PD-L1-specific chimeric antigen receptor and immune cells containing the same

Abstract

The present invention relates to a PD-L1-specific chimeric antigen receptor and an immune cell comprising the same. In particular, the PD-L1-specific chimeric antigen receptor contains a binding domain that specifically binds to PD-L1 (Programmed Death Ligand 1), and therefore not only has excellent PD-L1-specific targeting efficiency, but also has excellent anti-cancer effects against cancer cells expressing PD-L1, and can be effectively used for the prevention or treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to a PD-L1 (Programmed Death-Ligand 1)-specific chimeric antigen receptor and an immune cell comprising the same. In particular, the PD-L1-specific chimeric antigen receptor comprises a binding domain that specifically binds to PD-L1. [Background technology]

[0002] In the treatment of solid cancers, it is difficult to completely remove cancer stem cells, remaining cancer cells, or metastatic cancer cells by surgical resection alone, so combined surgical resection with radiation therapy, chemotherapy, targeted therapy, immunotherapy, etc. is used. In particular, immunotherapy targets specific genetic characteristics of tumor cells and utilizes the body's immune system, so it has almost no side effects like those of radiation therapy or chemotherapy and is effective against various types of cancer.

[0003] In general, immune cells have proteins called immune checkpoints on their cell membranes, which can suppress autoimmune reactions. Immune checkpoints are receptors that negatively regulate the proliferation and function of T cells, which play a central role in adaptive immunity. These include receptors such as CTLA4 (cytotoxic T-lymphocyte associated protein 4), PD-1 (programmed cell death protein 1), LAG3 (lymphocyte-activation gene-3), KIR (killer cell immunoglobulin-like receptor), TIM3 (T-cell immunoglobulin and mucin-domain containing-3), and VISTA (V-domain Ig suppressor of T cell activation).

[0004] Tumor cells suppress or evade immunity by expressing ligands for these immune checkpoints, suppressing immune function by modifying the tumor micro-environment, and evading immunity through immune tolerance or immune-editing of T cells.

[0005] It has been discovered that cancer cells can survive from immune cells by utilizing immune checkpoints, so active development of drugs that inhibit the binding of immune checkpoint proteins to ligands on cancer cells has been conducted. As a result, monoclonal antibodies that can specifically bind to the immune checkpoint proteins PD-1 / CTLA-4 proteins have been developed, and PD-1 antibodies or CTLA-4 antibodies bind to the PD-1 protein and CTLA-4 protein on the immune cell membrane, respectively, and can maintain the cancer cell killing function of immune cells by preventing these immune checkpoint proteins from binding to the ligands on cancer cells. Such antibody-based immunotherapeutic agents include Atezolizumab, Avelumab, and Ipilimumab.

[0006] In addition, unlike conventional immunotherapy, immune cell-based anticancer immunotherapy drugs have been developed that specifically bind to cancer cells and eliminate them through immune cells in the body. These include T cells (CAR T cells) that express chimeric antigen receptors (CARs) that can directly recognize tumor-associated antigens, or T cells that express specific antibody sequences against immune checkpoint receptors on cancer cells, enabling immune cells to specifically attack cancer cells.

[0007] The structure of the chimeric antigen receptors currently being developed is divided into an scFv (single chain variable fragment) portion that recognizes the antigen, a transmembrane domain, and a signaling domain that transmits a signal into the cell. Pharmaceutical companies and clinicians such as Novartis, Gilead, Celgene, and Abcloon have developed CAR T cells using scFv that specifically binds to the CD19 antigen of blood cancer cells as a treatment for blood cancer (Patent Documents 3 to 7).

[0008] However, even for solid tumors, there is a strong demand for CAR-based immune cell therapy that combines various gene manipulation technologies. However, in the case of solid tumors, research results are still insufficient due to the heterogeneity of tumors, which causes each patient to express different antigens, the complex characteristics of the tumor microenvironment such as hypoxia, and restrictions on the movement and activity of T cells.

[0009] In addition, the currently available antibodies avelumab and atezolizumab bind to antigens too strongly and have a slow dissociation rate, which can lead to binding to normal cells and causing severe side effects (on-target off-tumor toxicity). As a result, there have been frequent reports of CAR T clinical trials using the scFv of these antibodies being discontinued midway.

[0010] Therefore, the present inventors have endeavored to develop an immune cell-based immunoanticancer agent that can exert an excellent anticancer effect not only against blood cancers but also against solid cancers, and as a result have completed the present invention. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Korean Patent Publication No. 10-2020-0119891 [Patent Document 2] Korean Patent No. 10-2048477 [Patent Document 3] International Publication No. 2016 / 016473 [Patent Document 4] International Publication No. 2015 / 157252 [Patent Document 5] International Publication No. 2018 / 023025 [Patent Document 6] International Publication No. 2017 / 211900 [Patent Document 7] International Publication No. 2016 / 028896 [Patent Document 8] Korean Patent No. 10-1069146 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1.

[0013] Another object of the present invention is to provide an immune cell comprising the chimeric antigen receptor.

[0014] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the immune cells. [Means for solving the problem]

[0015] The present invention provides a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1.

[0016] The chimeric antigen receptor may further comprise at least one selected from the group consisting of a signal sequence, a hinge and transmembrane domain, and an intracellular domain.

[0017] The signal sequence may comprise the signal sequence of CD8α, preferably the amino acid sequence of SEQ ID NO:7.

