpm, with 100 μg / ml kanamycin or ampicillin added to all media to prepare the host cells. Single colonies of the resulting host cells were then pre-cultured in LB liquid medium (Difco Laboratories, USA), followed by 1% inoculation into SOB (Super Optimal Broth) medium. After culturing at 18°C ​​for 24 hours, the cells were washed with Inoue TB buffer, and the culture was complete.

[0141] (3) Preparation of morphogenetic cells for mass production of vectors

[0142] After mixing the prepared vectors with host cells, the mixture was incubated on ice for 10 minutes, treated at 42°C for 50 seconds, and then treated on ice for 3 minutes to prepare transgenic cells infused with the respective vectors. The prepared transgenic cells were then plated onto LB solid medium containing antibiotics and cultured at 37°C. This process yielded a large quantity of each vector.

[0143] 1-2. Preparation of recombinant lentiviral vectors expressing hydrophilic E1 monobody-chimeric antigen receptor (hpE1 CAR)

[0144] Using the two prepared lentiviral vectors (pLenti7.3::CD8α leader sequence-E1 Monobody-CD8 H&T-CD28-CD3ζ vector and pLenti7.3::CD8α leader sequence-E1 Monobody-CD8 H&T-4-1BB-CD3ζ vector) as templates, polymerase chain reaction (PCR) was performed using the CPL-forward primer and CPL-reverse primer listed in Table 3 below. The amplified vectors were isolated and purified to prepare two chimeric antigen receptor vectors without scFv.

[0145] The hpE1 monobody gene sequence was prepared as follows: PCR was performed using the hpE1-forward primer and hpE1-reverse primer listed in Table 3 below to amplify the pMX-hpE1 vector, followed by separation and purification.

[0146] The prepared vector and hpE1 Monobody sequence were used. Using an Overlap Clone DNA Cloning Kit (Elpisbio), the hpE1 Monobody sequence was inserted into the original site of the E1 Monobody sequence via homologous recombination to prepare two recombinant lentiviral vectors expressing chimeric antigen receptors containing the hpE1 Monobody (pLenti7.3::CD8α leader-hpEphA2 Monobody chimeric antigen receptor vector). The prepared recombinant lentiviral vectors were named hpEphA2 mCAR-1 (hpVEC-1) and hpEphA2 mCAR-2 (hpVEC-2), respectively, and their structures are shown below. Figure 1 As shown.

[0147] Table 3

[0148]

[0149] [Example 2]

[0150] Comparison of hpE1 Monobody and E1 Monobody

[0151] 2-1. Sequence and structural comparison of hpE1 Monobody and E1 Monobody

[0152] Amino acid sequence alignment between hpE1 Monobody and E1 Monobody was performed using Clustal Omega. For the aligned sequences, identical sequences between hpE1 Monobody and E1 Monobody were identified using Jalview.

[0153] AlphaFold was used to obtain the predicted structures of hpE1 Monobody and E1 Monobody. The predicted structures were used as the relaxed structures corresponding to the highest-ranking structures. The structural similarity between hpE1 Monobody and E1 Monobody was evaluated using the PyMOL superimposition method. Figure 2a and Figure 2b The protein sequence differences and overlaps between hpE1 and E1 Monobody are shown.

[0154] 2-2. Comparison of immunogenicity between hpE1 Monobody and E1 Monobody

[0155] Database analysis was performed on the monobody sequences binding to EphA2, and MHC-II epitope binding prediction was conducted using the Immune Epitope Database (IEDB). As shown in Table 4, according to the IEBDB MHC-II binding prediction method, the overall immunogenicity of hpE1 and E1 monobody was predicted to be low. However, due to the introduction of hydrophobic amino acids in the BC loop, E1 monobody was predicted to have a relatively high probability of antigen presentation at the Bβ chain-BC loop linkage site compared to hpE1 monobody.

[0156] Table 4

[0157]

[0158]

[0159]

[0160] 2-3. Determination of the binding affinity between hpE1 monobody and EphA2 antigen protein

[0161] 1) Purification of hydrophilic E1 monobody and E1 monobody protein in E. coli

