Hydrophilic monobody having improved properties targeting epha2 and multispecific chimeric antigen receptor and engineered immune cell comprising same

Abstract

The present invention relates to a monobody-based chimeric antigen receptor. The monobody-based chimeric antigen receptor comprises an extracellular ligand binding domain including a monobody. In addition, the present invention relates to an immune cell having the monobody-based chimeric antigen receptor expressed on the cell surface membrane thereof. The monobody-based CAR-immune cell: 1) can exhibit excellent effects in preventing or treating cancer by targeting a receptor expressed on the surface of a cancer cell; 2) uses human FN3-based monobodies as extracellular ligand binding domains unlike animal-derived scFv-based CAR-immune cells, thereby exhibiting low immunogenicity when administered to humans; 3) has a smaller size than scFv, thereby providing higher tissue penetration capability; 4) can minimize side effects (e.g. occurrence of autoimmune diseases due to non-specific binding) associated with conventional antibody therapies for cancer treatment; and 5) enables various monobody-based CAR-immune cells to be produced by using already-established monobody libraries for various cancer cell antigens. Accordingly, the present invention can be usefully employed in preventing or treating intractable solid tumors having various antigens

Description

Hydrophilic monobodies with improved properties targeting EphA2 and multispecific chimeric antigen receptors and engineered immune cells comprising the same

[0001] The present invention relates to a hydrophilic monobody in which a portion of the sequence of fibronectin (FN3), which targets EphA2, is replaced with a hydrophilic amino acid, and to a chimeric antigen receptor comprising the hydrophilic monobody, and more particularly, to a dual chimeric antigen receptor targeting both EphA2 and PD-L1. The present invention also relates to an isolated immune cell comprising the dual chimeric antigen receptor and its use in cancer treatment.

[0002]

[0003] Surgical resection alone is not sufficient to completely eliminate cancer stem cells, residual cancer cells, or metastatic cancer cells in the treatment of solid tumors. Therefore, combined treatments, combining surgical resection with radiation therapy, chemotherapy, targeted therapy, and immunotherapy, are often used. Immunotherapy, in particular, utilizes the body's immune system, so it rarely carries the side effects associated with radiation therapy or chemotherapy.

[0004] T cells, a representative example of immune cells, mediate the adaptive immune response and account for 75% of all lymphocytes. Most T cells exist in the blood in an inactive state, but upon antigen stimulation, they become activated and can eliminate cells harboring specific antigens. T cell activation can be triggered by antigen-recognizing receptors (T cell receptors; TCRs), co-stimulatory molecules, and cytokines.

[0005] Immune cells, including T cells, kill cancer cells by binding to MHC (major histocompatibility complex) molecules bound to tumor-specific antigens. However, cancer cells suppress the expression of MHC, change the function of immune checkpoints such as CTLA-4 (cytotoxic T-lymphocyte associated protein-4), PD-1 (programmed cell death protein-1), LAG-3 (lymphocyte-activation gene-3), KIR (killer cell immunoglobulin-like receptor), TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), and VISTA (V-domain Ig suppressor of T cell activation) expressed on the surface of immune cells, or express ligands for immune checkpoints (programmed cell death protein ligand-1). In addition, they change the tumor microenvironment to favor cancer growth and intravascular migration. In particular, one of the greatest challenges in treating solid tumors is the presence of diverse antigens, which, unlike hematological cancers, can differentiate into different cancer cells within the same tumor type, making it difficult to identify specific cancers. Consequently, therapies targeting at least two antigens have begun to be developed for solid tumors. Examples include catumaxomab (peritoneal cancer, withdrawn from the market in 2017), the first bispecific antibody approved in 2009; blinatumomab (hematological cancer), the second approved in 2014; and Kimtrak and Lunsumio, both approved in the US and Europe in 2022.

[0006] As cancer cells have discovered that immune checkpoints allow them to survive immune cells, the development of drugs that block the binding of immune checkpoint proteins to their ligands on cancer cells has been actively pursued. As a result, monoclonal antibodies that specifically bind to the immune checkpoint proteins PD-1 and CTLA-4 have been developed. PD-1 and CTLA-4 antibodies bind to PD-1 and CTLA-4, respectively, on the immune cell membrane. This prevents these immune checkpoint proteins from binding to their ligands on cancer cells, thereby maintaining the immune cell's ability to kill cancer cells. Examples of such antibody-based immunotherapies include atezolizumab, avelumab, and ipilimumab.

[0007] Ephrin receptor, a representative cancer antigen but for which no antibody treatment has yet been developed, is a membrane receptor tyrosine kinase (RTK) with a size of 108 kDa, which is divided into three parts: a ligand-binding domain, a cysteine-rich region, and two fibronectin III repeat structures on the outside of the cell membrane, a central transmembrane region, and a kinase activation domain on the cytoplasmic side (Labrador et al., 1997; Pasquale, 1997). Ephrin receptors are proteins that bind to their ligands, ephrins, and transmit extracellular signals into cells through a phosphorylation process. They are involved in cell proliferation, cell migration, differentiation, synaptic transmission of nerve cells, tissue remodeling, bone cell 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 ephrin receptors and ligand-receptor specificity, they are classified into nine types of group A (EphA1-8 and EphA10) and five types of group B (EphB1-4 and EphB6) (Ieguchi K. Endocr Metab Immune Disord Drug Targets. 15:119-128, 2015; Eph Nomenclature Committee. Cell. 90:403-404, 1997; Lindberg RA et al., Mol Cell Biol. 10(12):6316-6324, 1990; Davis S et al., Science. 266(5186):816-819, 1994).Among these, ephrin receptor A2 (EphA2) is expressed in extremely small amounts in normal cells, but is nonspecifically overexpressed in most cancer cells, including liver cancer, prostate cancer, pancreatic cancer, head and neck cancer, stomach cancer, colon cancer, lung cancer, cervical cancer, and ovarian cancer, and promotes carcinogenesis, metastasis, angiogenesis, and resistance of tumors. EphA2 shares approximately 65% ​​protein homology with EphA1 and is found in nearly identical locations in the mouse lung. It is overexpressed with EphA1 and its ligand, Ephrin-A1, in several malignancies, including non-small cell lung cancer, and is involved in carcinogenesis and malignant transformation through EphA2 / A1-Ephrin-A1 interaction (Naj AC et al., Nat Genet. 43:436-441, 2011; Finney AC et al., Circulation. 136:566-582, 2017).

[0008] Since 2017, immune cell-based immunotherapy has been developed, which, unlike conventional immunotherapy, specifically binds to cancer cells and eliminates them through the body's immune cells. These cancer treatments include T cells expressing chimeric antigen receptors (CARs) that can directly recognize tumor-associated antigens (CAR T cells), or T cells expressing antibody sequences specific to immune checkpoint receptors on cancer cells. These cancer treatments enable immune cells to specifically attack cancer cells.

[0009] The structure of currently developed chimeric antigen receptors is divided into a single-chain variable fragment (scFv) portion that recognizes the antigen, a transmembrane domain, and a signaling domain that transmits signals into the cell. Pharmaceutical companies such as Novartis, Gilead, Celgene, AbClon, and Janssen, as well as clinicians, have developed CAR-T therapies using scFvs that specifically bind to the CD19 antigen on blood cancer cells or nanobodies that specifically bind to the BCMA antigen of multiple myeloma.

[0010] For most types of cancer, solid tumors, CAR-based immune cell therapy that combines various genetic engineering technologies is absolutely required. However, solid tumors have heterogeneity, where each patient expresses different antigens, complex characteristics of the tumor microenvironment such as hypoxia, and restrictions on T cell migration and activity. According to recent clinical trial results reports, when dual targeting is used to overcome heterogeneity, a stronger immune response is ensued than when targeting a single antigen, resulting in serious side effects including cytokine storm and neurotoxicity. On the other hand, when the antibodies used in antibody drugs are used as they are, that is, when the antigen recognition site is directly utilized as a CAR immune cell therapy without considering the strong antigen binding characteristic of antibody drugs, serious side effects due to on-target and off-tumor effects have been reported.

[0011] Existing cancer immunotherapy is known as a treatment for blood cancer that recognizes and binds to human cancer antigens using mouse-based antibody molecules, specifically antibody fragments (scFv), and then removes cancer cells through the secretion of various cytokines or attacks by surrounding immune cells. However, it has problems such as side effects due to animal-based antibodies, low tissue penetration due to the size of scFv, and the need to recognize at least two antigens simultaneously due to the characteristic of expressing different antigens for each patient in the case of solid cancer (heterogeneity).