[0018] The binding domain that specifically binds to PD-L1 may be a single chain variable fragment (scFv) of an anti-PD-L1 antibody, and preferably comprises the amino acid sequence of SEQ ID NO:8.

[0019] The hinge and transmembrane domain may comprise the hinge and transmembrane domain of CD8α, preferably comprising the amino acid sequence of SEQ ID NO:9.

[0020] The intracellular domain may comprise an intracellular signal region sequence selected from the group consisting of CD28, 4-1BB, and CD3zeta, or a combination thereof, and preferably, the intracellular signal region sequence of CD28 may comprise the amino acid sequence of SEQ ID NO: 10, the intracellular signal region sequence of 4-1BB may comprise the amino acid sequence of SEQ ID NO: 11, and the intracellular signal region sequence of CD3zeta may comprise the amino acid sequence of SEQ ID NO: 12.

[0021] In addition, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1.

[0022] Also provided are expression vectors comprising the polynucleotides, and viruses comprising the expression vectors.

[0023] Additionally, the present invention provides immune cells that express a chimeric antigen receptor on their surface that comprises a binding domain that specifically binds to PD-L1.

[0024] The immune cells may be T cells, MIL cells, TIL cells, NK cells, NKT cells, DC cells or immune cells derived from iPSCs, and preferably T cells.

[0025] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising immune cells expressing on their surface a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1.

[0026] The cancer may be of various types, including blood cancer, liver cancer, glioblastoma, gastric cancer, or colon cancer. Effect of the Invention

[0027] The PD-L1-specific chimeric antigen receptor and immune cells containing the same of the present invention not only have excellent PD-L1-specific targeting efficiency, but also exhibit excellent anti-cancer effects against cancer cells expressing PD-L1, and can be effectively used in the prevention or treatment of cancer. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the vector structure of PD-L1-specific chimeric antigen receptor. [Diagram 2] This figure shows the results of confirming whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Diagram 3] This figure shows the results of confirming whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 4] This figure shows the results of confirming whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Diagram 5] This figure shows the results of measuring cell proliferation, viability, and maintenance of CAR expression in immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 6] This figure shows the results of identifying surface markers of immune cells that express PD-L1-specific chimeric antigen receptors. [Figure 7] This figure shows the results of investigating whether PD-L1 is expressed in the liver cancer cell lines HepG2 and Hep3B, and the glioblastoma cell line U251. [Figure 8] This figure shows the results of confirming the cancer cell-killing ability of immune cells expressing PD-L1-specific chimeric antigen receptors in the liver cancer cell lines HepG2 and Hep3B, and the glioblastoma cell line U251. [Figure 9]This figure shows the results of confirming the cancer cell-killing ability of immune cells expressing PD-L1-specific chimeric antigen receptors in the gastric cancer cell line SNU638. [Figure 10] This figure shows the results of confirming the cancer cell-killing ability of immune cells expressing PD-L1-specific chimeric antigen receptors in the gastric cancer cell line SNU638, and the colon cancer cell lines DLD1, LOVO, and HCT15. [Figure 11] This figure shows the results of confirming the cancer cell-killing ability of immune cells expressing PD-L1-specific chimeric antigen receptors in the gastric cancer cell line SNU638, the glioblastoma cell line U251, and the colon cancer cell line DLD1. [Figure 12] This figure shows the results of confirming the cell-killing ability of immune cells expressing PD-L1-specific chimeric antigen receptors in the gastric cancer cell line SNU638 and the normal kidney cell line HEK293T. [Figure 13] This figure shows the results of confirming the anti-tumor ability of immune cells expressing PD-L1-specific chimeric antigen receptors in a mouse model of liver cancer. [Figure 14] This figure shows the results of confirming the anti-tumor ability of immune cells expressing PD-L1-specific chimeric antigen receptors in a mouse model of liver cancer. [Figure 15] This figure shows the results of confirming the anti-tumor ability of immune cells expressing PD-L1-specific chimeric antigen receptors in a mouse model of gastric cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Hereinafter, the present application will be described in detail with reference to the accompanying drawings so that a person having ordinary skill in the art to which the present invention pertains can easily carry out the present application. However, since the present application can be carried out in various forms, it is not limited to the following embodiments and examples.

[0030] Throughout this specification, when a part is said to "comprise" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0031] The present invention provides a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to PD-L1 (Programmed Death-Ligand 1).

[0032] Preferably, the binding domain that specifically binds to PD-L1 is an anti-PD-L1 antibody or a fragment thereof, but is not limited thereto.

[0033] In the present invention, the term "fragment" of an antibody means a fragment that retains antigen-binding function, and is used to include scFv, Fab, F(ab')2, and Fv fragments.

[0034] The anti-PD-L1 antibody fragment comprised in the PD-L1-specific chimeric antigen receptor of the invention is in the form of an scFv and may comprise the amino acid sequence of SEQ ID NO:8 or have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0035] In the present invention, "PD-L1 (Programmed death-ligand 1)" refers to a ligand for "Programmed cell death receptor 1 (PD-1)", an immunosuppressive receptor mainly expressed on activated T cells and B cells, and when PD-1 binds to its ligand PD-L1 or PD-L2, it can negatively regulate antigen receptor signaling. Ligands for PD-1 can be constitutively expressed or induced in many cell types, including non-hematopoietic cell tissues and various tumor types. PD-L1 is weakly expressed on B cells, T cells, bone marrow cells, and dendritic cells (DCs), but is also expressed in peripheral cells, similar microvascular endothelial cells, and non-lymphoid organs such as the heart, lung, etc. However, overexpression in tumor cells is mainly induced by interferon gamma in the tumor microenvironment, or expression is induced in immune cells present in the tumor microenvironment by the immune evasion mechanism of tumor cells. In contrast, PD-L2 is only found on macrophages and dendritic cells. The expression pattern of PD-1 ligands indicates that PD-1 plays a role in maintaining peripheral tolerance and may contribute to regulating autoreactive T and B cell responses in the periphery.