[0162] To construct an *E. coli* expression vector for monobody protein, the hydrophilic E1 monobody and E1 monobody genes were amplified by PCR. The amplified PCR fragments were excised from both ends using NdeI and BamHI restriction endonucleases and then bound to the restriction endonuclease sites of the pETamh (AviTag-Myc-6xHis) expression vector. The pETamh expression vector, derived from pET (Novagen, USA), has an Nde1 restriction endonuclease site containing the ATG start codon at its 5' position and an amh tag site located at its 3' position, after the BamH1 restriction endonuclease site and before the stop codon. Therefore, the monobody expressing the amh tag with the C-terminus linked to the monobody was named hpE1-amh and E1-amh, respectively. The hpE1 monobody and E1 monobody inserted into the pETamh expression vector were transformed into BL21(DE3) for expression. Single colonies were inoculated into 10 ml LB medium containing kanamycin (50 μg / ml), cultured overnight, and harvested by centrifugation. The colonies were resuspended in 1 mL LB medium and 1 mL glycerol (50%), and then aliquoted into 1 L LB medium at 200 µL. The cultures were incubated at 37°C and 250 rpm until the OD value reached 0.5–0.7. 1 ml of 0.5 M IPTG was added to the cultured *E. coli*, and the cultures were incubated overnight at 20°C and 250 rpm. The cultured *E. coli* were centrifuged at 4°C and 8000 rpm for 10 minutes to obtain the *E. coli* precipitate. For every 1 gram of E. coli precipitate, 10 ml of cold decomposition buffer (20 mM Tris-HCl (7.4), 100 mM NaCl) was added to disperse the E. coli precipitate, followed by centrifugation at 13000 rpm for 30 minutes. hpE1-amh and E1-amh in the supernatant were separated by imidazole gradient elution using a Biorad Fast Protein Liquid Chromatography (FPLC) system equipped with a HisTrap column (EconoFit Nuvia IMAC column, Ni-charged 5 ml). The once-purified monobody was further separated in PBS buffer using size exclusion chromatography (Superdex™ 75 Increase 10 / 300 GL).

[0163] [Example 3]

[0164] Preparation and evaluation of transduction rates of immune cells expressing hpE1 monobody-chimeric antigen receptor on cell membrane surface (hpVEC-1 and VEC-1)

[0165] 3-1. Preparation of lentiviruses containing recombinant lentiviral vectors

[0166] Lentivirals containing hpEphA2 mCAR-1 and EphA2 mCAR-1 prepared in Example 1 were prepared. LV-MAX, provided by Thermo Scientific, was used. ™ The Lentiviral Production System (A35684) kit was used for the production and titration of lentiviruses, prepared according to the manufacturer's experimental method.

[0167] 3-2. Preparation of immune cells and transduction via viral infection

[0168] (1) Isolation of CD3+ T cells from PBMCs

[0169] Using lymphocyte separation agent (Lymphoprep) TM Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood using STEMCELL. Human CD3+ T cells were then isolated from the isolated PBMCs using a positive selection method with CD3 microbeads (Miltenyi Biotech).

[0170] (2) Activation of CD3+ T cells

[0171] Anti-CD3 / anti-CD28 magnetic beads (anti-CD3 / CD28 Dynabead; Gibco) were mixed with isolated human CD3+ T cells at a 1:1 ratio. After 24 hours of mixing, the activation beads were removed using a MACSiMAG separation device. The cells were then washed with RPMI-1640.

[0172] (3) Transduction to lentivirus

[0173] Lentiviral-infected T cells were prepared using a spinoculation technique via centrifugation and viral infection. Specifically, activated human CD3+ T cells were infected with lentiviruses containing hpEphA2 mCAR-1 and EphA2 mCAR-1, respectively, and the viral culture medium was replaced after 24 hours. Cell proliferation began 48 hours after infection. The engineered immune cells prepared in this way were named hpVEC-1 and VEC-1, respectively. The expression rate of the delivery vector (i.e., the hpE1 monobody-chimeric antigen receptor sequence) was analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). Figure 3 As shown, FACS analysis of GFP-expressing cell numbers confirmed that, under IL-2-added culture conditions, hpVEC-1 immune cells showed approximately 60% expression rate compared to the control group without vector, while E1 Monobody-chimeric antigen receptor VEC-1 immune cells showed approximately 23% expression rate. Furthermore, under IL-7 and IL-15-added culture conditions, hpVEC-1 showed approximately 56% expression rate compared to the control group without vector, while VEC-1 showed approximately 25% expression rate. These results confirm that the vector introduced into immune cells expresses well, meaning that the number of cells expressing hpE1 Monobody is more than twice that of E1 Monobody. These results suggest that, in treating patients with cells expressing the cancer antigen EphA2, hpVEC-1 and hpVEC-2 are likely to recognize EphA2-expressing cancer cells faster or more frequently than VEC-1 and VEC-2, indicating high potential.

[0174] [Example 4]

[0175] Comparative assessment of hpVEC-1 and VEC-1 cell viability

[0176] Attune NxT flow cytometry (Fluorescence-activated cell sorting) was used to confirm whether the hpEphA2 mCAR-1 and EphA2 mCAR-1 vectors introduced into the prepared hpVEC-1 and VEC-1 immune cells were expressed normally, and the activity status of these immune cells.

[0177] Anti-green fluorescent protein (GFP) antibodies were used to confirm the expression of the vector introduced into each immune cell. Since the corresponding vector also contains a GFP expression site, the analysis results based on anti-GFP antibodies will be highly accurate in immune cells that express a large number of this vectors. In addition, anti-CD62L-APC antibody (APCMouse Anti-Human CD62L, BD Pharmaceuticals) was used. TM ), anti-CD25-PE antibody (PE Mouse Anti-HumanCD25, BD Pharmingen TM ) and anti-CD69-APC antibody (APC Mouse Anti-Human CD69, BDPharmingen TM To confirm whether T cells are activated.