[0012] The present inventors, while conducting research to complement the shortcomings of scFv and hydrophobic monobodies, confirmed that when a hydrophilic monobody based on PD-L1 scFv and hydrophilic human fibronectin FN3 is used as an extracellular ligand binding site of a chimeric antigen receptor, it has an excellent effect in preventing and treating solid cancer, thereby completing the present invention.

[0013]

[0014] (Prior art literature)

[0015] (Patent Document)

[0016] (Patent Document 1) Korean Patent No. 10-2533540

[0017] (Patent Document 2) Korean Patent No. 10-2048477

[0018] (Patent Document 3) Korean Patent No. 10-2157197

[0019]

[0020] The present invention aims to provide a hydrophilic monobody that specifically binds to EphA2.

[0021] The present invention aims to provide a chimeric antigen receptor comprising the above monobody.

[0022] The present invention aims to provide a vector comprising a polynucleotide comprising a nucleic acid sequence encoding the monobody or chimeric antigen receptor.

[0023] The present invention aims to provide the above polynucleotide.

[0024] The present invention aims to provide an immune cell expressing an extracellular ligand binding domain including the above monobody or the above chimeric antigen receptor on the cell surface membrane.

[0025] The present invention aims to provide a pharmaceutical composition for preventing or treating cancer containing the above-mentioned immune cells.

[0026]

[0027] The present invention provides a hydrophilic monobody comprising an amino acid sequence of SEQ ID NO: 46, wherein one or more polar nonionic amino acids are substituted with hydrophobic amino acids. The monobody may comprise an amino acid sequence of SEQ ID NO: 8.

[0028] The above monobody may be linked directly or via a linker to a PD-L1 scFv. The PD-L1 scFv may comprise the amino acid sequence of SEQ ID NO: 27.

[0029] The present invention provides a chimeric antigen receptor comprising the above monobody as an extracellular ligand binding domain and further comprising at least one selected from the group consisting of a transmembrane domain and an intracellular signaling domain.

[0030] The transmembrane domain may be selected from the group consisting of T-cell receptor alpha chain, T-cell receptor beta chain, T-cell receptor zeta chain, CD8 alpha chain, CD8 beta chain, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a portion thereof, and any combination thereof.

[0031] The intracellular signaling domain may be selected from the group consisting of TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b, and CD66d, and any combination thereof.

[0032] The intracellular signaling domain may be at least one selected from the group consisting of an OX40 domain, a CD2 domain, a CD27 domain, a CD28 domain, a CDS domain, an ICAM-1 domain, an LFA-1 domain, and a 4-1BB domain, and any combination thereof.

[0033] The above chimeric antigen receptor may additionally comprise a hinge.

[0034] The present invention provides a polynucleotide comprising a nucleic acid sequence encoding the monobody or the chimeric antigen receptor.

[0035] The present invention provides a vector comprising the above polynucleotide.

[0036] The present invention provides an immune cell expressing an extracellular ligand binding domain comprising the above monobody or the above chimeric antigen receptor on the cell surface membrane.

[0037] The above-mentioned immune cells may be selected from the group consisting of leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, and dendritic cells.

[0038] The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the above-described immune cells.

[0039] The cancer may 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, hemangiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colon cancer, hematopoietic tumor, and metastatic cancers thereof.

[0040] The cancer may be pancreatic cancer, prostate cancer, or ovarian cancer.

[0041]

[0042] The hydrophilic monobody-based chimeric antigen receptor according to the present invention, the immune cell expressing the same on the cell surface membrane, and the composition using the same for preventing or treating cancer can exhibit the following effects.

[0043] 1) It can reduce side effects (e.g., hair loss and digestive disorders) that occur by suppressing the growth of normal cells in conventional radiation therapy or chemotherapy such as DNA nucleotide analogs (cisplatin series and 5-FU series) that only suppress rapid cell proliferation.

[0044] 2) By using a small-sized monobody based on human FN3, it is possible to minimize autoimmune disease side effects and cancer cell metastasis due to anticancer drug resistance that occur in targeted antibody therapy used as an antibody-based anticancer treatment derived from animals and chimeric T-cell therapy using scFv obtained from a library of mouse monoclonal antibodies.

[0045] 3) It overcomes the limitations of monobodies that were used only for diagnosis due to their inability to kill cancer, instability, and short lifespan, and can effectively suppress or eliminate solid tumors expressing cancer antigens (e.g., EphA2), enabling simultaneous diagnosis and treatment of cancer.

[0046] 4) Monobody-based CAR immune cells can exhibit superior anticancer effects compared to conventional T-cell-based solid cancer treatment, and can be used as an immuno-oncology agent to treat or prevent solid cancers such as pancreatic cancer, prostate cancer, and ovarian cancer that express EphA2 as an antigen.

[0047] 5) The FN3 monobody has the advantage of being able to produce a variety of monobodies targeting target antigens. This allows for the production of monobody-based CAR immune cells specific for various antigens, making them highly effective in treating cancer.

[0048] 6) Monobody-based CAR immune cells are smaller than other CAR immune cells (for example, the size of the monobody is about 1 / 3 the size of the scFv), so they can penetrate cancer tissues more easily than scFv-based CARs. In addition, when producing a multivalent CAR in a viral vector, up to four monobody sequences that recognize cancer antigens can be loaded into the vector considering viral packing, which has the advantage of overcoming the heterogeneity of cancer antigens, which is a characteristic of solid cancer. In addition, since it has sufficient selectivity and specificity for cancer antigens, it can block cancer immune cell evasion, etc., and effectively prevent or treat cancer.

[0049] 7) The existing hydrophilic fibronectin FN3 domain has been reported to complement its function as a biomarker by strongly binding to the target and absorbing it into the endosome within the cell (Sirois AR, Deny DA, Baierl SR, George KS, Moore SJ (2018) Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding. PLoS ONE 13(5): e0197029. https: / doi.org / 10.1371 / journal.pone.0197029), but it has not been used for therapeutic purposes, and in particular, there has been no report of its use as a cell gene therapy agent. The hpEphA2 monobody-based CAR-immune cell (hpVEC) using the hydrophilic fibronectin FN3 domain for which a patent application is being filed was confirmed to have an expression rate more than twice that of the existing EphA2 monobody CAR-immune cell (hpVEC), which means that the probability of recognizing target cancer cells in the body can be significantly increased. In silico immunogenicity prediction analysis showed that immunogenicity could be further reduced.

[0050] The two cancer antigen PD-L1 and EphA2 specific chimeric antigen receptors according to the present invention and the immune cells containing them not only have excellent PD-L1 and EphA2 specific targeting efficiency, but also exhibit excellent anticancer effects even on cancer cells expressing only a single PD-L1 or EphA2 antigen, even if the cancer cells do not express PD-L1 or EphA2 or remove them themselves for immune evasion, so that they can be usefully used for preventing or treating tumor growth and metastasis.

[0051]

[0052] Figure 1 is a schematic diagram showing the structure of a vector expressing a hydrophilic monobody-based chimeric antigen receptor.

[0053] Figure 2a is a schematic diagram comparing the amino acid sequences of a non-hydrophilic monobody (E1) and a hydrophilic monobody (hpE1).

[0054] Figure 2b is a schematic diagram showing structural similarity by superimposing the 3D structures of a nonhydrophilic monobody (E1) and a hydrophilic monobody (hpE1) based chimeric antigen receptor.

[0055] Figure 2c is a graph comparing the protein binding ability between a non-hydrophilic monobody (E1) and a hydrophilic monobody (hpE1) for human antigen EphA2.

[0056] Figure 3 is a graph comparing the transduction rates between T immune cells (hpVEC-1) containing a hydrophilic monobody-based chimeric antigen receptor or T immune cells (VEC-1) containing a non-hydrophilic monobody-based chimeric antigen receptor.

[0057] Figure 4 is a graph comparing activation markers between pVEC-1 and VEC-1.

[0058] Figures 5a and 5b are graphs comparing inhibition markers between hpVEC-1 and VEC-1.

[0059] Figure 6 is a graph comparing the cytotoxicity of hpVEC-1 and VEC-1 against the human cancer antigen EphA2 overexpressing HEK293 kidney cell line.

