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, surgical resection alone is difficult to completely eliminate cancer stem cells, residual cancer cells, or metastatic cancer cells, so combination therapies that combine surgical resection with radiation therapy, chemotherapy, targeted therapy, and immunotherapy are used. In particular, immunotherapy targets specific genetic characteristics of tumor cells and utilizes the body's immune system, resulting in virtually no side effects seen with radiation therapy or chemotherapy, and is effective against a variety of cancer types.

[0003] Immune cells generally possess 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 the immune system by expressing ligands for these immune checkpoints, by modifying the tumor microenvironment to suppress immune function, and by T cell immune tolerance or immuno-editing.

[0005] Because cancer cells have been found to survive immune cell attacks by utilizing immune checkpoints, active research is underway to develop drugs that inhibit the binding of immune checkpoint proteins to their ligands on cancer cells. As a result, monoclonal antibodies capable of specifically binding to the immune checkpoint proteins PD-1 and CTLA-4 have been developed. PD-1 and CTLA-4 antibodies bind to the PD-1 and CTLA-4 proteins, respectively, on the immune cell membrane, preventing 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 immunotherapeutics include atezolizumab, avelumab, and ipilimumab.

[0006] In addition, immune cell-based anticancer drugs have been developed that, unlike conventional immunotherapy, specifically bind to cancer cells and eliminate them via immune cells in the body. These cancer treatments 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 currently developed chimeric antigen receptors is divided into an scFv (single chain variable fragment) portion that recognizes the antigen, a transmembrane domain, and a signaling domain that transmits signals into the cell. Pharmaceutical companies and clinicians such as Novartis, Gilead, Celgene, and Abcron have developed CAR T cells that use scFv that specifically bind to the CD19 antigen on 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 heterogeneity, whereby different antigens are expressed by different patients, 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 dissociate slowly, potentially causing serious side effects (on-target off-tumor toxicity) by binding to normal cells. 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 excellent anticancer effects not only on blood cancers but also on 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 Registration 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] An object of the present invention is 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] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises 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, and preferably comprises 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, and preferably comprises 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 CD3ζ, or a combination thereof. 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 CD3ζ may comprise the amino acid sequence of SEQ ID NO: 12.

[0021] Furthermore, 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 an expression vector containing the polynucleotide, and a virus containing the expression vector.

[0023] Furthermore, the present invention provides immune cells that express on their surface a chimeric antigen receptor comprising 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 obtained 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. [Effects 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 PD-L1-expressing cancer cells, and can be effectively used in the prevention or treatment of cancer. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the vector structure of the PD-L1-specific chimeric antigen receptor. [Figure 2] This figure shows the results of examining whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 3] This figure shows the results of examining whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 4] This figure shows the results of examining whether the introduced vector was expressed in immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 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] Fig. 1 shows the results of identifying surface markers of immune cells expressing PD-L1-specific chimeric antigen receptors. [Figure 7] This figure shows the results of examining whether PD-L1 is expressed in the liver cancer cell lines HepG2 and Hep3B, and the glioblastoma cell line U251. [Figure 8] Fig. 1 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]Fig. 1 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] Fig. 1 shows the results of confirming the antitumor ability of immune cells expressing PD-L1-specific chimeric antigen receptors in a liver cancer mouse model. [Figure 14] Fig. 1 shows the results of confirming the antitumor ability of immune cells expressing PD-L1-specific chimeric antigen receptors in a liver cancer mouse model. [Figure 15] Fig. 1 shows the results of confirming the antitumor activity of immune cells expressing PD-L1-specific chimeric antigen receptors in a mouse model of gastric cancer. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. However, since the present invention can be embodied 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] The binding domain that specifically binds to PD-L1 is preferably, but not limited to, an anti-PD-L1 antibody or a fragment thereof.