[0036] In the present invention, "Chimeric Antigen Receptor (CAR)" refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. CARs can be expressed on T cells or NK cells to enhance cytotoxicity. In general, the extracellular antigen-binding domain is a single chain variable fragment (scFv) specific for an antigen found on the subject's cells. Based on the specificity of the scFv domain, cells expressing a particular antigen on the cell surface are targeted. The scFv domain can be engineered to recognize any antigen, including tumor-specific and virus-specific antigens. For example, PD-L1 CAR recognizes PD-L1, a cell surface marker expressed by some cancers.

[0037] The PD-L1-specific chimeric antigen receptor of the present invention may further comprise at least one selected from the group consisting of a signal peptide (SP), a hinge and transmembrane domain (TM), and an intracellular domain, in addition to the binding domain that specifically binds to PD-L1.

[0038] The signal sequence comprised in the PD-L1-specific chimeric antigen receptor of the present invention is preferably, but is not limited to, a CD8α signal sequence, and may preferably comprise the amino acid sequence of SEQ ID NO: 7, or may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0039] The signal sequence and the binding domain that specifically binds to PD-L1 according to the present invention constitute the extracellular domain of a chimeric antigen receptor. The extracellular domain is the site where the main signal is transmitted, is present on the outside of the cell membrane, and is the domain that specifically recognizes PD-L1.

[0040] The hinge and transmembrane domains comprised in the PD-L1-specific chimeric antigen receptor of the present invention are preferably, but not limited to, those derived from CD8α and may preferably comprise the amino acid sequence of SEQ ID NO:9, or may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0041] The intracellular signaling domain according to the present invention is a portion located inside the cell membrane of a T cell, i.e., in the cytoplasm, and refers to a site that activates the immune response of immune cells when an antibody bound to the extracellular domain binds to a target antigen.

[0042] The intracellular domain of the PD-L1-specific chimeric antigen receptor of the present invention may comprise at least two intracellular signalling domains, preferably but not limited to an intracellular signalling domain derived from CD28 or an intracellular signalling domain derived from 4-1BB linked to an intracellular signalling domain derived from CD3ζ, and preferably, the intracellular signalling domain derived from CD28 may comprise the amino acid sequence of SEQ ID NO: 10, the intracellular signalling domain derived from 4-1BB may comprise the amino acid sequence of SEQ ID NO: 11, and the intracellular signalling domain derived from CD3ζ may comprise the amino acid sequence of SEQ ID NO: 12, and may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0043] Additionally, the present invention provides immune cells expressing a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1.

[0044] The immune cells may be, but are not limited to, T cells, MIL cells, TIL cells, NK cells, NKT cells or DC cells, and iPSC-derived immune cells, and are preferably T cells.

[0045] According to one embodiment of the present invention, the immune cells may be described as "anti-PD-L1 CAR T cells" and have been named "VYPC1" or "VYPC2".

[0046] The immune cells of the present invention may exhibit killing ability against cancer (tumor) cells expressing PD-L1. According to one embodiment, the immune cells (e.g., T cells) of the present invention may exhibit killing ability against liver cancer cells or glioblastoma cells. For example, the liver cancer cells or glioblastoma cells may express PD-L1.

[0047] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising immune cells having a chimeric antigen receptor comprising a binding domain that specifically binds to PD-L1, more specifically, a pharmaceutical composition for preventing or treating tumor malignancy and metastasis.

[0048] The pharmaceutical composition comprising the immune cells expressing the chimeric antigen receptor of the present invention may further comprise a pharma- ceutically acceptable excipient.

[0049] The term "cancer (tumor)" as used in the present invention refers to or describes a physiological condition in a mammal that is typically characterized by uncontrolled cell growth and proliferation. Cancers (tumors), which are diseases to which the composition is applied, include cancers that typically respond to immunotherapy and cancers that have not been previously associated with immunotherapy. In the present invention, the cancers (tumors) are preferably PD-L1 positive cancers, and may be liver cancer cells, glioblastoma cells, gastric cancer cells or colon cancer cells. Furthermore, the present invention includes refractory or recurrent cancers whose growth can be inhibited using the antibody or fragment thereof of the present invention.

[0050] The present invention also provides a nucleic acid sequence encoding the chimeric antigen receptor of the present invention. The polynucleotide (nucleic acid) encoding the antigen receptor of the present invention may be modified by codon optimization, which will be well understood by those skilled in the art due to the degeneracy of codons, and there are many nucleotide sequences encoding a polypeptide or variant fragment thereof. Some of these polynucleotides (nucleic acids) have minimal homology to the nucleotide sequence of any naturally occurring gene.

[0051] Furthermore, the present invention provides a vector comprising a nucleic acid sequence according to the present invention, and a virus comprising a vector according to the present invention.