[0060] Figures 7a and 7b are graphs comparing the cancer cell killing ability (ovarian cancer, gastric cancer, glioblastoma, pancreatic cancer) of hpVEC-1 and VEC-1 in vitro using RTCA analysis technique.

[0061] Figure 8 is a graph showing the results of analyzing the expression levels of CD62L, CD25, and CD69, which are activating factors, and CTLA-4, TIGIT, TIM-3, LAG-3, and PD-1, which are inhibitory factors, using FACS, 24 days after the completion of manufacturing hpVEC-1 and VEC-1, of immune cells.

[0062] Figure 9 is a graph showing the cancer cell killing ability of hpVEC-1 and VEC-1 immune cells using RTCA analysis technique.

[0063] Figure 10 is a graph showing the killing ability of hpVEC-1 and VEC-1 against ovarian and gastric cancer cells by time using an ELISA analyzer and the amount of LDH released by dead cancer cells present in the 3D spheroid co-culture method and the medium.

[0064] Figure 11 is a graph showing changes in tumor size and mouse body weight recovery in an ovarian cancer xenograft mouse model.

[0065] Figure 12 is a schematic diagram illustrating the structure of a dual chimeric antigen receptor vector that simultaneously recognizes two antigens, PD-L1 and EphA2.

[0066] Figure 13 is a graph comparing the activating and suppressing factors that are characteristics of T lymphocyte immune cells containing heterologous chimeric antigen receptors.

[0067] Figure 14 is a graph comparing the killing efficacy of T lymphocyte immune cells containing a PD-L1 and EphA2 dual antigen recognition chimeric antigen receptor using RTCA analysis using a HEK293 cell line that simultaneously overexpresses PD-L1, EphA2, and two antigens.

[0068] Figure 15 is a graph comparing the killing efficacy of T lymphocyte immune cells containing PD-L1 and EphA2 dual antigen recognition chimeric antigen receptors against two types of subpopulations in the ovarian cancer cell line OVCAR-5 derived from gastric cancer, using RTCA analysis.

[0069] Figure 16 is a graph showing changes in tumor size and mouse body weight recovery in an ovarian cancer xenograft mouse model.

[0070] Figure 17 is a graph of a mouse survival curve drawn using the Kaplan-Meier formula in an ovarian cancer xenograft mouse model.

[0071]

[0072] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise specifically stated.

[0073] Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0074] Unless otherwise specified, the practice of the present invention may utilize conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which may be within the skill of the art.

[0075] The term "prevention" as used in the present invention means any action that inhibits or delays the onset of a disease by administering a composition, "treatment" means any action that improves or beneficially changes the symptoms of a subject suspected of or suffering from a disease by administering a composition, and "improvement" means any action that at least reduces a parameter related to a condition, for example, the degree of a symptom, by administering a composition.

[0076]

[0077] Chimeric antigen receptor based on PD-L1 scFv and hydrophilic monobody

[0078] The present invention relates to a chimeric antigen receptor (CAR) based on PD-L1 scFv and a hydrophilic monobody.

[0079] The PD-L1 scFv and hydrophilic monobody-based chimeric antigen receptor of the present invention may comprise an extracellular ligand binding domain.

[0080] The term "extracellular ligand binding domain" as used herein may refer to an oligopeptide or polypeptide capable of binding a ligand. Preferably, the domain can interact with cell surface molecules. For example, the extracellular ligand binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with any disease.

[0081] The term "linker" used in the present invention refers to amino acids that connect protein domains. The "linker" of the present invention may have at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 19, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99%.

[0082] The term "hinge" as used in the present invention may be a naturally occurring hinge region or a synthetic amino acid sequence, and preferably may be a part of the CD8 alpha chain.

[0083] The PD-L1 scFv sequence of the present invention may 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 18.

[0084] The term "hydrophilic monobody (hp)" used in the present invention may be a stable form between proteins, such as hydrogen bonding, that specifically binds to a ligand or antigen present on the surface of a target cell by substituting a polar uncharged amino acid with a hydrophobic amino acid sequence in the backbone of the EphA2 monobody.

[0085] Hydrophilic monolayers can be composed of amino acids with uncharged polar side chains. Therefore, they can be charged depending on the pH, and the side chains of serine (S) and threonine (T) contain hydroxyl groups that can form hydrogen bonds, which are important for stabilizing protein structure and other biochemical reactions. They can also play a crucial role in protein-protein interactions and interactions between proteins and other molecules. Asparagine (N) and glutamine (Q), in particular, may be important for targeting and protein recognition.

[0086] Hydrophilic monobodies can be prepared by substituting 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), asparagine (D), and glutamic acid (E), or by adding hydrophilic amino acids. For example, hydrophilicity can be increased by substituting hydrophobic valine (V) with hydrophilic amino acids such as serine (S) or threonine (T), or by substituting hydrophobic leucine (L) with hydrophilic threonine (T), or by adding a sequence of 1 to 1000 amino acids containing hydrophilic amino acids (E, S).

[0087] Monobodies can be selected in a variety of ways depending on the desired target. For example, the monobody may specifically bind to a protein selected from the group consisting of CRL1, ephrin receptor, EphA2, ß-galactosidase, RAS, Abl kinase, VEGFR2, MBP, SARS-CoV-2, Bcr-Abl kinase, STAT3, EGFR, VEGFR2, SH3 domain of human Lyn tyrosine kinase, MLKL, Fluc homologues, mitogen-activated protein kinase (MAPK), ERK2, MAPK14, kinase SH2 domain, SUMO, AurA, WDR5, Fluc family, GFP, receptor binding domain of SARS-CoV-2, SH3 domain of FYN, SH2 domain of ABL, SUMO1, Bcr-Abl, GPR56 ECR, PD-L1, glypican-3, PCSK9, Gp41, CD4, and IL-23. It may be combined, but is not limited to:

[0088] Additionally, the monobody may be known.

[0089] The monobody may be one that specifically binds to the ephrin receptor, EphA2, or human EphA2.

[0090] The monobody may 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 SEQ ID NO: 8.

[0091] The monobody-based CAR may further comprise one or more selected from the group consisting of a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0092] Additionally, monobody-based CARs are expressed on the surface membrane of cells. Accordingly, monobody-based CARs may comprise a transmembrane domain. A distinguishing characteristic of a suitable transmembrane domain is that it is expressed on the surface of a cell, preferably an immune cell (particularly a lymphocyte or NK cell) in the present invention, and that it interacts with the cell to induce a cellular response of the immune cell against a predetermined target cell. The transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0093] The transmembrane domain may be at least one selected from the group consisting of a T-cell receptor alpha chain, a T-cell receptor beta chain, a T-cell receptor zeta chain, a CD8 alpha chain, a CD8 beta chain, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, portions thereof, and combinations thereof.

[0094] The hinge and transmembrane domains may comprise a human CD8 alpha chain or a portion thereof. The hinge and transmembrane domain may comprise the amino acid sequence of SEQ ID NO: 15, and preferably may comprise an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 22.

[0095] The intracellular signaling domain of a monobody-based CAR can trigger intracellular signaling after binding the extracellular ligand-binding domain to a target, thereby activating immune cells and an immune response. The signaling domain can trigger the activation of at least one of the normal effector functions of an immune cell expressing the monobody-based CAR. For example, the effector function of a T cell can be cytotoxic activity, including the secretion of cytokines, or helper activity.

[0096] The term "signaling domain" as used in the present invention may mean a portion of a protein that transduces an effector function signal within an immune cell and induces the cell to perform a specific function.

[0097] The intracellular signaling domain may comprise one or more selected from the group consisting of TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b and CD66d.

[0098] The intracellular signaling domain may include at least one selected from the group consisting of an OX40 domain, a CD2 domain, a CD27 domain, a CD28 domain, a CDS domain, an ICAM-1 domain, an LFA-1 domain, and a 4-1BB domain.

[0099] The intracellular signaling domain may comprise one or more from the group consisting of a CD28 domain, a 4-1BB domain, and a CD3 zeta domain, preferably a CD28 domain and a CD3 zeta domain, or a 4-1BB domain and a CD3 zeta domain.

[0100] The CD28 domain may comprise the amino acid sequence of SEQ ID NO: 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 thereto.

[0101] The 4-1BB domain may comprise the amino acid sequence of SEQ ID NO: 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 thereto.

