Chimeric antigen receptor modified myeloid cells and their use for anti-cancer therapy - Patents.com

Abstract

The present invention relates to modified myeloid cells or iPS or HSCs comprising a chimeric antigen receptor (CAR), or modified induced pluripotent stem cells (iPS) or hematopoietic stem cells (HSCs) comprising a CAR, the CAR comprising an extracellular antigen binding domain that binds to a tumor antigen or a tumor microenvironment (TME) antigen, a transmembrane domain, and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ. The present invention also relates to therapeutic uses thereof.

Description

[Technical field]

[0001] The present invention relates to modified myeloid cells or iPS or HSCs comprising a chimeric antigen receptor (CAR), or modified induced pluripotent stem cells (iPS) or hematopoietic stem cells (HSCs) comprising a CAR, the CAR comprising an extracellular antigen binding domain that binds to a tumor antigen or an antigen present on a cell of the tumor microenvironment (TME), optionally a hinge region, a transmembrane domain, and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ. The present invention also relates to therapeutic uses thereof.

[0002] Solid tumors and their metastases are the most common and difficult-to-treat type of cancer today. The tumor microenvironment (TME) is a complex and heterogeneous mixture of cell populations that interact with each other and with tumor cells. The TME is immunosuppressive, evades the immune system, and prevents efficient elimination of malignant cells by therapeutic intervention. Myeloid cells within the TME play a key role, contributing to immune evasion by exhibiting potent immunosuppressive and pro-tumorigenic properties.

[0003] Tumor-associated macrophages (TAMs) are important cellular components of the TME in various cancers. The prevailing consensus is that tumor-derived cytokines direct the recruitment of myeloid cells at the monocytic stage, and the TME then influences their development into polarized macrophages. TAMs can represent a significant portion of the tumor burden, up to 50% in some breast tumors. TAMs develop into immunosuppressive macrophages, which then express antitumor CD8 +They prevent T cells from infiltrating tumors and attract or induce regulatory T cells (Tregs). TAMs secrete growth factors such as VEGF or TGFβ, which promote tumor growth and invasive behavior. TAMs are generally associated with poor prognosis, but recent studies have shown that the prognostic impact of TAMs may vary depending on their localization and polarization (Ramos et al, 2022, Cell 185, 1-19, Tissue-resident FOLR2 + macrophages associate with tumor-infiltrating CD8 + T cells and with increased survival of breast cancer patients).

[0004] In recent years, there have been remarkable successes in treating certain malignancies with autologous immune cell-based therapies. The most advanced approach relies on T lymphocytes genetically engineered to express chimeric receptors that combine antigen-binding and T cell activation activities in one receptor, known as CAR T cells. Adoptively transferred CAR T cells have shown considerable promise in the fight against hematological malignancies.

[0005] However, CAR T cell therapy has so far failed to treat solid tumors. These failures are likely due to a combination of factors. First, it remains difficult to identify precise tumor-specific antigens, which raises concerns about possible off-target effects. Second, before reaching cancer cells in tumor tissue, CAR T cells may encounter physical barriers in the form of TAMs and cancer-associated fibroblasts that produce vast amounts of extracellular matrix. Third, CAR T cells cannot successfully infiltrate the TME due to lack of metabolic resources and signals generated by TME cellular components. Finally, the continuous antigenic stimulation they receive via tumor cells can cause CAR T cells to become “exhausted,” i.e., dysfunctional, lose their effector functions, and fail to develop into effector memory T cells.

[0006] Macrophages are antigen-presenting cells that can stimulate T cells locally, thus promoting adaptive antitumor responses. Macrophages produce proteases that can dramatically modify the extracellular matrix within the tumor mass and thus the architecture of tumor tissue. Macrophages also possess antitumor capabilities, including the ability to phagocytose whole tumor cells and perform antibody-dependent cellular phagocytosis. These unique properties make macrophages an ideal candidate to overcome the limitations of CAR T cells.

[0007] Various strategies have been employed to harness the antitumor potential of myeloid cells through genetic engineering. One of the most promising strategies has been to recruit the tumor cell phagocytic capacity of macrophages by virally transducing them with a CAR construct. For example, in WO 2017 / 019848, macrophages were transduced with an adenoviral vector encoding a CAR construct composed of an anti-HER2 scFv fused to a transmembrane domain and a CD3z intracellular domain. In vitro, these CAR macrophages were able to specifically phagocytose SKOV3 cells, a HER2-expressing cell line. In vivo, these CAR macrophages inhibited SKOV3 tumor growth, prolonged mouse survival, and reduced lung metastasis burden when injected into tumor-bearing NSG (NOD / SCID / Gchainnull) mice. Adenoviral vector transduction of macrophages enhanced interferon-related gene expression and polarized macrophages toward a proinflammatory phenotype.