[0052] As used herein, the term "prevention" refers to any action of administering a composition to inhibit or delay the onset of a disease, and the term "treatment" refers to any action of administering a composition to improve or beneficially alter the symptoms of an individual suspected of or suffering from a disease.

[0053] The present invention will be described in more detail below with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. EXAMPLES

[0054] Generation of recombinant lentiviral vectors The following strains and vectors were prepared to construct recombinant plasmid vectors expressing chimeric antigen receptors containing scFv that specifically bind to PD-L1 (anti-PD-L1 CAR).

[0055] 1-1. Preparation of vector Phagemid DNA containing the nucleic acid sequence (SEQ ID NO: 2) of scFv that specifically binds to human PD-L1 was provided by the research team at YBiologics. The sequence information of the phagemid DNA containing the nucleic acid sequence of the scFv and the production method are described in detail in Patent Document 8, as are the phagemid DNA sequence information of the VH and VL chains used as scFv below. Specifically, the basic structure of the expression vector was the pLenti7.3 / V5-DEST vector from Invitrogen, and nucleic acid sequences expressing two types of chimeric antigen receptors consisting of [CD8α signal sequence-anti-PD-L1 scFv-CD8α H&TM-CD28 or 4-1BB signaling domain-CD3ζ signaling domain] were incorporated between the 5'UTR and 3'UTR of the pLenti7.3 / V5-DEST vector (pLenti7.3::CD8α signal sequence-anti-PD-L1 scFv-CD8 H&T-CD28-CD3ζ vector, VYPC1; pLenti7.3::CD8α signal sequence-anti-PD-L1 scFv-CD8 H&T-4-1BB-CD3ζ vector, VYPC2).

[0056] The nucleic acid sequence constituting the PD-L1-specific chimeric antigen receptor in the recombinant plasmid vector is shown in Table 1 below.

[0057] [Table 1] JPEG2025508020000003.jpg86164

[0058] The nucleic acid sequences in Table 1 can be expressed in immune cells to express a PD-L1-specific chimeric antigen receptor consisting of the amino acid sequence in Table 2 below on the cell surface.

[0059] [Table 2]

[0060] 1-2. Preparation of cells for vector replication E. coli Stbl3(F-mcrB mrrhsdS20(rB-,mB-)recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R Host cells of )xyl-5 λ-leumtl-1) (Invitrogen) were cultured in LB (Luria-Bertani) solid medium (Difco Laboratories, USA) at 37°C and 200 rpm, and 100 μg / mL ampicillin was added to all media to prepare host cells. Then, a single colony of each cultured host cell was inoculated into LB liquid medium (Difco Laboratories, USA) and cultured, which was then inoculated into LB liquid medium at 1% and cultured at 37°C until OD 0.4-0.6, and then washed with CaCl2 buffer to prepare.

[0061] 1-3. Preparation of transformed cells for mass production of vectors The prepared vectors were mixed with host cells, and then the mixture was placed on ice for 10 minutes, and then treated with ice for 90 seconds at 42° C. and 3 minutes to prepare transformed cells into which the vectors had been incorporated. The prepared transformed cells were smeared on LB solid medium containing an antibiotic and then cultured at 37° C., thereby producing each vector in large quantities. EXAMPLES

[0062] Construction of recombinant lentiviral vectors expressing PD-L1-specific chimeric antigen receptors The lentiviral vector of Example 1 (pLenti7.3:: CD8α signal sequence-MSLN scFv-CD8 H&T-CD28-CD3ζ vector with the MSLN scFv sequence removed) was prepared. First, the vector was used as a template to perform inverse PCR using the CPL-F primer, CPL-R primer, and CloneAmp HiFi PCR premix (Takara) in Table 3 below. The vector thus amplified was separated and purified to prepare an MSLNscFv-free chimeric antigen receptor vector.

[0063] The PD-L1 scFv sequence was prepared by amplifying the phagemid DNA sequence provided by YBiologics by PCR using the VYPC-F and VYPC-R primers in Table 3 below, followed by isolation and purification.

[0064] [Table 3]

[0065] The vector and PD-L1 scFv sequence were inserted into the site where the MSLN scFv sequence was located by homologous recombination using the Overlap cloner DNA cloning Kit (Elpsibio) to generate a recombinant lentiviral vector expressing a chimeric antigen receptor containing PD-L1 scFv (pLenti7.3-anti-PD-L1 chimeric antigen receptor vector). The PD-L1-specific chimeric antigen receptor vectors (recombinant lentiviral vectors for anti-PD-L1 CAR T) that were generated were named pLenti7.3::VYPC1 and pLenti7.3::VYPC2, and their structures are shown in Figure 1. EXAMPLES

[0066] Generation of immune cells (VYPC1 and VYPC2) expressing PD-L1-specific chimeric antigen receptors 3-1. Preparation of lentiviruses containing recombinant lentiviral vectors Lentiviruses containing the PD-L1-specific chimeric antigen receptor vectors (pLenti7.3::VYPC1 and pLenti7.3::VYPC2) prepared in Example 2 were prepared.

[0067] 3-2. Preparation of immune cells and transduction by viral infection Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood using Lymphoprep (STEMCELL), and human CD3+ T cells were isolated from the isolated PBMCs by positive selection using CD3 microbeads (Miltenyi Biotech).