[0033] In the present invention, the term "fragment" of an antibody refers to 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 may be in the form of an scFv and may comprise the amino acid sequence of SEQ ID NO:8, or may have at least 70%, preferably at least 80%, and 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 the ligand for "Programmed Death Receptor 1 (PD-1)," an immunosuppressive receptor primarily expressed on activated T cells and B cells. Binding of PD-1 to its ligand, PD-L1 or PD-L2, negatively regulates antigen receptor signaling. PD-1 ligands are constitutively expressed or can be induced on many cell types, including non-hematopoietic tissues and various tumor types. PD-L1 is weakly expressed on B cells, T cells, myeloid cells, and dendritic cells (DCs), but is also expressed on peripheral cells, similar microvascular endothelial cells, and non-lymphoid organs such as the heart and lungs. However, overexpression on tumor cells is primarily induced by interferon gamma in the tumor microenvironment, or its expression is induced on immune cells present in the tumor microenvironment as a tumor cell immune evasion mechanism. In contrast, PD-L2 is found only 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, the term "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. Typically, the extracellular antigen-binding domain is a single-chain variable fragment (scFv) specific for an antigen found on the target cell. Based on the specificity of the scFv domain, cells expressing a specific antigen on their 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 CARs recognize 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, the signal sequence of CD8α, and may preferably comprise the amino acid sequence of SEQ ID NO: 7, or may have at least 70%, preferably at least 80%, and 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 PD-L1-specific binding domain of the present invention constitute the extracellular domain of the chimeric antigen receptor, which is the site where the main signal is transmitted, is present on the outside of the cell membrane, and is responsible for specifically recognizing 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 of the present invention is a portion located inside the cell membrane of a T cell, i.e., in the cytoplasm, and refers to the 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 signaling domains, preferably, but not limited to, a CD28-derived intracellular signaling domain or a 4-1BB-derived intracellular signaling domain linked to a CD3ζ-derived intracellular signaling domain. Preferably, the CD28-derived intracellular signaling domain may comprise the amino acid sequence of SEQ ID NO: 10, the 4-1BB-derived intracellular signaling domain may comprise the amino acid sequence of SEQ ID NO: 11, and the CD3ζ-derived intracellular signaling domain may comprise the amino acid sequence of SEQ ID NO: 12, and may share 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: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0043] Furthermore, 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 are named "VYPC1" or "VYPC2".

[0046] The immune cells of the present invention may exhibit the ability to kill cancer (tumor) cells that express PD-L1. According to one embodiment, the immune cells (e.g., T cells) of the present invention may exhibit the ability to kill 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 containing a binding domain that specifically binds to PD-L1, more particularly for preventing or treating tumor malignant progression and metastasis.

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

[0049] The term "cancer (tumor)" as used herein refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth and proliferation. Cancers (tumors), which are diseases to which the composition is applicable, include cancers that typically respond to immunotherapy and cancers that have not previously been associated with immunotherapy. In the present invention, the cancer (tumor) is preferably a PD-L1-positive cancer, 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 nucleic acid sequences encoding the chimeric antigen receptors of the present invention. Polynucleotides (nucleic acids) encoding the antigen receptors of the present invention may be modified by codon optimization. This is due to codon degeneracy, and those skilled in the art will appreciate that there are many nucleotide sequences encoding polypeptides or variant fragments 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 that inhibits or delays the onset of a disease by administering a composition, and the term "treatment" refers to any action that improves or beneficially alters the symptoms of an individual suspected of or suffering from a disease by administering a composition.

[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. [Example]

[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 of an scFv that specifically binds to human PD-L1 (SEQ ID NO: 2) was provided by the research team at YBiologics. The sequence information and production method of the phagemid DNA containing the nucleic acid sequence of the scFv are described in detail in Patent Document 8, as are the phagemid DNA sequences of the VH and VL chains used as scFv below. Specifically, the basic framework of the expression vector was Invitrogen's pLenti7.3 / V5-DEST vector, and nucleic acid sequences expressing two types of chimeric antigen receptors (chimeric antigen receptors), consisting of either CD8α signal sequence-anti-PD-L1 scFv-CD8α H&TM-CD28 or 4-1BB signaling domain-CD3ζ signaling domain, were inserted 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 sequences that make up the PD-L1-specific chimeric antigen receptor in the recombinant plasmid vector are shown in Table 1 below.

[0057] [Table 1] JPEG0007810471000002.jpg86164

[0058] The nucleic acid sequences in Table 1 can be expressed in immune cells to produce PD-L1-specific chimeric antigen receptors on the cell surface, which consist of the amino acid sequences in Table 2 below.

[0059] [Table 2]

[0060] 1-2. Preparing 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. All media were supplemented with 100 μg / mL ampicillin. A single colony of each host cell was then inoculated into LB liquid medium (Difco Laboratories, USA) and cultured. This was then inoculated into LB liquid medium at 1% and cultured at 37°C until an OD of 0.4-0.6 was reached. The colony was then washed with CaCl2 buffer.