[0008] However, there is a need for therapeutic cells that are not only capable of activating their own superior tumor cell phagocytic ability upon binding to a given antigen, but also capable of recruiting the antigen-presenting and costimulatory capabilities of macrophages upon tumor cell encounter.

[0009] The present invention addresses this need. Summary of the Invention

[0010] That is, the present invention relates to a modified cell comprising a chimeric antigen receptor (CAR), the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells in the tumor microenvironment (TME); optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising (i) STING or one of its fragments, and / or (ii) the intracellular domain of CD3ζ; Including, The modified cells are myeloid cells. Concerning modified cells.

[0011] That is, the present invention relates to a modified cell comprising a chimeric antigen receptor (CAR), the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; Including, The modified cells are myeloid cells. Concerning modified cells. The myeloid cells modified with the CAR are referred to herein as "CAR myeloid cells." Preferably, the CAR comprises a hinge domain between the extracellular antigen-binding domain and the transmembrane domain.

[0012] The present invention also relates to a modified induced pluripotent stem cell (iPS) or hematopoietic stem cell (HSC) comprising a chimeric antigen receptor (CAR), the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; The present invention relates to a modified iPS or HSC comprising: Said CAR modified iPS or HSC are referred to herein as "CAR iPS" or "CAR HSC", respectively.

[0013] The present invention also relates to a pharmaceutical composition comprising the modified CAR myeloid cells, or modified CAR iPS or CAR HSC, and a pharma- ceutically acceptable carrier.

[0014] The present invention also relates to the use of modified CAR myeloid cells, or modified CAR iPS or CAR HSC, in the treatment of cancer or inflammatory diseases.

[0015] The invention also relates to products comprising modified CAR myeloid cells, modified CAR iPS or CAR HSC and CAR-T cells as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or inflammatory diseases.

[0016] The present invention also relates to products comprising modified CAR myeloid cells, modified CAR iPS or CAR HSC and an immune checkpoint inhibitor (ICI) as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or inflammatory diseases.

[0017] The present invention further provides a method of producing a modified CAR myeloid cell, modified CAR iPS or CAR HSC, comprising: Providing at least one cell selected from an isolated myeloid cell, an induced pluripotent stem cell (iPS), and a hematopoietic stem cell (HSC); transducing the cells with a vector, preferably a lentiviral vector, comprising a nucleic sequence encoding the CAR; The present invention relates to a method comprising the steps of:

[0018] The present invention further provides an in vitro assay for co-culturing a tumor cell line and a myeloid cell line, comprising: a) culturing at least one tumor cell line or at least one tumor cell derived from a primary tumor together with at least one myeloid cell line in ultra-low attachment surface plates such that all cell lines or cells grow in spheroid form; b) Tracking the growth of co-cultured cell lines or 3D spheroids by time-lapse microscopy; c) Optionally, periodically collecting samples of the co-cultured cell line or cells, the three-dimensional spheroids and / or the supernatant, analyzing their composition and performing three-dimensional imaging; The present invention relates to an in vitro assay method comprising:

[0019] The present invention also provides a modified cell comprising a CAR, the CAR comprising: an extracellular antigen-binding domain with antigen specificity for a tumor antigen or an antigen present on cells of the TME; optionally, a hinge domain; A transmembrane domain; and an intracellular signaling domain comprising STING or one of its fragments; Including, The modified cells are myeloid cells. Concerning modified cells.

[0020] The present invention also relates to a modified cell comprising a CAR, the CAR comprising: an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; Optionally, a hinge domain, A transmembrane domain; and an intracellular signaling domain comprising STING or one of its fragments fused to (i) the cytoplasmic tail of CD40, and / or (ii) the intracellular domain of CD3ζ; Including, The modified cells are myeloid cells. Concerning modified cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present inventors have now synthesized a novel CAR myeloid cell lineage that contains the cytoplasmic domain of the CD40 molecule and CD3ζ. CD3ζ can activate the phagocytosis pathway, and CD40 is expressed by all macrophages and their precursors, enhancing the proinflammatory function of macrophages upon interaction with its ligand (CD40L) expressed by activated T lymphocytes. CD40 signaling triggers the secretion of proinflammatory cytokines, chemokines, and the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinases.