[0068] Anti-CD3 / anti-CD28 magnetic beads (anti-CD3 / CD28 Dynabeads; Gibco) were mixed with the isolated human CD3+ T cells at a ratio of 1:1. 24 hours after mixing, the activated magnetic beads were removed using a MACSiMAG separation device, and the cells were washed with RPMI-1640 to activate the CD3+ T cells.

[0069] Lentivirus-infected T cells were generated by a virus infection method using polybrene (Sigma Aldrich). Specifically, the activated human CD3+ T cells were infected with a lentivirus containing a PD-L1-specific chimeric antigen receptor vector, and the virus medium was replaced 24 hours later. Cell proliferation began 48 hours after infection, and the generated immune cells (anti-PD-L1 CAR T cells) were named VYPC1 and VYPC2. EXAMPLES

[0070] Evaluation of immune cells expressing PD-L1-specific chimeric antigen receptors (VYPC1 and VYPC2)1 4-1. Transduction rate of anti-PD-L1 CAR T cells (VYPC1) We used FACS (fluorescence-activated cell sorting) to confirm whether the PD-L1-specific chimeric antigen receptor vector introduced from the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 was correctly expressed.

[0071] The experiment was performed using Attune NxT flow cytometry (Thermo Fisher Scientific). For each immune cell, the presence or absence of expression of the introduced vector was confirmed using an anti-GFP (Green Fluorescent Protein) antibody. Since the vector also contains a GFP expression site, the analysis results using the anti-GFP antibody are measured highly in immune cells expressing a large amount of such a vector.

[0072] As shown in Figure 2, the expression rate of the introduced vector (PD-L1-specific chimeric antigen receptor sequence) was comparatively analyzed by FACS, and under general IL-2 culture conditions, the anti-PD-L1 CAR T cells (VYPC1) showed an expression rate of about 85.5%. In addition, as shown in Figure 3, the transduction rates after treatment with the cytokines IL-2, IL-7, and IL-15 were analyzed by FACS, and the anti-PD-L1 CAR T cells (VYPC1) showed all of 90% or more.

[0073] Therefore, we confirmed that the PD-L1-specific chimeric antigen receptor was appropriately expressed in the generated anti-PD-L1 CAR T cells (VYPC1).

[0074] 4-2. Transduction rate of anti-PD-L1 CAR T cells (VYPC1 and VYPC2) by cytokines Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood using Lymphoprep (STEMCELL) in the same manner as in Example 3-2. Human CD3+ T cells were then isolated from the isolated PBMCs by positive selection using CD3 microbeads (Miltenyi Biotech).

[0075] Anti-CD3 / anti-CD28 magnetic beads (anti-CD3 / CD28 Dynabead; Gibco) were mixed with the isolated human CD3+ T cells at a ratio of 1:1. 24 hours after mixing, the activated magnetic beads were removed using a MACSiMAG separation device, the cells were washed with RPMI-1640 to activate the CD3+ T cells, and the cells were cultured in three groups (group 1, cultured with IL-2; group 2, cultured with IL-7 and IL-15; group 3, cultured with IL-2, IL-7, and IL-15).

[0076] Lentivirus-infected T cells were prepared by a virus infection method using polybrene (Sigma Aldrich). Specifically, the activated human CD3+ T cells were infected with a lentivirus containing a PD-L1-specific chimeric antigen receptor vector, and 24 hours later, the virus medium was replaced in three groups. Then, on the sixth day, the presence or absence of expression of the introduced vector was confirmed for each immune cell using an Attune NxT flow cytometer (Thermo Fisher Scientific) with an anti-GFP (Green Fluorescent Protein) antibody.

[0077] As a result, as shown in Figure 4, both VYPC1 and VYPC2 showed high expression rates, and VYPC1 in particular consistently showed a high CAR transduction rate of 80% or more in all cytokine cultures.

[0078] Therefore, we confirmed that the PD-L1-specific chimeric antigen receptor was appropriately expressed in the generated anti-PD-L1 CAR T cells (VYPC1 and VYPC2). EXAMPLES

[0079] Evaluation of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor 2 To measure the cell proliferation, viability, and CAR expression maintenance of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3, the following procedures were performed.

[0080] Human peripheral blood T cells were transduced with lentivirus containing anti-PD-L1 scFv CAR one day after activation with CD3 and CD28 magnetic beads, and then total and viable cell counts were measured every 2 days using a NucleoCounterNC-250TM automated cell counter (ChemoMetec), and GFP-positive cell counts were measured using an Attune FACS instrument.

[0081] As a result, as shown in Figure 5, the anti-PD-L1 CAR T cells (VYPC1) showed a superior cell proliferation rate compared to the control group, and the survival rate was at a similar level to that of the control group. Furthermore, it was found that the expression of the chimeric antigen receptor (CAR) was maintained at a high level of more than 80%.

[0082] Therefore, it was confirmed that the generated anti-PD-L1 CAR T cells (VYPC1) maintained excellent cell proliferation and expression of the chimeric antigen receptor. EXAMPLES

[0083] Evaluation of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor 3 To confirm the surface marker of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3, the following was performed.