[0178] like Figure 4 As shown, in vitro experiments confirmed that in engineered immune cells hpVEC-1 and VEC-1, the expression of CD62L, a marker of cell growth activity, was similar to that of the control group, reaching over 85%, while CD25 was expressed at a rate more than 20% higher than that of the control group.

[0179] 2) Determine the binding affinity (Kd dissociation constant) of the purified monobody protein.

[0180] The binding affinity of the hpE1 monobody to hEphA2 was assessed by determining the dissociation constant (Kd) using ELISA. 1 μg / mL of recombinant EphA2 protein (R&D Systems) was aliquoted into each well of a 96-well ELISA plate (Costar) and coated overnight at 4°C. After coating, each well was washed three times with PBST containing 0.05% Tween 20 and treated for 2 hours at room temperature with 200 µL of blocking buffer (PBST containing 5% bovine serum albumin (BSA)). Each well was washed three times with PBST. Monobody proteins for hpE1-amh and E1-amh were added sequentially at half concentration, starting at 500 nM, and bound for 2 hours at room temperature. After washing each well with PBST, treat with 100 µl of horseradish peroxidase-conjugated c-Myc monoclonal antibody (HRPconjugated c-Myc monoclonal antibody) (Invitrogen) at room temperature for 1 hour. After washing, react each well with 100 µl of substrate [a 1:1 mixture of substrate (TMB) and hydrogen peroxide, BDBiosciences] at room temperature for 5 minutes. Stop the reaction with 50 µl of 1M H₂SO₄ and read the color development at 450 nm using an ELISA reader. Figure 2c As shown, the binding force between hpE1 and hEphA2 is confirmed to be stronger than that between E1 and hpEphA2.

[0181] [Example 5]

[0182] Comparative assessment of hpVEC-1 and VEC-1 cytosolic inhibitor expression

[0183] To compare the expression of inhibitory factors in T cells expressing hydrophilic VEC-1 and existing VEC-1, anti-CTLA-4-APC antibody, anti-TIGIT-APC antibody, anti-TIM-1-BV421 antibody, anti-LAG-1-PE antibody, and anti-PD-1-PE antibody were diluted 1:100 with FACS buffer and mixed with 1x10 6 Each CAR-T cell was incubated at 4°C for 30 minutes, then washed twice with 200 µl of FACS buffer, and the expression of each protein was analyzed by FACS. Figure 5a and Figure 5bAs shown, the expression of CTLA4 or PD-1 was very low in all immune cells expressing existing VEC-1 or hydrophilic VEC-1, including the control group without virus introduction. Furthermore, the expression of TIGIT and TIM-3 in immune cells expressing hydrophilic VEC-1 was about 5-10% lower than that of existing VEC-1, while the expression of LAG-3 was about 5.73% lower than that in cells expressing existing VEC-1.

[0184] [Example 6]

[0185] In vitro killing ability assessment of hpVEC-1 and VEC-1 in EphA2-overexpressing HEK293 cell lines

[0186] To confirm the cytotoxic ability of T cells expressing hydrophilic VEC-1 against cells expressing EphA2, a stable HEK293 cell line overexpressing EphA2 was prepared. A plasmid containing EphA2 cDNA was purchased from Addgen (#116732) and introduced into HEK293 cells using Lipofectamine 2000. After 48 hours, cells with plasmid insertion into the HEK293 cell genome were selected by treatment with G418 at 800 μg / ml. Single cells were then transferred to 96-well plates for culture. For each clone proliferating in single cells, EphA2 protein expression in HEK293 cells was confirmed by FACS analysis using an EphA2-PE antibody. Real-time cell analysis (RTCA) was performed to determine the cytotoxic ability of T cells expressing each VEC-1 against HEK293 cells overexpressing EphA2. HEK293 cells (1 x 10^4 / 100 μL) were cultured in microplates and monitored for 24 hours. After 24 hours, the same number of T cells expressing hydrophilic VEC-1 were treated, and the cytotoxic ability of the T cells expressing hydrophilic VEC-1 was then confirmed by RTCA. Figure 6 As shown, T cells that do not express CAR (control group) are unaffected, but T cells expressing hydrophilic VEC-1 exhibit the effect of killing almost all HEK293 cells expressing EphA2.

[0187] [Example 7]

[0188] Evaluation of in vitro cancer cell killing ability

[0189] To compare cytotoxicity, ovarian cancer cell lines (OVCAR-5), gastric cancer cell lines (SNU638), glioblastoma cell lines (U251), and pancreatic cancer cell lines (ASPC1, Panc-1), purchased from the Korean Cell Line Bank, were treated with CAR-T cells and then RTCA was performed in the same manner as in Example 6. Figure 7a and Figure 7b As shown, T cells expressing hydrophilic VEC-1 exhibited cytotoxicity against all cancer cell lines, confirming that their efficacy was almost identical to that of T cells expressing existing VEC-1.