[0061] 1-3. Preparation of transformed cells for mass production of vectors Each of the prepared vectors was mixed with host cells, then left to stand on ice for 10 minutes, then incubated at 42°C for 90 seconds and then on ice for 3 minutes to produce transformed cells into which each vector had been integrated. The transformed cells were then smeared on LB solid medium containing antibiotics and cultured at 37°C, thereby producing each vector in large quantities. [Example]

[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, minus the MSLN scFv sequence) was prepared. First, using the vector as a template, inverse PCR was performed using the CPL-F primer, CPL-R primer, and CloneAmp HiFi PCR Premix (Takara) listed in Table 3 below. The amplified vector was then isolated 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 combined using the Overlap cloner DNA cloning Kit (Elpsibio) for homologous recombination to insert the PD-L1 scFv sequence into the site where the MSLN scFv sequence had been located, generating a recombinant lentiviral vector expressing a chimeric antigen receptor containing PD-L1 scFv (pLenti7.3-anti-PD-L1 chimeric antigen receptor vector). The resulting PD-L1-specific chimeric antigen receptor vectors (recombinant lentiviral vectors for anti-PD-L1 CAR T) were named pLenti7.3::VYPC1 and pLenti7.3::VYPC2, and their structures are shown in Figure 1. [Example]

[0066] Generation of immune cells (VYPC1 and VYPC2) expressing PD-L1-specific chimeric antigen receptors 3-1. Preparation of lentivirus 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), followed by positive selection of human CD3+ T cells 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 1:1 ratio. 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 using a viral infection method using polybrene (Sigma-Aldrich). Specifically, the activated human CD3+ T cells were infected with lentivirus containing a PD-L1-specific chimeric antigen receptor vector, and the viral 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. [Example]

[0070] Evaluation of immune cells (VYPC1 and VYPC2) expressing PD-L1-specific chimeric antigen receptors 1 4-1. Transduction rate of anti-PD-L1 CAR T cells (VYPC1) The PD-L1-specific chimeric antigen receptor vector introduced from the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 was examined for correct expression using fluorescence-activated cell sorting (FACS).

[0071] The experiment was performed using an Attune NxT flow cytometer (Thermo Fisher Scientific). Expression of the introduced vector was confirmed for each immune cell type using an anti-GFP (Green Fluorescent Protein) antibody. Because the vector also contains a GFP expression site, immune cells expressing large amounts of the vector showed high levels of expression using the anti-GFP antibody.

[0072] As shown in Figure 2, the expression rates of the introduced vector (PD-L1-specific chimeric antigen receptor sequence) were comparatively analyzed by FACS. Under typical IL-2 culture conditions, anti-PD-L1 CAR T cells (VYPC1) showed an expression rate of approximately 85.5%. Furthermore, as shown in Figure 3, the transduction rates after treatment with the cytokines IL-2, IL-7, and IL-15 were analyzed by FACS. In both cases, anti-PD-L1 CAR T cells (VYPC1) showed transduction rates of over 90%.

[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 Dynabeads; Gibco) were mixed with the isolated human CD3+ T cells at a 1:1 ratio. 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. The cells were then divided into 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 generated using a viral infection method using polybrene (Sigma-Aldrich). Specifically, the activated human CD3+ T cells were infected with lentivirus containing a PD-L1-specific chimeric antigen receptor vector. 24 hours later, the viral medium was replaced in three groups. On day 6, each immune cell was examined for expression of the introduced vector 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). [Example]

[0079] Evaluation of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptors 2 The cell proliferation, viability, and CAR expression maintenance of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3 were measured as follows.

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

[0081] As shown in Figure 5, the anti-PD-L1 CAR T cells (VYPC1) showed superior cell proliferation rates compared to the control group, and their survival rates were similar to those of the control group. Furthermore, they maintained a high level of chimeric antigen receptor (CAR) expression of over 80%.

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

[0083] Evaluation of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptors3 To confirm the surface markers of the anti-PD-L1 CAR T cells (VYPC1) prepared in Example 3, the following procedure 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). 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). Because the vector also contains a GFP expression site, the anti-GFP antibody assay yielded high results in immune cells expressing large amounts of the vector.