[0084] T cell activation was confirmed using anti-CD25-PE antibody (PE Mouse Anti-Human CD25, BD Pharmingen) and anti-CD69-APC antibody (APC Mouse Anti-Human CD69, BD Pharmingen), and the presence or absence of inhibition was predicted using immune cell inhibitors anti-LAG-3-PE antibody (Thermofisher), anti-PD-1-PE-Cy7 antibody (Thermofisher), anti-CTL-4-APC antibody (Thermofisher), TIGIT-APC-Cy7 antibody (Thermofisher), and anti-TIM-3-BV421 (Thermofisher). Since the vector also contains a GFP expression site, the analysis results using anti-GFP antibody are high in immune cells expressing such vectors in large amounts.

[0085] As a result, as shown in Figure 6, in the case of anti-PD-L1 CAR T cells (VYPC1), CD25, a marker of cell proliferation activity, was expressed higher than in the control group, and CD69, a marker of cytotoxic T cell activity, was increased in the same way as in the control group. Meanwhile, the inhibitors LAG-3 and TIGIT, immune cell inhibitors, were found to be as low as in the control group. Furthermore, the inhibitors PD-1, CTLA4, and TIM-3 were at the same level as or slightly higher than in the control group, but had no significant effect on the cancer killing effect.

[0086] Therefore, the generated anti-PD-L1 CAR T cells (VYPC1) were confirmed to be in an activated state. EXAMPLES

[0087] Evaluation of the in vitro cancer cell killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor against liver cancer and glioblastoma To evaluate the anti-cancer ability of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 against PD-L1-specific antigens, the liver cancer cell lines HepG2 and Hep3B, which express PD-L1, and the glioblastoma cell line U251 were co-cultured with the anti-PD-L1 CAR T cells (VYPC1), respectively, to measure their cancer cell-killing ability.

[0088] 7-1. Evaluation of PD-L1 expression in cancer cell lines The liver cancer cell lines HepG2 and Hep3B, and the glioblastoma cell line U251 were purchased from ATCC (American Type Culture Collection). To confirm whether PD-L1 is expressed in these cancer cell lines and whether PD-L1 expression is dependent on IFNγ secreted as the immune response from immune cells becomes stronger, the presence or absence and the level of PD-L1 antigen expression were analyzed by FACS using an anti-PD-L1 antibody.

[0089] As shown in Figure 7, the liver cancer cell lines HepG2 and Hep3B and the glioblastoma cell line U251 were found to have higher PD-L1 expression by IFNγ, confirming the cancer cell killing ability of anti-PD-L1 CAR-T (VYPC1) cells targeting PD-L1.

[0090] 7-2. Evaluation of the cancer cell killing ability of anti-PD-L1 CAR T cells (VYPC1) The cancer cell lines for which the expression of PD-L1 antigen was confirmed in Example 5-1 were placed in each well at 1 × 10 4 The cancer cells were seeded in a 96-well plate at 37°C and 5% CO for 30 min. 2 The cells were cultured in a cell incubator under the conditions for 24 hours. Then, 4 × 10 cells were cultured in each well. 4 Anti-PD-L1 CAR T cells (VYPC1) were treated and co-cultured for 72-90 hours, during which cell viability and cell motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). RTCA measures the resistance value, called impedance, that occurs while cells are growing, allowing automatic kinetic measurement of various cell responses (changes in cell number, proliferation rate, cell size, cell shape, and substrate attachment quality). The resistance value detected by the cells is expressed as an index called the Cell Index (CI), and changes in the CI value indicate changes in cell shape.

[0091] As shown in Figure 8, the cancer cell-killing ability of the transduced anti-PD-L1 CAR T cells (VYPC1) was compared with that of the non-transduced human T cells (control group), and it was confirmed that treatment with VYPC1 of the present invention completely killed cancer cells in both liver cancer cell lines HepG2 and Hep3B, and glioblastoma cell line U251.

[0092] Therefore, it is clear that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have excellent cancer cell-killing ability against malignant tumor cells that express PD-L1, including liver cancer and glioblastoma. EXAMPLES

[0093] Evaluation of the in vitro cancer cell killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor against gastric cancer To evaluate the anti-cancer ability of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 against PD-L1-specific antigens, the PD-L1-expressing gastric cancer cell line SNU638 was co-cultured with the anti-PD-L1 CAR T cells (VYPC1) to measure the cancer cell-killing ability.

[0094] Gastric cancer cell line SNU638, which has been confirmed to express PD-L1 antigen, was added to each well at 1 × 10 4 The cancer cells were seeded in a 96-well plate at 37°C and 5% CO for 30 min. 2 The cells were cultured in a cell incubator under the conditions for 24 hours. Then, 0.5 × 10 cells were cultured in each well. 4 pieces (0.5:1)~2×10 4 The cells were treated with anti-PD-L1 CAR T cells (VYPC1) at a ratio of 2:1 and co-cultured for 72-90 hours, during which the cell viability and cell motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). Different cytokines (IL-2 and IL-7 + IL-15) were added to the culture medium used for the co-culture. RTCA can automatically measure various cell responses (cell number, proliferation rate, cell size, cell shape, and changes in substrate attachment quality) kinetically by measuring the resistance value called impedance that occurs during cell growth. The resistance value detected by the cells is expressed as an index called cell index (CI), and changes in the CI value indicate changes in cell shape.

[0095] As shown in Figure 9, the cancer cell killing ability of transduced anti-PD-L1 CAR T cells (VYPC1) was compared with that of non-transduced human T cells (control group), and it was confirmed that cancer cells were killed by treatment with VYPC1 of the present invention. It was also confirmed that the cancer cells died more rapidly as the ratio of VYPC1 to cancer cells increased, and that the cancer cells died more rapidly when cultured with IL-7 and IL-15 than when cultured with IL-2.