[0190] [Example 8]

[0191] Stability assessment of hpVEC-1 and VEC-1 cell efficacy (Persistency)

[0192] To understand the persistence of various marker factors in T cells expressing existing VEC-1 and T cells expressing hydrophilic VEC-1, T cells expressing various chimeric antigen receptors were prepared, cultured for 25 days, and then the ratio of CD4+ to CD8+ cells, activity factor markers, and repressor factor markers were compared using the antibodies used in Examples 3 and 4. Figure 8 As shown, after 25 days, the CD4+ ratio was relatively higher in T cells compared to those without CAR (control group), but not significantly different from that of existing VEC-1. Regarding biomarkers of active factors, T cells expressing hydrophilic VEC-1 showed higher CD62L levels compared to those expressing existing VEC-1, while CD25 and CD69 were slightly lower. Regarding repressive factors, TIGIT, Tim3, and LAG-3 were expressed lower in T cells expressing hydrophilic VEC-1 compared to those expressing existing VEC-1.

[0193] [Example 9]

[0194] Evaluation of the in vitro cancer cell killing ability of hpVEC-1 and VEC-1 24 days after preparation

[0195] The killing ability of gastric cancer cells by transduced hpE1 monobody CAR-T cells (hpVEC-1 or hpVEC-2), E1 monobody CAR-T cells (VEC-1 or VEC-2), and untransduced human T cells (control group) was analyzed using RTCA. The results confirmed, as shown in Example 7, that the killing ability of hpVEC-1 (or hpVEC-2) produced after a 9-day preparation period was significantly higher than that of the control group, but very similar to that of VEC-1 (or VEC-2). However, after an additional 24-day culture period, the killing ability was re-examined by co-culturing with cancer cells using RTCA equipment. Figure 9 As shown, hpVEC-1 (or hpVEC-2) with a 24-day culture period exhibited higher killing capacity compared to the control group, and also maintained higher killing capacity compared to VEC-1 (or VEC-2). These results confirm that immune cells engineered with the hpE1 monobody according to the present invention can maintain stable activity more persistently.

[0196] [Example 10]

[0197] The killing effects of hpVEC-1 and VEC-1 on ovarian and gastric cancer cells were compared using three-dimensional (3D) co-culture.

[0198] Real-time cell analysis methods have limitations when using two-dimensional culture methods. Therefore, a three-dimensional spheroid culture method, which better simulates tumors, was used to compare the functions of two VEC-1 chimeric antigen receptor T cell lines. 0.5 x 10^4 cells of gastric cancer cells and ovarian cancer cells were mixed with RPMI medium containing 2.5% artificial basement membrane (Matrigel, Corning), and placed in 96-well round-bottomed dishes without cell adhesion. After centrifugation at 1000 x g for 10 minutes, the cells were cultured for 48 hours. Since the artificial basement membrane solidifies at room temperature, the entire process was performed on ice. After confirming the formation of spheroids through the reaction of the artificial basement membrane with the cells, the VEC-1-expressing T cells were treated. The culture medium was collected at 16, 24, 48, and 72 hours, and the amount of lactate dehydrogenase (LDH) (Promega) in the medium was measured. Figure 10As shown, in spheroids using the ovarian cancer cell line (OVCAR5), LDH levels gradually increased over time, but the difference between the two cell types was not significant. However, in spheroids using the gastric cancer cell line (SNU638), the LDH levels measured in the group treated with T cells expressing hydrophilic VEC-1 were higher than those in the group treated with existing VEC-1. This indicates that T cells expressing hydrophilic VEC-1 have a stronger cytotoxic capacity compared to T cells expressing existing VEC-1.

[0199] [Example 11]

[0200] Evaluation of the antitumor capacity of immune cells in xenograft mouse models

[0201] All animal experiments were conducted with the approval of the Bioethics Committee (IACUC, Chonnam National University, South Korea). Four-week-old female NOG mice were provided by Kootech SPF Animal Laboratory.

[0202] Ovary cancer cells OVCAR-5 were used at 2x10 5 OVCAR-3 at a concentration of 100 μL / cell was suspended in a 1:1 mixture of PBS (Welgene) and a high-concentration artificial basement membrane (Corning). In the experiment, OVCAR-3 (2 x 10⁻⁶ cells / 100 μL) was subcutaneously injected into the right rib area of ​​mice. 5 cell).

[0203] When the tumor size reaches 50 to 100 mm 3 At that time, 5x10 6 Administer 200 μL of prepared PBS, control group T cells, VEC-1, and hpVEC-1 immune cells intravenously once. Use 4 to 7 mice per group, and measure tumor size and mouse weight twice a week.