[0085] As shown in Figure 6, the anti-PD-L1 CAR T cells (VYPC1) showed higher expression of CD25, a marker of cell proliferation activity, than the control group, and CD69, a marker of cytotoxic T cell activity, increased at a similar level to the control group. Meanwhile, the inhibitory factors LAG-3 and TIGIT, immune cell inhibitors, were found to be low, similar to the control group. Furthermore, the inhibitory factors PD-1, CTLA4, and TIM-3 were at levels similar to or slightly higher than the control group, but this did not significantly affect the cancer killing effect.

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

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

[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 determine whether these cancer cell lines express PD-L1 and whether PD-L1 expression is dependent on IFNγ secretion as the immune response from immune cells becomes stronger, the presence and level of PD-L1 antigen expression were analyzed by FACS using an anti-PD-L1 antibody.

[0089] As shown in Figure 7, IFNγ induced increased PD-L1 expression in both the liver cancer cell lines HepG2 and Hep3B and the glioblastoma cell line U251, 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 PD-L1 antigen expression was confirmed in Example 5-1 were placed in each well at 1 × 10 4 The cells were seeded in a 96-well plate and placed in a BSC for 30 minutes. After that, the cancer cells were incubated at 37°C, 5% CO 2 The cells were cultured in a cell incubator under the conditions described above for 24 hours. Then, 4 × 10 cells were added to each well. 4 Cells were treated with anti-PD-L1 CAR T cells (VYPC1) and co-cultured for 72-90 hours. Cell viability and motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). RTCA measures the resistance (impedance) generated during cell growth, enabling automated kinetic measurement of various cellular responses (cell number, proliferation rate, cell size, cell shape, and changes in substrate attachment quality). The resistance detected by the cells is expressed as a cell index (CI), and changes in CI indicate changes in cell shape.

[0091] As shown in Figure 8, 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). Treatment with VYPC1 of the present invention completely killed cancer cells in both the liver cancer cell lines HepG2 and Hep3B, and the glioblastoma cell line U251.

[0092] Therefore, it can be seen 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. [Example]

[0093] Evaluation of the in vitro cancer cell-killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptors 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 their cancer cell-killing ability.

[0094] Gastric cancer cell line SNU638, which has been confirmed to express PD-L1 antigen, was placed in each well at 1 × 10 4 The cells were seeded in a 96-well plate and placed in a BSC for 30 minutes. After that, the cancer cells were incubated at 37°C, 5% CO 2 The cells were cultured in a cell incubator under the conditions described above for 24 hours. Then, 0.5 × 10 cells were added to each well. 4 pieces (0.5:1)~2×10 4 The cells were treated with anti-PD-L1 CAR T cells (VYPC1) at a 2:1 ratio and co-cultured for 72-90 hours. Cell viability and cell motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). Different cytokines (IL-2, IL-7, and IL-15) were added to the culture medium. RTCA measures the resistance (impedance) generated during cell growth, enabling automated kinetic measurement of various cellular responses (cell number, proliferation rate, cell size, cell shape, and changes in substrate attachment quality). The resistance detected by the cells is expressed as a parameter called the cell index (CI), and changes in CI 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). Treatment with VYPC1 of the present invention demonstrated that cancer cells died. Furthermore, the greater the ratio of VYPC1 to cancer cells, the more rapidly cancer cells died. Cultures containing IL-7 and IL-15 killed cancer cells more rapidly than those containing 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. [Example]

[0097] Evaluation of the cancer cell-killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptors 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 PD-L1-expressing gastric cancer cell line SNU638 and the colon cancer cell lines DLD1, LOVO, and HCT15 were co-cultured with the anti-PD-L1 CAR T cells (VYPC1), and their cancer cell-killing ability was measured.

[0098] Cancer cell lines that were confirmed to express PD-L1 antigen were placed in each well at 1 × 10 4 The cells were seeded in a 96-well plate and placed in a BSC for 30 minutes. After that, the cancer cells were incubated at 37°C, 5% CO 2 The cells were cultured in a cell incubator under the conditions described above for 24 hours. Then, 4 × 10 cells were added to each well. 4 Cells were treated with anti-PD-L1 CAR T cells (VYPC1) and co-cultured for 72-90 hours. Cell viability and motility were analyzed using RTCA (xCELLigence, ACEA Biosciences, Inc.). RTCA measures the resistance (impedance) generated during cell growth, enabling automated kinetic measurement of various cellular responses (cell number, proliferation rate, cell size, cell shape, and changes in substrate attachment quality). The resistance detected by the cells is expressed as a parameter called the cell index (CI), and changes in CI indicate changes in cell shape.