[0096] Therefore, it is clear that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have excellent cancer cell-killing ability against malignant tumor cells, including gastric cancer, that express PD-L1. EXAMPLES

[0097] Evaluation of the cancer cell killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor against gastric and colorectal cancer To evaluate the anti-cancer ability of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 against PD-L1-specific antigens, the gastric cancer cell line SNU638, which expresses PD-L1, and the colon cancer cell lines DLD1, LOVO, and HCT15 were co-cultured with the anti-PD-L1 CAR T cells (VYPC1), respectively, to measure their cancer cell-killing ability.

[0098] Cancer cell lines with confirmed PD-L1 antigen expression were placed in each well at 1 × 10 4 The cancer cells were seeded in a 96-well plate at 37°C and 5% CO for 30 min. 2 The cells were cultured in a cell incubator under the conditions for 24 hours. Then, 4 × 10 cells were cultured in each well. 4 Anti-PD-L1 CAR T cells (VYPC1) were treated and co-cultured for 72-90 hours, during which cell viability and cell motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). RTCA measures the resistance value, called impedance, that occurs while cells are growing, allowing automatic kinetic measurement of various cell responses (changes in cell number, proliferation rate, cell size, cell shape, and substrate attachment quality). The resistance value detected by the cells is expressed as an index called cell index (CI), and changes in the CI value indicate changes in cell shape.

[0099] As shown in Figure 10, the cancer cell-killing ability of the transduced anti-PD-L1 CAR T cells (VYPC1) was compared with that of the non-transduced human T cells (control group). It was confirmed that treatment with VYPC1 of the present invention completely killed cancer cells in all of the colon cancer cell lines DLD1, LOVO, and HCT15, and the gastric cancer cell line SNU638.

[0100] Therefore, it is clear that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have excellent cancer cell-killing ability against malignant tumor cells that express PD-L1, including gastric cancer and colon cancer. EXAMPLES

[0101] Evaluation of the cancer cell-killing ability of immune cells (VYPC1 and VYPC2) expressing PD-L1-specific chimeric antigen receptors against gastric cancer and glioblastoma To evaluate the anti-cancer ability of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 against PD-L1-specific antigens, the gastric cancer cell line SNU638, the glioblastoma cell line U251, and the colon cancer cell line DLD1 were co-cultured with the anti-PD-L1 CAR T cells (VYPC1 and VYPC2), respectively, to measure the cancer cell-killing ability.

[0102] As a result, as shown in Figure 11, the cancer cell-killing ability of the transduced anti-PD-L1 CAR T cells (VYPC1 and VYPC2) was compared with that of the non-transduced human T cells (control group), and it was confirmed that treatment with VYPC1 or VYPC2 of the present invention resulted in the death of cancer cells in parental cells or in the gastric cancer cell line SNU638, glioblastoma cell line U251, and colon cancer cell line DLD1 that overexpressed PD-L1 (PD-L1 O / E).

[0103] Therefore, it is clear that the anti-PD-L1 CAR T cells of the present invention (VYPC1 and VYPC2) have excellent cancer cell-killing ability against gastric cancer, colon cancer, and glioblastoma. EXAMPLES

[0104] Evaluation of the in vitro cell-killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor against normal cells To evaluate the anti-cancer ability of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 against PD-L1-specific antigens, the normal kidney cell line HEK293T was co-cultured with the anti-PD-L1 CAR T cells (VYPC1) to measure the cell-killing ability.

[0105] In the same manner as in Example 8, gastric cancer cell line SNU638, normal renal cell line HEK293T (transformed with SV40 Large T) that does not express PD-L1, and renal cell line HEK293T PDL1 O / E that overexpresses PD-L1 antigen were co-cultured with anti-PD-L1 CAR T cells (VYPC1). At that time, VYPC1 was cultured and co-cultured with cytokines IL-2 and IL-7+IL-15, respectively.

[0106] As a result, as shown in Figure 12, in the case of gastric cancer cell line (SNU638), the cancer cells were killed, and in particular, they were completely killed when co-cultured with twice the amount of VYPC1 compared to the cancer cell line. In addition, in the case of normal cell line (HEK293 T) that does not express PD-L1, it was found that the cells were not affected by VYPC1-induced cell death, and in the case of HEK293T overexpressing PD-L1 (HEK 293T PDL1 O / E), the cells were completely killed by relatively large amounts of VYPC1, unlike the killing effect shown in the cancer cells.

[0107] Therefore, it has been confirmed that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have absolutely no killing ability against normal cells that do not express PD-L1, and in the presence of normal cells that express PD-L1, their killing ability is lower than their reliable killing ability against cancer cells, so they have weak side effects against on-target off-tumors and are safe. EXAMPLES

[0108] Evaluation of the antitumor potential of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor in a mouse model of liver cancer All animal experiments were performed with the approval of the Institutional Bioethics Committee (IACUC, Chonnam National University, Republic of Korea). Four-week-old female NSG (NOD SCID gamma) mice were provided by the SPF animal laboratory of the Hwasun Vaccine Center, and human hepatocellular carcinoma cell lines HepG2 and Hep3B were provided by the Korea Cell Line Bank.