[0204] 11-1. Cell thawing and cell culture

[0205] Experimental materials: RPMI 1640 (Gibco), FBS (Gibco), P / S (Gibco), L-glutamine 200mM (Gibco), 100 μL cell culture flasks, baskets, tumor cell line (OVCAR-5), Solution 18 AODAPI (ChemoMetec), PBS (Welgene), TrypLE TM (Gibco). The human ovarian cell line was provided and used by the Korea Cell Bank.

[0206] Cell thawing: The tumor cell line was removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. When a thin layer of ice floated to the surface, it was transferred to a clean bench and placed into a 15 mL conical tube. 9 mL of cell culture medium (RPMI 1640 + 10% FBS + 1% P / S + 2 mM L-glutamine) was added to the 15 mL tube and centrifuged at 350g for 3 minutes. The supernatant was removed, and 1 mL of cell culture medium was added to completely resuspend the cells. Then, 9 mL of cell culture medium was added to bring the total volume to 10 mL, and the cells were resuspended again. The cell count was calculated using PBS: Solution 18 = 90:5, and the cell density was adjusted to 0.5 x 10⁻⁶ cells / mL. 6 / 10 mL and 1x10 6 / 10 mL, and spread the cells in a 100∮ flask. Then subculture every 3 days (when the cell density reaches 70-80%).

[0207] Subculture: After removing the supernatant from the flask, wash with 10 mL of PBS to remove cell debris and other impurities, then discard the supernatant. To remove the cells from the flask, use TrypLE... TM (2 mL or 4 mL) were placed in 100∮ and 150∮ flasks, respectively. Under a microscope, if the cells appeared round and floating, 10 mL or 30 mL of cell culture medium (RPMI 1640 + 10% FBS + 1% P / S + 2 mM L-glutamine) was added. The cells were then loaded into 50 mL conical tubes and centrifuged at 500 g for 3 to 5 minutes. After removing the supernatant, 1 mL of cell culture medium was added to resuspend the cells, followed by 9 mL of cell culture medium. Cell counts were determined using PBS: Solution 18 = 90:5. Cancer cells (e.g., ovarian cancer cells OVCAR-5: 1 x 10⁻⁵) were then counted. 6 (10 mL) was placed in a culture container for incubation.

[0208] 11-2. Preparing the mice

[0209] Experimental materials: PICO 5053 (Ausstar Biotech), 18% γ-irradiated β-pad (beta-chip) (Ausstar Biotech), sterilized tap water and water bottle, autoclave, razor (JEUNGDO BIO&PLANT), wet wipes, isoflurane solution (Hanna Pharmaceutical), depilatory agent (Veet), inhalation anesthesia machine and anesthesia chamber.

[0210] Experimental Methods: Feed (PICO 5053), bedding (r-irradiated 18% β-bedding), sterilized tap water, and cages were changed twice weekly. Hair was removed 3 days before tumor implantation and removed again during the experiment when hair grew to the point of obscuring the tumor. The concentration of isoflurane was adjusted to 2-3%, and mice were anesthetized by inhalation. Hair on the right rib area of ​​the mice was shaved with a razor, followed by application of depilatory agent and leaving it on for approximately 1 minute. The depilatory agent was then wiped off with a damp paper towel.

[0211] 11-3. Preparation of a mouse model of human tumor xenograft

[0212] Experimental materials: Cells, 1ml syringe (Korea Vaccine Corporation), 27 ½ G needle (Korea Vaccine Corporation), high-concentration artificial basement membrane (Corning Corporation), ice box, EP tubing, inhalation anesthesia machine and anesthesia chamber, isoflurane solution (Hanna Pharmaceutical Corporation). The artificial basement membrane was placed in a refrigerator overnight the night before xenografting. Because the artificial basement membrane gels at room temperature, experiments were often conducted on ice.

[0213] Experimental method: Mice were anesthetized by inhalation with 2-3% isoflurane, and then 100 μL of OVCAR-5 (2 x 10⁻⁶) was subcutaneously injected into the right rib area of ​​the mice. 5 A solution of cells and artificial basement membrane mixed in a 1:1 ratio.

[0214] 11-4. Injection of chimeric antigen receptor T cells

[0215] Experimental materials: 27 ½ G needles (Jung Lin Pharmaceutical Co., Ltd.), 1 ml syringe (Korea Vaccine Co., Ltd.), PBS, normal T cells (control group), E1 Monobody CAR-T cells (VEC-1), hydrophilic E1 Monobody CAR-T cells (hpVEC-1), mouse restraint device (JEUNGDO BIO & PLANT), heat irradiator (JEUNGDO BIO & PLANT), 70% alcohol swabs, vernier calipers (JEUNGDO BIO & PLANT), weighing scale, camera.