[0102] The CD3 zeta domain may comprise the amino acid sequence of SEQ ID NO: 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 thereto.

[0103]

[0104] Polynucleotides and vectors

[0105] The present 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, the vector may mean an expression vector.

[0106] The polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 2. The polynucleotide may comprise a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 45.

[0107] The nucleic acid sequence encoding the hydrophilic monobody-based CAR may be codon-optimized for expression in human cells.

[0108] As used herein, "vector" may refer to a construct capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest into a host cell. Examples thereof include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0109] Viral vectors can include retroviruses, adenoviruses parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as finornaviruses 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., vaccinia, fowlpox, and canarypox). Other viruses that can be used as vectors can be, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses may include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentiviruses, and spumaviruses.

[0110]

[0111] engineered immune cells

[0112] The present invention may relate to an isolated cell or cell line obtainable by the method for producing or manipulating the above-described immune cells. The isolated cell may comprise a monobody-based CAR. The monobody-based CAR may preferably be a CAR comprising a hydrophilic monobody according to the present invention and a PD-L1 scFv.

[0113] The isolated cells of the present invention may comprise a population of CARs comprising different extracellular ligand binding domains. In particular, the isolated cells may comprise an exogenous polynucleotide sequence encoding a CAR.

[0114] The immune cells of the present invention may refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The immune cells of the present invention may be derived from stem cells. The stem cells may be adult stem cells, non-human embryonic stem cells, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, pluripotent stem cells, or hematopoietic stem cells.

[0115] The immune cells of the present invention may be human CD3+ T cells. In one embodiment, the isolated cells may be T cells selected from the group consisting of dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and helper T lymphocytes. In one embodiment, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0116] Cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0117] Any T cell line known and available to those skilled in the art may be used. The cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. The cells may be part of a mixed population of cells exhibiting different phenotypic characteristics. Cell lines obtained from T cells engineered according to the above-described method may be used, and the immune cells according to the present invention may be resistant to immunosuppressive treatment.

[0118] The above immune cells may be, but are not limited to, leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, dendritic cells, or a combination thereof.

[0119]

[0120] Treatment using PD-L1 scFv and hydrophilic monobody-based dual antigen recognition CAR immune cells

[0121] PD-L1 scFv and hydrophilic monobody-based dual antigen recognition CAR-immune cells, engineered immune cells or populations of these cells can be used as pharmaceutical compositions for preventing or treating cancer.

[0122] The present invention may relate to a pharmaceutical composition for preventing or treating cancer, comprising a PD-L1 scFv and a hydrophilic monobody-based dual antigen recognition CAR-immune cell or an engineered immune cell.

[0123] The present invention may relate to a method for preventing or treating cancer in a subject in need thereof, comprising administering to the subject a PD-L1 scFv and hydrophilic monobody-based dual antigen recognition CAR-immune cell.

[0124] The present invention may relate to the use of PD-L1 scFv and hydrophilic monobody-based dual antigen recognition CAR-immune cells for the manufacture of a medicament for preventing or treating cancer.

[0125] The present invention may be a dual antigen recognition CAR-immune cell based on PD-L1 scFv and hydrophilic monobody for use in preventing or treating cancer.

[0126] The term "subject" as used herein refers to a mammal, more preferably a human. Mammals include, but are not limited to, livestock, pets, primates, horses, dogs, cats, mice, and rats.

[0127] In the present invention, the treatment may be part of autologous immunotherapy or allogeneic immunotherapy. "Autologous" may mean that the cells, cell lines, or populations of cells used to treat the subject are obtained from the subject or from a donor compatible with human leukocyte antigen (HLA). "Allogeneic" may mean that the cells or populations of cells used to treat the subject are obtained from a donor rather than from the subject.

[0128] The cancer may be, but is not limited to, 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, colon cancer, hematopoietic tumor, or metastases thereof. In addition, the cancer may be a non-solid tumor or a solid tumor.

[0129] Non-solid tumors or non-solid cancers may include, but are not limited to, myeloma, lymphoma, or leukemia.

[0130] The solid cancer may be one or more selected from the group consisting of, but is not limited to, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lung cancer.

[0131] Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various forms and is not limited to the embodiments and examples described herein.

[0132]

[0133] [Example 1]

[0134] Preparation of vectors expressing chimeric antigen receptors based on hydrophilic monobodies

[0135]

[0136] 1-1. Preparation for production of recombinant lentiviral vectors

[0137] The following strains and vectors were prepared for the production of recombinant plasmid vectors expressing hydrophilic monobody-based chimeric antigen receptors.

[0138]

[0139] (1) Vector to be copied

[0140] A pMX-hpE1 vector containing the hpE1 monobody nucleic acid sequence of SEQ ID NO: 2 that specifically binds to human EphA2 was synthesized by Thermo Fisher (GeneArt), and a CAR vector expressing a chimeric antigen receptor was prepared. Specifically, the expression vector used was the pLenti7.3 / V5-DEST-vector of Invitrogen, and between the 5' UTR and 3' UTR of the pLenti7.3 / V5-DEST vector, the following sequences were included: a CD8α leader sequence of SEQ ID NO: 1 - an E1 monobody sequence of SEQ ID NO: 45 - a CD8 H&TM sequence of SEQ ID NO: 3 - a CD28 sequence of SEQ ID NO: 4 - a CD3ζ sequence of SEQ ID NO: 6; Or, it was made to include a nucleic acid sequence expressing a chimeric antigen receptor consisting of a CD8α leader sequence of sequence number 1 - an E1 monobody sequence of sequence number 45 - a CD8 H&TM sequence of sequence number 3 - a 4-1BB sequence of sequence number 5 - a CD3ζ sequence of sequence number 6. Two lentiviral vectors were prepared in this way (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).

[0141] The nucleic acid sequences constituting the hydrophilic monobody-based chimeric antigen receptor in the recombinant plasmid vector are as shown in Table 1 below.

[0142]

[0143]

[0144]

[0145] The nucleic acid sequence of Table 1 above can be expressed in immune cells and expressed on the cell surface membrane as a monobody-based chimeric antigen receptor comprising the amino acid sequence of Table 2 below.

[0146]

[0147]

[0148]

[0149] (2) Preparation of cells for vector replication

[0150] Host cell for pMX-hpE1 vector: E. coliDH5α(F-endA1glnV44 thi-1 recA1 relA1gyrA96 deoR nupG purB20φ80dlacZ△M15 △(lacZYA-argF)U169, hsdR17(rK-mK+), λ-)

[0151] Host cells for p lentiviral vectors: E. coli Stbl3(F-mcrB mrrhsdS20(rB-, mB-)recA13supE44ara-14galK2lacY1proA2rpsL20(Str R )xyl-5 λleumtl-1), Invitrogen™

[0152] The host cells were cultured using LB (Luria-Bertani) solid medium (Difco Laboratories, USA) at 37°C and 200 rpm, and 100 μg / ml kanamycin or ampicillin was added to all media to prepare the host cells. Afterwards, a single colony of each cultured host cell was inoculated into LB liquid medium (Difco Laboratories, USA) for pre-culture, and this was inoculated into SOB (Super Optimal Broth) medium at 1%, cultured at 18°C ​​for 24 hours, and then washed with Inoue TB buffer to prepare.

[0153]

[0154] (3) Production of transformed cells for mass production of vectors

[0155] After mixing the prepared vectors and host cells, the cells were placed on ice for 10 minutes, then treated at 42°C for 50 seconds and then on ice for 3 minutes to produce transformed cells introduced with each vector. The transformed cells were plated on LB solid medium containing antibiotics and cultured at 37°C. Through this, each vector was prepared in large quantities.

[0156]

[0157] 1-2. Production of a recombinant lentiviral vector expressing hydrophilic E1 monobody-chimeric antigen receptor (hpE1 CAR)

[0158] Using the two lentiviral vectors prepared above (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, PCR was performed using the CPL-F primer and CPL-R primer in Table 3 below. The vectors thus amplified were isolated and purified to prepare two types of scFv-free chimeric antigen receptor vectors.

[0159] The hpE1 monobody gene sequence was prepared by amplifying the pMX-hpE1 vector by PCR using the hpE1-F primer and hpE1-R primer in Table 3 below, and then isolating and purifying it.