[0022] Surprisingly, as shown in the Examples, the inventors have demonstrated that the CD40 cytoplasmic domain in the CAR construct of the invention enhances the antigen-dependent immune response of the resulting CAR macrophages through the secretion of proinflammatory cytokines. These cells are also capable of phagocytosis of tumor cells in two and three dimensions. The inventors have also demonstrated that in the preclinical setting of human tumor growth in NSG mice, the CAR myeloid cells of the invention induce efficient tumor regression and provide mice partially reconstituted with human T cells.

[0023] That is, the present invention relates to a modified cell comprising a chimeric antigen receptor (CAR), the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells in the tumor microenvironment (TME); optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising (i) STING or one of its fragments, and / or (ii) the intracellular domain of CD3ζ; Including, The modified cells are myeloid cells. Concerning modified cells.

[0024] That is, the present invention also relates to a modified cell comprising a CAR, the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; The modified cells are myeloid cells. The present invention relates to a modified cell comprising: The myeloid cells modified with the CAR are referred to herein as "CAR myeloid cells."

[0025] The present invention also provides a modified cell comprising a CAR, the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells in the tumor microenvironment (TME); optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused, preferably at its C-terminus, to a) a second intracellular signaling domain comprising STING or one of its fragments, or b) the intracellular domain of CD3ζ; Including, the intracellular domain of CD3ζ is fused, preferably at its C-terminus, to a second intracellular signaling domain comprising STING or a fragment thereof; The modified cells are myeloid cells. Concerning modified cells.

[0026] The present invention also relates to a modified induced pluripotent stem cell (iPS) or hematopoietic stem cell (HSC) comprising a chimeric antigen receptor (CAR), the CAR comprising: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; The present invention relates to a modified iPS or HSC comprising:

[0027] Said CAR modified iPS or HSC are referred to herein as "CAR iPS" or "CAR HSC", respectively.

[0028] Myeloid cells A myeloid cell of the present invention is any cell type that is derived from or resembles bone marrow tissue (bone marrow).

[0029] Preferably, the myeloid cell is a monocyte, macrophage, or dendritic cell, more preferably a monocyte.

[0030] The myeloid cells are modified in that they express a CAR.

[0031] Induced pluripotent stem cells (iPS) or hematopoietic stem cells (HSC) The term "stem cell" refers to a cell that can give rise to specialized cells by successive divisions. The term "pluripotent stem cell" refers to a stem cell that has the ability to differentiate into any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). Pluripotent stem cells can give rise to both fetal and adult cell types, but cannot give rise to an entire organism. "Pluripotent stem cells" can be identified by expression of one or more of the cell markers Klf4, Sox2, Oct4, cMyc, Nanog, and SSEA1. A cell is considered a pluripotent stem cell if it is capable of giving rise to cells from any of the three germ layers: endoderm (identified by expression of alpha-fetoprotein); mesoderm (identified by expression of desmin and / or alpha smooth muscle actin), and ectoderm (identified by expression of class III beta tubulin (=Tuj1) and / or E-cadherin-N-cadherin). Assays for assessing the pluripotency of a cell are known in the art.

[0032] The term "induced pluripotent stem cells" or "iPS" refers to pluripotent cells artificially derived from non-pluripotent cells, typically adult somatic cells, by inducing forced expression of certain genes. "Induced pluripotent stem cells" are defined by the expression of several transcription factors, including one or more of Klf4, Sox2, Oct4, and cMyc. iPS cells are typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved via a viral vector, such as a retrovirus, and transfected genes include Oct-3 / 4 (Pou5fl) and Sox2. Additional genes include certain members of the Klf family (Klfl, Klf2, Klf4, and Klf5), certain members of the Myc family (c-myc, L-myc, N-myc), and Nanog and LIN28 have been identified to increase induction efficiency. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated through morphological sorting, doubling time, or through reporter gene and antibiotic selection. Protocols for iPS culture are disclosed in Mochiduki and Okita, 2012. Non-pluripotent cells that can be used to obtain iPS include, but are not limited to, fibroblasts, keratinocytes, and adipocytes. These cells can be obtained from adults by methods well known in the art (Mochiduki and Okita, 2012).

[0033] "Hematopoietic stem cells" (HSCs) have the capacity to completely reconstitute the immune system of the lethally irradiated host from which they are derived. Hematopoietic stem cells give rise to all blood and immune cells.

[0034] The iPS or HSCs are modified in that they express a CAR.

[0035] The CAR myeloid cells, CAR iPS or CAR HSC comprise a CAR as detailed below.

[0036] Chimeric antigen receptors (CARs) The CAR of the present invention comprises, from its N-terminus to its C-terminus: an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; Includes.