[0216] Experimental methods: Tumor size and body weight were measured in mice, and the tumor size and body weight of mice in each group were adjusted to be similar. When the tumor size reached 50 to 100 mm... 3 At that time, 200µl of PBS and cells (5x10) were added. 6 The control group (normal T cells, VEC-1, and hpVEC-1) was injected intravenously into the tail vein of mice (200 μL of normal T cells, VEC-1, and hpVEC-1).

[0217] 11-5. Mouse monitoring (tumor size and body weight measurement)

[0218] Experimental materials: Inhalation anesthesia machine and anesthesia chamber, isoflurane solution (Hanna Pharmaceutical Co., Ltd.), vernier calipers (JEUNGDO BIO & PLANT), weighing scale, camera.

[0219] Experimental methods: Mice were anesthetized by inhalation with 2-3% isoflurane. Tumor size and body weight were measured twice a week. The long axis (L), short axis (W), and height (H) of the mouse tumor were measured with calipers and then multiplied by 0.52 to calculate the size of the mouse tumor. The body weight of the mice was measured using an electronic scale.

[0220] 11-6. Confirmation of the killing ability of ovarian cancer cell lines

[0221] 2x10 ovarian cancer cell line (OVCAR-5) 5 In a mouse model where cells / 100µl were xenografted into subcutaneous tissue on the right back of mice, tumors grew for 14 days. Afterward, each group was intravenously injected with PBS, control T cells (normal T cells from the activated control group without viral transduction; Cont-T), VEC-1 cells, and hpVEC-1 cells. Tumor size was then measured using a ruler. The results are as follows: Figure 11 As shown.

[0222] like Figure 11 As shown, in mice injected with VEC-1 or hpVEC-1, the tumor size was significantly smaller from 14 days after tumor growth compared with mice injected with only control group normal T cells or PBS.

[0223] After intravenous administration of E1 monobody CAR-T cells, the body weight of mice was measured to assess whether their physical condition had recovered and whether the administration caused any side effects. The results are as follows: Figure 11 As shown.

[0224] like Figure 11 As shown, compared with the control group of mice with normal T cells and VEC-1, mice given hpVEC-1 began to recover to a healthy state more quickly starting from 8 days after administration.

[0225] [Example 12]

[0226] Preparation of a dual-target vector for PD-L1 single-chain fragment antibody (VYPC) and hydrophilic E1 monobody antibody.

[0227] 12-1. Preparation for the preparation of recombinant lentiviral vectors

[0228] Prepare the following strains and vectors to prepare recombinant plasmid vectors expressing chimeric antigen receptors based on hydrophilic monobody antibodies.

[0229] Thermo Fisher Scientific (GeneArt) synthesized and used the pLenti7.3-VYPC vector containing the nucleic acid sequence (Y-Biologics) of a PD-L1 single-chain fragment antibody (VYPC) with sequence number 18 that specifically binds to human PD-L1, and the pMX-hpE1 vector containing the nucleic acid sequence (sequence number 20) of a hpE1 monobody antibody that specifically binds to human EphA2, to prepare vectors for expressing chimeric antigen receptors. Specifically, the expression vector used was Invitrogen's pLenti7.3 / V5-DEST vector. Between the 5'UTR and 3'UTR of the pLenti7.3 / V5-DEST vector was a nucleic acid sequence for expressing a chimeric antigen receptor, which consisted of either a CD8α leader sequence-E1 Monobody sequence-CD8 H&TM-CD28-CD3ζ or a CD8α leader sequence-E1 Monobody sequence-CD8 H&TM-4-1BB-CD3ζ. The nucleic acid sequences constituting the Monobody-based chimeric antigen receptor in the recombinant plasmid vector are shown in Table 5 below.

[0230] Table 5

[0231]

[0232]

[0233]

[0234]

[0235] The nucleic acid sequences in Table 5 can be expressed in immune cells, and the chimeric antigen receptor based on PD-L1 and hydrophilic EphA2 monobody, containing the amino acid sequences in Table 6 below, can be expressed on the cell surface membrane.

[0236] Table 6

[0237]

[0238]

[0239] 12-2. Preparation of a recombinant lentiviral vector expressing a dual-target chimeric antigen receptor of PD-L1 single-chain fragment antibody (VYPC) and hydrophilic E1 monobody antibody (Monobody).

[0240] Using the two prepared lentiviral vectors (pLenti7.3::CD8α leader sequence-E1 monobody-CD8H&T-CD28-CD3ζ vector and pLenti7.3::CD8α leader sequence-E1 monobody-CD8 H&T-4-1BB-CD3ζ vector) as templates, PCR was performed using the CPL-F and CPL-R primers listed in Table 7. The amplified vectors were then isolated and purified to prepare two chimeric antigen receptor vectors that do not contain monobody antibodies.