[0160] Two types of recombinant lentiviral vectors (pLenti7.3::CD8α leader-hpEphA2 monobody chimeric antigen receptor vectors) expressing chimeric antigen receptors including hpE1 monobody were produced by inserting the hpE1 monobody sequence into the site where the E1 monobody sequence existed through homologous recombination using the prepared vector and hpE1 monobody sequence using an Overlap cloner DNA cloning kit (Elpisbio). The produced recombinant lentiviral vectors were named hpEphA2 mCAR-1 (hpVEC-1) and hpEphA2 mCAR-2 (hpVEC-2), respectively, and their structures are shown in Fig. 1.

[0161]

[0162]

[0163]

[0164] [Example 2]

[0165] Comparison of hpE1 monobody and E1 monobody

[0166]

[0167] 2-1. Sequence and structural alignment of hpE1 monobody and E1 monobody

[0168] Amino acid sequence alignment between the hpE1 monobody and the E1 monobody was performed using Clustal Omega. The aligned sequences were then run through the Jalview program, and identical sequences between the hpE1 monobody and the E1 monobody were indicated.

[0169] AlphaFold was used to obtain predicted structures of the hpE1 monobody and E1 monobody. The predicted structures were based on the relaxed structure of the highest-ranked structure. The superimposition method of pymol was used to assess the structural similarity between the hpE1 monobody and E1 monobody. Figures 2a and 2b show the protein sequence differences and overlaps between the hpE1 and E1 monobodies.

[0170]

[0171] 2-2. Comparison of immunogenicity between hpE1 monobody and E1 monobody

[0172] We performed database analysis of the monobody sequences that bind to EphA2 and predicted MHC-II epitope binding of IEDB. As shown in Table 4 below, according to the IEDB MHC-II binding prediction method, the hpE1 and E1 monobodies are predicted to have low immunogenicity overall. However, due to the hydrophobic amino acids introduced into the BC Loop, the E1 monobodies are predicted to have a relatively higher antigen presentation potential than the hpE1 monobodies at the junction of the B beta-strand and the BC Loop.

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] 2-3. Measurement of binding affinity of hpE1 monobody to EphA2 antigen protein

[0179] 1) Purification of hydrophilic E1 monobody and E1 monobody protein from E. coli

[0180] To construct an E. coli expression vector for the monobody protein, the hydrophilic E1 monobody and E1 monobody genes were amplified by PCR. The amplified PCR fragments were cleaved at both ends with NdeI and BamHI restriction enzymes and ligated into the restriction sites of the pETamh (AviTag-Myc-6xHis) expression vector. The pETamh expression vector, derived from pET (Novagen, USA), has an Nde1 restriction site containing an ATG initiation codon at the 5' end and a BamH1 restriction site followed by an amh tag site before the stop codon at the 3' end. Therefore, the monobodies expressed with the amh tag linked to the C-terminus of the monobodies were designated hpE1-amh and E1-amh, respectively. The hpE1 monobody and E1 monobody inserted into the pETamh expression vector were transformed into BL21(DE3) and expressed. A single colony was inoculated into 10 ml of LB medium containing kanamycin (50 ug / ml), cultured overnight, and harvested by centrifugation. The culture was resuspended in 1 ml of LB medium and 1 ml of glycerol (50%). 200 μL of the strain was inoculated into 1 L of LB medium and cultured 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 culture, and the culture was further cultured overnight at 20°C and 250 rpm. The cultured E. coli was centrifuged at 4°C and 8000 rpm for 10 minutes to obtain an E. coli precipitate. The E. coli pellet was disrupted by adding 10 ml of cold lysis buffer (20 mM Tris-HCl (7.4), 100 mM NaCl) per 1 g of E. coli pellet, and centrifuged at 13,000 rpm for 30 min. hpE1-amh and E1-amh in the obtained supernatant were separated by imidazole gradient elution on a Biorad FPLC equipped with a HisTrap column (EconoFit Nuvia IMAC Columns, Ni-charged 5 ml).The primary purified monobody protein was further separated in PBS buffer using size exclusion chromatography (Superdex™ 75 Increase 10 / 300 GL).

[0181]

[0182] [Example 3]

[0183] Production of immune cells expressing hpE1 monobody-chimeric antigen receptors on their cell membrane surfaces (hpVEC-1 and VEC-1) and comparative evaluation of transduction rates.

[0184]

[0185] 3-1. Production of lentivirus containing recombinant lentiviral vector

[0186] Lentiviruses containing hpEphA2 mCAR-1 and EphA2 mCAR-1 manufactured in Example 1 were prepared. Lentivirus production and titration were performed using LV-MAX provided by Thermo Scientific. TM The Lentiviral Production System (A35684) kit was used and manufactured according to the manufacturer's experimental method.

[0187]

[0188] 3-2. Preparation of immune cells and transduction through viral infection

[0189] (1) Isolation of CD3+ T cells from PBMC

[0190] Peripheral blood mononuclear cells (PBMCs) from human donor blood were prepared using Lymphoprep TM , STEMCELL). Afterwards, human CD3+ T cells were isolated from the separated PBMCs using the positive selection method using CD3 microbeads (Miltenyi Biotech).

[0191] (2) Activation of CD3+ T cells

[0192] Anti-CD3 / anti-CD28 magnetic beads (anti-CD3 / CD28 Dynabead; Gibco) were mixed with isolated human CD3+ T cells in a 1:1 ratio. After 24 hours of mixing, the activated magnetic beads were removed using a MACSiMAG separator device. The cells were then washed with RPMI-1640.

[0193] (3) Transduction with lentivirus

[0194] Lentivirus-infected T cells were prepared using a spinoculation technique using a centrifuge. Specifically, activated human CD3+ T cells were infected with lentiviruses containing hpEphA2 mCAR-1 and EphA2 mCAR-1, respectively, and the viral medium was replaced 24 hours later. Cell proliferation began 48 hours after infection. The engineered immune cells prepared in this manner were designated hpVEC-1 and VEC-1, respectively. An Attune NxT flow cytometer (Thermo Fisher Scientific) was used for the expression rate experiment of the introduced vector (i.e., hpE1 monobody-chimeric antigen receptor sequence). As shown in Fig. 3, the results of comparative analysis of the number of GFP-expressing cells by FACS showed that, under culture conditions supplemented with IL-2, hpVEC-1 immune cells showed an expression rate of about 60% compared to the control group not introducing the vector, and E1 monobody-chimeric antigen receptor VEC-1 immune cells showed an expression rate of about 23%. In addition, under culture conditions supplemented with IL-7 and IL-15, hpVEC-1 showed an expression rate of about 56% compared to the control group not introducing the vector, and VEC-1 showed an expression rate of about 25%. These results confirm that the vector introduced into the immune cells is well expressed, and mean that the number of cells expressing hpE1 monobody is more than twice as high as that of E1 monobody. These results suggest that hpVEC-1 and hpVEC-2 are likely to recognize cancer cells expressing EphA2 much earlier or in greater numbers than VEC-1 and VEC-2 in the treatment of patients with cells expressing the tumor antigen EphA2.

[0195]

[0196] [Example 4]

[0197] Comparative evaluation of cell activity of hpVEC-1 and VEC-1

[0198] Whether the hpEphA2 mCAR-1 and EphA2 mCAR-1 vectors introduced in the hpVEC-1 and VEC-1 immune cells manufactured above were properly expressed and the activity status of these immune cells was confirmed using an Attune NxT flow cytometer (fluorescence-activated cell sorting).

[0199] For each immune cell, the expression of the introduced vector was confirmed using an anti-GFP (Green Fluorescent Protein) antibody. Since the vector contains a GFP expression site, the analysis result by the anti-GFP antibody will be measured higher in immune cells that express a lot of this vector. In addition, an anti-CD62L-APC antibody (APC Mouse Anti-Human CD62L, BD Pharmingen) TM ), anti-CD25-PE antibody (PE Mouse Anti-Human CD25, BD Pharmingen TM ) and anti-CD69-APC antibody (APC Mouse Anti-Human CD69, BD Pharmingen TM ) was used to confirm whether T cells were activated.

[0200] As shown in Fig. 4, in the case of the in vitro test, it was confirmed that CD62L, a cell growth activity marker, was expressed more than 85% higher in the manipulated immune cells hpVEC-1 and VEC-1, similar to the control group, and CD25 was expressed more than 20% higher than the control group.