[0109] Human hepatocellular carcinoma cell line HepG2 (2.5 × 10 6 cells) and Hep3B (2.5 × 10 6 A 100uL solution of each of the 1:1 mixture of 100- and 100-μL high-concentration Matrigel (Corning) was subcutaneously injected into the right flank of each mouse to create a human tumor xenograft mouse model. 3 At the time of reaching the target age, each group was treated with PBS, control T cells (activated control normal T cells not transduced with virus; Cont-T), or 1 × 10 anti-PD-L1 CAR T cells (VYPC1). 7 200μL / mL was injected intravenously once. Four to five mice were used per group, and the tumor size and mouse weight were measured twice a week. From the mouse weight, it is possible to know whether the mouse's physical condition has recovered or not, and whether there are any side effects associated with the administration.

[0110] As shown in Figure 13, tumor size was measured and it was confirmed that the tumor size of the mice administered with the anti-PD-L1 CAR T cells of the present invention (VYPC1) was significantly reduced compared to the control group from day 18 onwards in the case of HEPG2 and from day 15 onwards in the case of HEP3B.

[0111] Furthermore, as shown in Figure 14, when the body weight of the mice was measured, it was confirmed that the group of mice administered with the anti-PD-L1 CAR T cells of the present invention (VYPC1) recovered from the weight loss 10 days after administration and their health condition was quickly restored.

[0112] Therefore, it is clear that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have excellent anti-cancer effects. EXAMPLES

[0113] Evaluation of the antitumor potential of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptor in a mouse model of gastric cancer All animal experiments were performed with the approval of the Institutional Bioethics Committee (IACUC, Chonnam National University, Republic of Korea). Four-week-old female NSG (NOD SCID gamma) mice were provided by the SPF animal laboratory of the Hwasun Vaccine Center, and the human gastric cancer cell line SNU638 was provided by the Korea Cell Line Bank.

[0114] Human gastric cancer cell line SNU638 (2 × 10 6 A 100 μL mixture of 1:1 IgG4 cells and high-concentration Matrigel (Corning) was subcutaneously injected into the right flank of each mouse to create a human tumor xenograft mouse model. 3 At that time, each group received either PBS, a high dose of control T cells (10 × 10 activated control normal T cells not transduced with virus), or 6 cells / 200uL; Cont-T), PD-L1 CAR T cells (VYPC1) at a low dose (1 x 10 6 cells / 200uL), medium dose (5×10 6 cells / 200uL), high dose (10×10 6 The mice were divided into three groups and injected intravenously once (100 μL / 200 μL of cells). Seven mice were used per group, and the tumor size and mouse weight were measured twice a week. The mouse weight can be used to determine whether the mouse's condition has improved or not, and whether there are any side effects associated with the administration.

[0115] As shown in Figure 15, tumor size was measured and it was confirmed that the tumor size of the mice administered with the anti-PD-L1 CAR T cells of the present invention (VYPC1) was significantly reduced from day 13 onwards compared to the control group. In particular, when administered at a high dose, it showed a more excellent effect.

[0116] Therefore, it is clear that the anti-PD-L1 CAR T cells (VYPC1) of the present invention have excellent anti-cancer effects even in experiments using small animals.

Claims

1. A chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to PD-L1 (programmed death-ligand 1).

2. The chimeric antigen receptor of claim 1, further comprising at least one selected from the group consisting of a signal sequence, a hinge and a transmembrane domain (TM), and an intracellular domain.

3. The chimeric antigen receptor according to claim 1, wherein the binding domain that specifically binds to PD-L1 is a single-chain variable fragment (scFv) of an anti-PD-L1 antibody.

4. The chimeric antigen receptor of claim 3, wherein the single chain variable fragment (scFv) of the anti-PD-L1 antibody comprises the amino acid sequence of SEQ ID NO:

8.

5. The chimeric antigen receptor of claim 2 , wherein the signal sequence comprises a CD8α signal sequence.

6. The chimeric antigen receptor according to claim 5, characterized in that the signal sequence of CD8α comprises the amino acid sequence of SEQ ID NO:

7.

7. The chimeric antigen receptor of claim 2, characterized in that the hinge and transmembrane domains comprise the hinge and transmembrane domains of CD8α.

8. The chimeric antigen receptor of claim 7, characterized in that the hinge and transmembrane domain of CD8α comprises the amino acid sequence of SEQ ID NO:

9.

9. The chimeric antigen receptor according to claim 2, wherein the intracellular domain comprises an intracellular signal region sequence selected from the group consisting of CD28, 4-1BB, and CD3ζ, or a sequence selected from the group consisting of a combination thereof.

10. The chimeric antigen receptor according to claim 9, wherein the intracellular signal region sequence of CD28 comprises the amino acid sequence of SEQ ID NO: 10, the intracellular signal region sequence of 4-1BB comprises the amino acid sequence of SEQ ID NO: 11, and the intracellular signal region sequence of CD3ζ comprises the amino acid sequence of SEQ ID NO:

12.

11. A polynucleotide comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 1 to 10.

12. An expression vector comprising the polynucleotide of claim 11.

13. A virus comprising the expression vector of claim 12.

14. An immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 10 on its surface.

15. The immune cell according to claim 14, characterized in that the immune cell is a T cell, an MIL cell, a TIL cell, an NK cell, an NKT cell, a DC cell, or a cell derived from an iPSC.

16. A pharmaceutical composition for preventing or treating cancer, comprising the immune cell according to claim 14.

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