[0099] As shown in Figure 10, 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). 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 can be seen 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. [Example]

[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 each co-cultured with the anti-PD-L1 CAR T cells (VYPC1 and VYPC2), and their cancer cell-killing ability was measured.

[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 non-transduced human T cells (control group). Treatment with VYPC1 or VYPC2 of the present invention resulted in the death of parental or PD-L1 overexpressed (PD-L1 O / E) gastric cancer cell line SNU638, glioblastoma cell line U251, and colon cancer cell line DLD1 cancer cells.

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

[0104] Evaluation of the in vitro cell-killing ability of immune cells (VYPC1) expressing PD-L1-specific chimeric antigen receptors 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] 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 overexpressing PD-L1 antigen were co-cultured with anti-PD-L1 CAR T cells (VYPC1) using the same method as in Example 8. VYPC1 cells were cultured and co-cultured with either cytokine IL-2 or IL-7 + IL-15, respectively.

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

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

[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 approved by the Institutional Bioethics Committee (IACUC, Chonnam National University, South Korea). Four-week-old female NSG (NOD SCID gamma) mice were provided by the Special Purpose Pathogen Free (SPF) Animal Laboratory at the Hwasun Vaccine Center. 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 100 μL solution of each of the cells mixed with high-concentration Matrigel (Corning) at a 1:1 ratio was subcutaneously injected into the right flank of each mouse to create a human tumor xenograft mouse model. 3 At this time, each group received 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). 7 200 μL of the drug was injected intravenously once. Four to five mice were used per group, and tumor size and mouse weight were measured twice a week. Mouse weight was used to determine whether the mice were recovering and whether there were 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 weight loss in the group of mice administered with the anti-PD-L1 CAR T cells (VYPC1) of the present invention recovered from 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. [Example]

[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 approved by the Institutional Bioethics Committee (IACUC, Chonnam National University, South Korea). Four-week-old female NSG (NOD SCID gamma) mice were provided by the Special Purpose Pathogen Free (SPF) Animal Laboratory at the Hwasun Vaccine Center. 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 solution of 1:1 mixture of 100 μL of 100 μL of 100 μL of high-concentration Matrigel (Corning) was subcutaneously injected into the right flank of mice to create a human tumor xenograft mouse model. 3 At this time, each group received PBS, a high dose of control T cells (10 × 10 activated control normal T cells that were 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 (200µL of cells / 200µL). Seven mice were used per group, and tumor size and body weight were measured twice a week. Mouse weights were used to determine whether the mice were recovering and whether there were any side effects associated with the treatment.

[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 (VYPC1) of the present invention was significantly reduced from day 13 onwards compared to the control group. In particular, administration at a high dose 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. It contains a binding domain that specifically binds to PD-L1 (Programmed Death-Ligand 1), the binding domain that specifically binds to PD-L1 is a single-chain variable fragment (scFv) of an anti-PD-L1 antibody; A chimeric antigen receptor (CAR), wherein the single-chain variable fragment (scFv) of the anti-PD-L1 antibody comprises the amino acid sequence of SEQ ID NO:

8.

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 transmembrane domain (TM), and an intracellular domain.

3. The chimeric antigen receptor according to claim 2, wherein the signal sequence comprises the signal sequence of CD8α.

4. The chimeric antigen receptor according to claim 3, wherein the signal sequence of CD8α comprises the amino acid sequence of SEQ ID NO:

7.

5. The chimeric antigen receptor according to claim 2, characterized in that the hinge and transmembrane domain comprises the hinge and transmembrane domain of CD8α.

6. The chimeric antigen receptor of claim 5, wherein the hinge and transmembrane domain of CD8α comprises the amino acid sequence of SEQ ID NO:

9.

7. 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.

8. The chimeric antigen receptor according to claim 7, 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.

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

10. An expression vector comprising the polynucleotide of claim 9.

11. A virus comprising the expression vector of claim 10.

12. An immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 8 on its surface.

13. The immune cell according to claim 12, 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.

14. A pharmaceutical composition for preventing or treating cancer, comprising the immune cells of claim 12.

Images