[0201]

[0202] 2) Measurement of binding affinity (Kd dissociation constant) of purified monobody proteins

[0203] The binding affinity of hpE1 monobody to hEphA2 was evaluated by measuring the dissociation constant (Kd) using ELISA analysis. 1 μg / mL of recombinant EphA2 protein (R&D systems) was dispensed 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 with 200 μL of blocking buffer (PBST containing 5% BSA) for 2 h at room temperature. Each well was washed three times with PBST, and hpE1-amh and E1-amh monobody proteins were added at serially half-diluted concentrations starting from 500 nM and allowed to bind for 2 h at room temperature. Each well was washed with PBST and then treated with 100 μl of HRP-conjugated c-Myc monoclonal antibody (Invitrogen) for 1 h at room temperature. After washing, each well was incubated with 100 μl of substrate [a 1:1 mixture of substrate (TMB) and hydrogen peroxide, BD Biosciences] for 5 min at room temperature. The reaction was stopped with 50 μl of 1 M H2SO4, and the color development was read at 450 nm using an ELISA reader. As shown in Figure 2c, hpE1 was confirmed to have stronger binding affinity to hEphA2 than E1.

[0204]

[0205] [Example 5]

[0206] Comparative evaluation of cytostatic factor expression in hpVEC-1 and VEC-1

[0207]

[0208] To compare the expression of inhibitory factors in T cells expressing hydrophilic VEC-1 and conventional 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 using FACS buffer, and 1 x 106 After reacting with each CAR-T cell at 4℃ for 30 minutes, the cells were washed twice with 200 μl of FACS buffer, and then the expression of each protein was analyzed by FACS. As shown in Figures 5a and 5b, the expression of CTLA4 or PD-1 was very low in all immune cells expressing conventional VEC-1 or hydrophilic VEC-1, including the control group in which the virus was not introduced, and the expression of TIGIT and TIM-3 was about 5-10% lower in immune cells expressing hydrophilic VEC-1 compared to conventional VEC-1, whereas the expression of LAG-3 showed a difference of about 5.73% compared to cells expressing conventional VEC-1.

[0209]

[0210] [Example 6]

[0211] In vitro killing activity of hpVEC-1 and VEC-1 in EphA2-overexpressing HEK293 cells

[0212]

[0213] To confirm the killing ability of T cells expressing the hydrophilic VEC-1 against cells expressing EphA2, a stable HEK293 cell line overexpressing EphA2 was created. The plasmid containing the cloned EphA2 cDNA was purchased from Addgen (#116732) and introduced into HEK293 cells using lipofectamine 2000. After 48 hours, only cells in which the plasmid was integrated into the HEK293 cell genome were selected by treating with 800 ug / ml of G418, and single cells were transferred one by one to 96-well plates and cultured. Each clone grown from a single cell was analyzed by FACS using the EphA2-PE antibody to confirm that the EphA2 protein was expressed in HEK293 cells. Real-time cell analysis (RTCA) was performed to measure the cytotoxicity of T cells expressing each VEC-1 against HEK293 cells overexpressing EphA2. 1 x 10^4 / 100 ul of HEK293 cells were cultured in microtiter plates and monitored for 24 hours. After 24 hours, the same number of T cells expressing hydrophilic VEC-1 were treated, and the killing ability of T cells expressing hydrophilic VEC-1 was confirmed through RTCA. As shown in Figure 6, T cells that did not express CAR (control) had no effect, but T cells expressing hydrophilic VEC-1 showed the effect of killing almost all HEK293 cells expressing EphA2.

[0214]

[0215] [Example 7]

[0216] Evaluation of cancer cell killing ability in vitro

[0217]

[0218] To compare the killing ability, CAR-T cells were treated with 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, and then RTCA was performed in the same manner as in Example 6. As shown in Figures 7a and 7b, T cells expressing hydrophilic VEC-1 showed toxicity to all cancer cell lines, and almost the same efficacy as T cells expressing conventional VEC-1 was confirmed.

[0219]

[0220] [Example 8]

[0221] Stability Evaluation of hpVEC-1 and VEC-1 Cellular Efficacy (Persistency)

[0222]

[0223] In order to investigate the persistence of each marker factor of T cells expressing conventional VEC-1 and T cells expressing hydrophilic VEC-1, T cells expressing each chimeric antigen receptor were generated and cultured for 25 days. The ratio of CD4+: CD8+ cells, activator markers, and inhibitory factor markers were compared using the antibodies used in Examples 3 and 4. As shown in Fig. 8, compared to T cells without CAR introduction (control group), after 25 days, the ratio of CD4+ showed a relatively higher value than the control group, and there was no significant difference compared to conventional VEC-1. Regarding the activator marker, CD62L was expressed higher in T cells expressing hydrophilic VEC-1 than in T cells expressing conventional VEC-1, while CD25 and CD69 were expressed slightly lower. Regarding the inhibitory factors, TIGIT, Tim3, and LAG-3 were expressed lower in T cells expressing hydrophilic VEC-1 than in T cells expressing conventional VEC-1.

[0224]

[0225] [Example 9]

[0226] Evaluation of in vitro cancer cell killing activity of hpVEC-1 and VEC-1 24 days after production

[0227]

[0228] The killing ability of transduced hpE1 monobody CAR-T cells (hpVEC-1 or hpVEC-2), E1 monobody CAR-T cells (VEC-1 or VEC-2), and non-transduced human T cells (control) against gastric cancer cells was analyzed by RTCA. As confirmed in Example 7, the killing ability of hpVEC-1 (or hpVEC-2) produced 9 days after manufacturing was much higher than that of the control group, but the killing ability was confirmed to be almost similar to that of VEC-1 (or VEC-2). However, after an additional culture period of 24 days after manufacturing, each was co-cultured with cancer cells using RTCA equipment, and the killing ability was re-examined. As shown in Fig. 9, hpVEC-1 (or hpVEC-2) cultured for 24 days showed higher killing capacity compared to the control group, and also maintained higher killing capacity compared to VEC-1 (or VEC-2). From these results, it was confirmed that immune cells engineered with the hpE1 monobody according to the present invention stably maintain activity for a long period of time.

[0229]

[0230] [Example 10]

[0231] Comparison of the killing capacity of hpVEC-1 and VEC-1 against ovarian and gastric cancer cells using 3D coculture.

[0232]

[0233] Since real-time cell analysis has limitations in two-dimensional culture, the functions of two VEC-1 chimeric antigen receptor T cells were compared using a three-dimensional spheroid culture method that better mimics tumors. 0.5 x 10^4 cells of each gastric cancer cell line and ovarian cancer cell line were mixed with RPMI medium containing 2.5% Matrigel (Corning), placed in a non-attachable round-bottom 96-well culture dish, centrifuged at 1000 x g for 10 minutes, and cultured for 48 hours. Because Matrigel solidifies at room temperature, all procedures were performed on ice. After confirming that the reaction between Matrigel and cells formed spherical spheroids, T cells expressing each VEC-1 were treated, and after 16, 24, 48, and 72 hours, the medium was collected and the amount of LDH contained in the medium was measured (Promega). As shown in Fig. 10, in spheroids using an ovarian cancer cell line (OVCAR5), the amount of LDH gradually increased over time, but there was little difference between the two cells. However, in spheroids using a gastric cancer cell line (SNU638), a higher amount of LDH was measured in the group treated with T cells expressing hydrophilic VEC-1 than in the group treated with conventional VEC-1. This shows that the function of T cells expressing hydrophilic VEC-1 has a more enhanced cell killing ability than T cells expressing conventional VEC-1.

[0234]

[0235] [Example 11]

[0236] Evaluation of the antitumor activity of immune cells in xenograft mouse models

[0237]

[0238] All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC, Chonnam National University, Republic of Korea). Four-week-old female NOG mice were provided by the Cortech SPF Animal Laboratory.

[0239] Ovarian cancer cells OVCAR-5 were cultured at 2 x 10 in a 1:1 mixture of PBS (Welgene) and high-concentration Matrigel (Corning). 5 cells / 100ul concentration. In the experiment, OVCAR-3 (2 x 10 5 cells) were injected subcutaneously.

[0240] Tumor size 50 to 100 mm 3 When reached, 5 x 10 prepared PBS, control T cells, VEC-1, hpVEC-1 immune cells 6 / 200ul was injected intravenously once. 4 to 7 mice were used per group, and tumor size and mouse body weight were measured twice a week.