[0241] For the VYPC ScFv-linker peptide gene sequence used for the dual-target chimeric antigen receptor VYPC ScFv-linker peptide-hpE1 monobody (Monobody)-Flag tag (hereinafter referred to as VYPC-linker peptide-hpE1-Flag), PCR amplification was performed on the pLenti7.3-VYPC vector using the VYPC-forward primer and VYPC-linker peptide-reverse primer listed in Table 3. For the hpE1 monobody (Monobody)-Flag gene sequence, PCR amplification was performed on the pMX-hpE1 vector using the linker peptide-hpE1-forward primer and hpE1-Flag-reverse primer listed in Table 7. The amplified gene sequences were isolated and purified, and overlap PCR was performed using the VYPC-forward primer and hpE1-Flag-reverse primer listed in Table 7. Similar to the above method, the hpE1 monobody-linker-VYPC ScFv-Flag tag (hereinafter referred to as hpE1-linker-VYPC-Flag) was amplified using hpE1-F and hpE1-linker-reverse primers listed in Table 7. For the VYPC-Flag gene sequence, the pLenti7.3-VYPC vector was amplified using the linker-VYPC-forward primer and VYPC-Flag-reverse primer listed in Table 7. The amplified gene sequence was isolated and purified, and then amplified using overlap PCR with the hpE1-forward primer and VYPC-Flag-reverse primer listed in Table 3.

[0242] For the prepared vector, VYPC-linker peptide-hpE1-Flag sequence, and hpE1-linker peptide-VYPC-Flag sequence, using the Overlap cloner DNA cloning kit (Elpisbio), the sequences were inserted into the original site of the E1 Monobody sequence via homologous recombination to prepare four recombinant lentiviral vectors expressing chimeric antigen receptors containing dual-target antibodies and Monobody (pLenti7.3::CD8α leader-VYPC ScFv-hpE1 Monobody chimeric antigen receptor vector and pLenti7.3::CD8α leader-hpE1 Monobody-VYPC ScFv chimeric antigen receptor vector). The prepared recombinant lentiviral vectors were named PD-L1-hpEphA2 mCAR-1 (VYPC-hpVEC-1), PD-L1-hpEphA2 mCAR-2 (VYPC-hpVEC-2), hpEphA2-PD-L1 mCAR-1 (hpVEC-VYPC-1), and hpEphA2-PD-L1 mCAR-2 (hpVEC-VYPC-2), respectively, and their structures are as follows. Figure 12 As shown.

[0243] Table 7

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] [Example 13]

[0262] Comparative assessment of the activities of VYPC and hpVEC-1, as well as dual-target CAR cells containing both.

[0263] The expression of active and repressive factors in the created bichiral antigen receptor T cells was compared with that in single chiral antigen receptor T cells at a set time difference. Figure 13 As shown, CD62L expression was high in both CAR-T cells after preparation, but decreased after one month, especially compared to the two single CAR-T cell lines, while maintaining a higher level in dual CAR-T cells. The same was observed with CD25; its expression was generally higher in dual CAR-T cells compared to single CAR-T cells. Regarding repressors, CTLA-4 and PD-1 were almost not expressed after preparation, while TIGIT expression was roughly the same. Except for dual CAR-T cells expressing VYPC_hpVEC-1, TIM-3 expression was higher than in single CAR-T cells. After one month, CTLA-4, TIGIT, and TIM-3 all showed a high expression trend in dual CAR-T cells.

[0264] [Example 14]

[0265] Comparison of in vitro cell-killing capabilities of dual-target CAR cells containing PD-L1 scFv and hydrophilic EphA2 monobody in cell lines overexpressing antigens PD-L1 and EphA2.

[0266] The study investigated the cytotoxicity of dual-target CAR-T cells in HEK293 cells expressing PD-L1, HEK293 cells expressing EphA2, and a mixture of cells expressing both PD-1 and EphA2. To create HEK293 cells expressing PD-L1, a plasmid containing EphA2 cDNA was purchased from AddGene (#121486) and introduced into HEK293 cells using Lipofectamine 2000. After 48 hours, cells were screened for plasmid insertion into the HEK293 cell genome by treatment with puromycin (5 μg / ml). Single cells were then transferred to 96-well plates and cultured. For each clone proliferating in single cells, EphA2 protein expression in HEK293 cells was confirmed by FACS analysis using a PD-L1-PE antibody. HEK293 cells overexpressing PD-L1 and HEK293 cells overexpressing EphA2 were cultured in RTCA plates at a concentration of 1 x 10^4 / 100 μL. 0.5 x 10^4 PD-L1 overexpressing cells and 0.5 x 10^4 EphA2 overexpressing cells were co-cultured. Single-CAR-T and dual-CAR-T cells were then treated with 1 x 10^4 / 100 μL, followed by RTCA analysis. The results are shown below. Figure 14 As shown, the anti-cancer ability of VYPC_hpVEC-1 is significantly higher than that of hpVEC-1.