[0241]

[0242] 11-1. Cell thawing and cell culture

[0243] Experimental materials: RPMI 1640 (Gibco), FBS (Gibco), P / S (Gibco), L-glutamine 200 mM (Gibco), 100∮ cell culture flask, basket, tumor cell line (OVCAR-5), Solution 18 AODAPI (ChemoMetec), PBS (Welgene), TrypLE TM (Gibco). Human ovarian cell lines were supplied and used from the Korea Cell Line Bank.

[0244] Cell thawing: After quickly thawing the tumor cell line in a 37℃ constant temperature water bath from the liquid nitrogen tank, it was moved to a clean bench while the ice was floating, and placed in 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 350 g for 3 minutes. The supernatant was removed, 1 mL of cell culture medium was added, and resuspended completely. 9 mL of cell culture medium was added to make 10 mL and resuspended. The cell number was counted using a cell counting solution (PBS: Solution 18 = 90:5) and the cell density was 0.5 x 10 6 / 10 mL and 1 x 10 6 / 10 mL was adjusted and cells were spread in a 100∮ flask. Subculture was performed at 3-day intervals (when the cell density reached 70-80%).

[0245] Subculture: After removing the supernatant from the flask, add 10 mL of PBS to wash to remove impurities such as cell debris, and discard the supernatant. TrypLE was used to detach the cells from the flask. TM (2 mL or 4 mL) were placed in 100∮ and 150∮ flasks, respectively. When cells were observed under a microscope to be 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, and the cells were placed in a 50-ml conical tube and centrifuged at 500 g for 3 to 5 minutes. After removing the supernatant, 1 mL of cell culture medium was added and resuspended, and then 9 mL of cell culture medium was added. The cell number was measured using a cell counting solution (PBS: Solution 18 = 90:5) and cancer cells (e.g., ovarian cancer cells OVCAR-5: 1x10 6 / 10 mL) was placed in a culture vessel and cultured.

[0246]

[0247] 11-2. Preparing the mouse

[0248] Experimental materials: PICO 5053 (Orient Bio), γ-irradiated 18% beta-chip (Orient Bio), sterilized tap water and water bottle, high-pressure steam sterilizer, clipper (Jeongdo B&P), wet tissue, Ipran liquid (Hana Pharmaceutical), hair removal agent (Veet), respiratory anesthesia machine and chamber

[0249] Experimental method: Feed (PICO 5053), litter (irradiated 18% beta-chip), sterilized tap water, and cage were changed twice a week. Hair was removed 3 days before tumor implantation, and during the experiment, when the tumor had grown to the point where it was no longer visible, it was removed. Mice were anesthetized with 2-3% isoflurane, and the hair on the right flank of the mouse was removed with clippers, a depilatory agent was applied, left on for about 1 minute, and the depilatory agent was wiped off with a wet tissue.

[0250]

[0251] 11-3. Production of human tumor xenograft mouse models

[0252] Experimental materials: cells, 1 ml syringes (Korea Vaccine), 27 1 / 2 G needles (Korea Vaccine), high-concentration Matrigel (Corning), icebox, EP tubing, anesthesia machine and chamber, and Ipran solution (Hana Pharmaceutical). Matrigel was refrigerated overnight the day before xenografting. Because Matrigel gels at room temperature, experiments were always conducted on ice.

[0253] Experimental method: OVCAR-5 (2 x 10) was injected into the right flank of mice anesthetized with 2-3% isoflurane. 5 100 ul of a solution containing 1:1 mixture of cells and Matrigel was injected subcutaneously.

[0254]

[0255] 11-4. Chimeric antigen receptor T cell infusion

[0256] Experimental materials: 27 ½ G needle (Jeonglimsa), 1 ml syringe (Korea Vaccine), PBS, normal T cells (control), E1 monobody CAR-T cells (VEC-1), hydrophilic E1 monobody CAR-T cells (hpVEC-1), mouse compensator (Jeongdo B&P), heat probe (Jeongdo B&P), 70% alcohol swab, vernier caliper (Jeongdo B&P), scale, camera.

[0257] Experimental method: The tumor size and body weight of the mice were measured, and the tumor size and body weight of the mice were adjusted to be similar for each group. The tumor size was 50 to 100 mm. 3 When reached, 200 ul of PBS and cells (5 x 10 6 / 200ul of control normal T cells, VEC-1, and hpVEC-1 were injected intravenously into the tail vein of mice.

[0258]

[0259] 11-5. Mouse monitoring (measuring tumor size and weight)

[0260] Experimental materials: anesthesia machine and chamber, Ipran solution (Hana Pharmaceutical), vernier caliper (Jeongdo B&P), scale, camera.

[0261] Experimental Method: Mice were anesthetized with 2–3% isoflurane, and tumor size and body weight were measured twice a week. The size of mouse tumors was calculated by measuring the long axis (L), short axis (W), and height (H) with a Vernier caliper and multiplying by 0.52. The body weight of the mice was measured using an electronic scale.

[0262]

[0263] 11-6. Confirmation of ovarian cancer cell line killing ability

[0264] 2 x 10 ovarian cancer cell line (OVCAR-5) 5Fourteen days after tumor formation in a mouse model in which cells / 100ul were xenografted into the subcutaneous tissue of the right dorsal region, each group was intravenously injected with PBS, control T cells (activated control normal T cells not transduced with a virus; Cont-T), VEC-1 cells, and hpVEC-1 cells. The tumor size was measured using a scale bar, and the results are shown in Fig. 11.

[0265] As shown in Figure 11, after 14 days of tumor formation in mice injected with VEC-1 or hpVEC-1, the tumor size was significantly reduced compared to mice injected with only control normal T cells or PBS.

[0266] To determine whether the body condition of the mice recovered after intravenous administration of E1 monobody CAR-T cells and whether there were any side effects due to administration, the body weights of the mice were measured, and the results are shown in Figure 11.

[0267] As shown in Figure 11, the health status of mice administered hpVEC-1 recovered more quickly than that of mice administered control normal T cells and VEC-1 8 days after administration.

[0268]

[0269] [Example 12]

[0270] Preparation of PD-L1 single-chain fragment antibody (VYPC)-hydrophilic E1 monobody dual-targeting vector

[0271]

[0272] 12-1. Preparation for production of recombinant lentiviral vectors

[0273] The following strains and vectors were prepared for the production of recombinant plasmid vectors expressing hydrophilic monobody-based chimeric antigen receptors.

[0274] A vector expressing a chimeric antigen receptor was prepared by using the pLenti7.3-VYPC vector containing the PD-L1 single chain fragment antibody (VYPC) nucleic acid sequence of SEQ ID NO: 18 (Y-Biologics) that specifically binds to human PD-L1 and the pMX-hpE1 vector containing the hpE1 monobody nucleic acid sequence (SEQ ID NO: 20) that specifically binds to human EphA2, synthesized by Thermo Fisher (GeneArt). Specifically, the expression vector used was Invitrogen's pLenti7.3 / V5-DEST-vector, and the nucleic acid sequence expressing the chimeric antigen receptor composed of CD8α leader sequence-E1 monobody sequence-CD8 H&TM-CD28-CD3ζ or CD8α leader sequence-E1 monobody sequence-CD8 H&TM-4-1BB-CD3ζ was included between the 5' UTR and 3' UTR of the pLenti7.3 / V5-DEST vector. The nucleic acid sequences constituting the monobody-based chimeric antigen receptor in the recombinant plasmid vector are as shown in Table 5 below.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] The nucleic acid sequence of Table 5 above can be expressed in immune cells, and a chimeric antigen receptor based on PD-L1 and hydrophilic EphA2 monobody consisting of the amino acid sequence of Table 6 below can be expressed on the cell surface membrane.

[0281]

[0282]

[0283]

[0284] 12-2. Production of a recombinant lentiviral vector expressing a PD-L1 single-chain fragment antibody (VYPC)-hydrophilic E1 monobody dual-targeting chimeric antigen receptor

[0285] Using the two lentiviral vectors prepared above (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, PCR was performed using the CPL-F primer and CPL-R primer in Table 7 below. The vectors thus amplified were isolated and purified to prepare two types of monobody-free chimeric antigen receptor vectors.

[0286] The VYPC ScFv - Linker gene sequence for the dual-target chimeric antigen receptor VYPC ScFv - Linker - hpE1 monobody -Flag tag (hereinafter referred to as VYPC-Linker-hpE1-Flag) was amplified by PCR using the VYPC-F primer and VYPC-Linker-R primer in Table 3 in the pLenti7.3-VYPC vector. The hpE1 monobody - Flag gene sequence was amplified by PCR using the Linker-hpE1-F primer and hpE1-Flag-R primer in Table 7 in the pMX-hpE1 vector. The amplified gene sequence was isolated and purified, and overlap PCR was performed using the VYPC-F primer and hpE1-Flag-R primer in Table 7 in the following. hpE1 monoobdy - Linker - VYPC ScFv - Flag tag (hereinafter referred to as hpE1-Linker-VYPC-Flag) was amplified by PCR using the hpE1-F and hpE1-Linker-R primers in Table 7 below in a similar manner to the above method. The hpE1-Linker gene sequence was amplified by PCR using the pMX-hpE1 vector and the VYPC-Flag gene sequence was amplified by PCR using the Linker-VYPC-F and VYPC-Flag-R primers in Table 7 below in a pLenti7.3-VYPC vector. The amplified gene sequences were isolated and purified, and amplified by overlap PCR using the hpE1-F and VYPC-Flag-R primers in Table 3 above.

[0287] The prepared vector and VYPC-Linker-hpE1-Flag sequence and hpE1-Linker-VYPC-Flag sequence were inserted into the site where the E1 monobody sequence existed through homologous recombination using an Overlap cloner DNA cloning kit (Elpisbio), thereby producing four types of recombinant lentiviral vectors (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) expressing a chimeric antigen receptor including a dual targeting antibody and a monobody. The manufactured 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), and their structures are shown in Figure 12.

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] [Example 13]

[0308] Comparative evaluation of the activity of VYPC and hpVEC-1 and dual-targeting CAR cells containing them.

[0309]

[0310] The expression of activating and suppressing factors in the manufactured dual chimeric antigen receptor T cells was compared with that of single chimeric antigen receptor T cells over time. As shown in Figure 13, CD62L showed high expression rates immediately after production, but when compared one month later, CD62L expression decreased in all cases and was observed to be maintained higher in dual CAR-T cells than in both single CAR-T cells. CD25 was also observed to be expressed higher overall in dual CAR-T cells than in single CAR-T cells. Regarding suppressing factors, CTLA-4 and PD-1 were barely expressed immediately after production, while TIGIT was expressed at a similar level. TIM-3 showed higher expression than single CAR-T cells in all cases except for dual CAR-T cells expressing VYPC_hpVEC-1. After one month, CTLA-4, TIGIT, and TIM-3 all tended to be expressed higher in dual CAR-T cells.

[0311]

[0312] [Example 14]

[0313] Comparison of the in vitro cell killing capacity of dual-targeting CAR cells containing PD-L1 scFv and hydrophilic EphA2 monobody in cell lines overexpressing the antigens PD-L1 and EphA2.

[0314]

[0315] We investigated whether the developed dual-target CAR-T had cell killing ability in HEK293 cells expressing PD-L1, HEK293 cells expressing EphA2, and mixed cells expressing PD-1 and EphA2. To generate HEK293 cells expressing PD-L1, a plasmid containing the cloned EphA2 cDNA was purchased from Addgene (#121486) and introduced into HEK293 cells using lipofectamine2000. After 48 hours, only cells in which the plasmid was integrated into the HEK293 cell genome were selected by treating with 5 ug / ml of puromycin, and then single cells were transferred one by one to a 96-well plate and cultured. Each clone expanded from a single cell was analyzed by FACS using a PD-L1-PE antibody to confirm that the EphA2 protein was expressed in HEK293 cells. PD-L1 overexpressing HEK293 cells and EphA2 overexpressing HEK293 cells were cultured in RTCA plates at 1 x 10^4 / 100 ul each, and PD-L1 overexpressing cells and EphA2 overexpressing cells were mixed and cultured at 0.5 x 10^4 each. After 24 hours, single CAR-T and dual CAR-T were treated at 1 x 10^ / 100 ul each, and RTCA analysis was performed. As a result of the analysis, as shown in Fig. 14, it was confirmed that VYPC_hpVEC-1 had significantly higher anticancer ability than hpVEC-1.

[0316]

[0317] [Example 15]

[0318] Comparison of antitumor activity of dual-targeting CAR cells containing PD-L1 scFv and hydrophilic EphA2 monobody against two subtypes of OVCAR5 derived from ovarian cancer metastasized from gastric cancer.

[0319]

[0320] To confirm the efficacy of the manufactured dual CAR-T in cancer cell lines and compare it with single CAR-T, RTCA was performed using ovarian cancer cell lines (OVCAR5) using the same method as mentioned above. OVCAR5 cells were cultured in microtiter plates at 1 x 10^4 / 100 ul each, and after 24 hours, each single and dual CAR-T was treated at 1 x 10^4 / 100 ul each, and then monitored for 72 hours. As a result of the test, as shown in Fig. 15, VYPC_hpVEC-1 or hpVEC_VYPC-2 dual-target immune cells showed a superior killing ability than VEC-1 and hpVEC-1 single-target immune cells.

[0321]

[0322] [Example 16]

[0323] Evaluation of the antitumor activity of single- and dual-target immune cells in xenograft mouse models

[0324]

[0325] Animal testing was performed in the same manner as in Example 11, and as a result of the test, as shown in Figures 16 and 17, in the ovarian cancer cell line OVCAR-5 xenograft disease model, both single-target (hpVEC-1, VYPC-1) and VYPC_hpVEC-1 dual-target CAR cells showed similarly high killing abilities, but in terms of toxicity, VYPC-1 and VYPC_hpVEC-1 showed no toxicity, and the mice's body weight recovery rate was fast and good. On the other hand, mice injected with hpVEC-1 showed continuous weight loss, decreased fur luster, and two out of seven mice died on the 15th day of the experiment.

[0326] The VYPC_hpVEC-1 monobody-based dual-targeting chimeric antigen receptor according to the present invention and the immune cells (e.g., cytotoxic T cells) expressing the same on the cell membrane surface have an excellent anticancer effect that significantly inhibits the proliferation of cancer cells such as ovarian cancer and gastric cancer and significantly increases the survival rate of mice, and it has been confirmed that the toxicity of the hpVEC-1 single target is significantly improved. Therefore, solid cancer can be prevented or treated using immune cells containing PD-L1 scFv and a hydrophilic monobody-based chimeric antigen receptor.

Claims

1. A monobody that specifically binds to EphA2, wherein one or more hydrophobic amino acids of the monobody consisting of the amino acid sequence of SEQ ID NO: 46 are replaced with hydrophilic amino acids and / or one or more hydrophilic amino acids are added.

2. A monobody comprising the amino acid sequence of sequence number 8 in the first paragraph.

3. In the first paragraph, a monobody directly linked to PD-L1 scFv or via a linker.

4. A chimeric antigen receptor comprising a monobody according to any one of claims 1 to 3 as an extracellular ligand binding domain, and further comprising at least one selected from the group consisting of a transmembrane domain and an intracellular signaling domain.

5. A chimeric antigen receptor in claim 4, wherein the transmembrane domain is selected from the group consisting of T-cell receptor alpha chain, T-cell receptor beta chain, T-cell receptor zeta chain, CD8 alpha chain, CD8 beta chain, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a part thereof, and any combination thereof.

6. A chimeric antigen receptor in claim 4, wherein the intracellular signaling domain is selected from the group consisting of TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d, and any combination thereof.

7. A chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain is at least one selected from the group consisting of an OX40 domain, a CD2 domain, a CD27 domain, a CD28 domain, a CDS domain, an ICAM-1 domain, an LFA-1 domain, and a 4-1BB domain, and any combination thereof.

8. A chimeric antigen receptor according to claim 4, characterized in that it further comprises a hinge.

9. A polynucleotide comprising a nucleic acid sequence encoding 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.

10. A vector comprising a polynucleotide according to Article 9.

11. An isolated immune cell expressing 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 on its cell surface membrane.

12. An isolated immune cell selected from the group consisting of leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, and dendritic cells in the 11th paragraph.

13. A pharmaceutical composition for preventing or treating cancer, comprising an isolated immune cell according to Article 11.

14. A pharmaceutical composition according to claim 13, characterized in that the cancer is at least one 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, hemangiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colon cancer, hematopoietic tumor, and metastatic cancer thereof.

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

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