[0267] [Example 15]

[0268] Comparison of the anti-cancer efficacy of dual-target CAR cells containing PD-L1 scFv and hydrophilic EphA2 monobody against two subtypes of OVCAR5 derived from ovarian cancer metastasized from gastric cancer.

[0269] To confirm the efficacy of the dual CAR-T cells in cancer cell lines and compare them with single CAR-T cells, RTCA was performed using the ovarian cancer cell line (OVCAR5) following the same method described above. OVCAR5 cells were cultured in microplates at 1x10^4 / 100 µl for 24 hours, followed by treatment with both single and dual CAR-T cells at 1x10^4 / 100 µl, and monitoring was conducted for 72 hours. The results are as follows: Figure 15 As shown, compared with single-target immune cells of VEC-1 and hpVEC-1, dual-target immune cells of VYPC_hpVEC-1 or hpVEC_VYPC-2 have significantly higher killing ability.

[0270] [Example 16]

[0271] Evaluation of the antitumor capabilities of single-target and dual-target immune cells in xenograft mouse models

[0272] Animal experiments were performed using the same method as in Example 11, and the results are as follows: Figure 16 and Figure 17 As shown, in the OVCAR-5 xenograft disease model of ovarian cancer cell line, both single-target (hpVEC-1, VYPC-1) and dual-target VYPC_hpVEC-1 CAR cells exhibited similar levels of high killing ability. However, regarding the toxic component, neither VYPC-1 nor VYPC_hpVEC-1 showed toxicity, and the mice recovered their weight rapidly and well. Conversely, mice injected with hpVEC-1 experienced a continuous decline in weight and decreased coat luster, and 2 out of 7 mice died at the end of the experiment (day 15).

[0273] Therefore, it can be seen that the dual-target chimeric antigen receptor based on VYPC_hpVEC-1 monobody according to the present invention, and the immune cells (e.g., cytotoxic T cells) expressing it on the cell membrane surface, have excellent anti-cancer effects, significantly inhibiting the proliferation of cancer cells such as ovarian cancer and gastric cancer, and significantly improving the survival rate of mice. It has also been confirmed that it can significantly improve the toxicity of the hpVEC-1 single-target receptor. Therefore, immune cells containing PD-L1 scFv and the chimeric antigen receptor based on the hydrophilic monobody can be used to prevent or treat solid cancers.

Claims

1. A monobody that specifically binds to liver glycoprotein receptor A2, wherein one or more hydrophobic amino acids of the monobody with sequence number 46 are replaced by hydrophilic amino acids and / or one or more hydrophilic amino acids are added.

2. The Monobody according to claim 1, comprising the amino acid sequence of sequence number 8.

3. The Monobody according to claim 1, wherein the Monobody is directly or via a linker peptide linked to a single-chain variable region fragment of programmed cell death protein ligand 1.

4. A chimeric antigen receptor comprising an extracellular ligand-binding domain of a monobody according to any one of claims 1 to 3, and further comprising one or more of a group consisting of a transmembrane domain and an intracellular signal transduction domain.

5. The chimeric antigen receptor according to claim 4, wherein the transmembrane domain is selected from the group consisting of: T cell receptor α chain, T cell receptor β chain, T cell receptor ζ chain, CD8 α chain, CD8 β chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a portion thereof, and any combination thereof.

6. The chimeric antigen receptor according to claim 4, wherein the intracellular signal transduction domain is selected from the group consisting of: TCR ζ, FcR γ, FcR β, FcR ε, CD3 γ, CD3, CD3 ε, CD3 ζ, CD5, CD22, CD79a, CD79b and CD66d and any combination thereof.

7. The chimeric antigen receptor according to claim 4, wherein the intracellular signal transduction domain is selected from one or more of the following groups: OX40 domain, CD2 domain, CD27 domain, CD28 domain, CDS domain, ICAM-1 domain, LFA-1 domain and 4-1BB domain, and any combination thereof.

8. The chimeric antigen receptor of claim 4, wherein It also includes the hinge area.

9. A polynucleotide comprising a nucleic acid sequence encoding a Monobody of any one of claims 1 to 3 or a chimeric antigen receptor of any one of claims 4 to 8.

10. A carrier comprising the polynucleotide of claim 9.

11. An isolated immune cell expressing, on its cell surface membrane, an extracellular ligand-binding domain comprising a Monobody according to any one of claims 1 to 3 or a chimeric antigen receptor according to any one of claims 4 to 8.

12. The immune cells isolated according to claim 11 are selected from a group consisting of leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, and dendritic cells.

13. A pharmaceutical composition comprising the isolated immune cells of claim 11 for the prevention or treatment of cancer.

14. The pharmaceutical composition of claim 13, wherein, The cancer mentioned is selected from one or more cancers in the group consisting of melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colorectal cancer, hematopoietic system tumors and their metastatic cancers.

15. The pharmaceutical composition according to claim 14, wherein the cancer is pancreatic cancer, prostate cancer, or ovarian cancer.

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