[0037] Between each domain, there may be a linker, which may be the same or different.Preferably, CAR does not contain any linker between different domains.In other words, CAR is obtained by direct fusion between different domains.

[0038] Antigen-binding domain The CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, comprise an extracellular antigen binding domain at the N-terminus of the CAR that binds to a tumor antigen or an antigen present on cells of the TME (i.e., a TME antigen).

[0039] An "antigen-binding domain" may be any polypeptide or fragment thereof, such as an antibody fragment variable domain, naturally derived or synthetic, that binds to an antigen. Antigen-binding domains include, inter alia, antibody-derived polypeptides such as single chain variable fragments (scFv), Fab, Fab', F(ab')2, Fv fragments, and nanobodies; T-cell receptor (TCR) derived polypeptides such as TCR variable domains; and any ligand or receptor fragment that binds to an antigen. The antigen-binding domain has antigen specificity for a tumor antigen or a TME antigen. An "antigen-binding domain with antigen specificity for a tumor antigen" is an antigen-binding domain that binds to an antigen on a tumor. An "antigen-binding domain with antigen specificity for a TME antigen" is an antigen-binding domain that binds to an antigen present on cells in the tumor microenvironment (TME). The TME includes tissues and cells surrounding the tumor; in particular, surrounding blood vessels, immune cells such as Treg cells and immunosuppressive macrophages, fibroblasts, signaling molecules, and extracellular matrix.

[0040] Preferably, the tumor antigen is selected from antigens that are expressed at a higher level on the surface of tumor cells than on other cell types. Preferably, the tumor antigen is selected from CD19, MUC16, MUC1, CA1X, carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD2Ac, GD3, ITER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, protein ase3 (PR1), tyrosinase, survivin, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-l, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-l, CD70, Tim3, CLEC12A, ER, human telomerase reverse transcriptase (hTERT), mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, p95HER2, Wilms tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), prostate-specific membrane antigen (PSMA), cyclin (Dl), mesothelin, B-cell maturation antigen (BCMA), and tumor-associated calcium transducer and activator of signaling 2 (TROP2).

[0041] Preferably, the TME antigen is selected from an antigen expressed by activated CAFs, such as FAP, an antigen expressed by regulatory T cells, and an antigen expressed by pre-neoplastic myeloid cells, such as TREM-2. Preferably, the TME antigen is selected from FAP, an antigen expressed by regulatory T cells, and TREM-2.

[0042] Preferably, the tumor antigen or TME antigen is CD 19. More preferably, the extracellular antigen binding domain that binds to the tumor antigen or TME antigen is an anti-CD19 binding domain, preferably an anti-CD19 scFV.

[0043] Preferably, the extracellular antigen binding domain comprises the following amino acid sequence: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 1).

[0044] Hinge Domain The CAR may comprise a hinge domain, which confers flexibility to the resulting CAR.

[0045] The hinge domain may be any hinge domain present in an immunoglobulin or in a CD molecule.

[0046] Preferably, the hinge domain is the hinge domain of CD8. CD8 comprises an alpha chain (CD8a) and a beta chain (CD8b). Preferably, the hinge domain is the hinge domain of the CD8a chain.

[0047] The human version of CD8a may be found in UniProt under the accession number Q8TAW8. CD8a contains 235 amino acids. The hinge domain is a fragment of said sequence consisting of amino acids 138 to 182, which corresponds to SEQ ID NO:2.

[0048] Preferably, the hinge domain is that of CD8a, preferably human CD8a.

[0049] Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:2).

[0050] Transmembrane domain CAR comprises a transmembrane domain, which may be a single or multiple transmembrane sequence.

[0051] Single-pass transmembrane domains are present in certain CD molecules, tyrosine kinase receptors, serine-threonine kinase receptors, TGF, BMP, activin, and phosphatases. Single-pass transmembrane domains often contain a signal peptide domain and a transmembrane domain of about 20 to about 25 amino acids, many of which are hydrophobic and capable of forming an alpha helix. A short sequence of positively charged amino acids often follows the transmembrane domain to anchor the protein to the membrane.

[0052] Multi-spanning membrane domains are present in proteins such as ion pumps, ion channels, and transporters and contain two or more helices that traverse the membrane multiple times.

[0053] The sequences of single and multiple transmembrane domains are known and can be selected for incorporation into the CAR.

[0054] The transmembrane domain may be selected from wild-type transmembrane domains and mutant transmembrane domains. The mutant transmembrane domain may be modified by mutations such as amino acid substitutions (e.g., typically charged amino acids are replaced by hydrophobic residues). Preferably, the transmembrane domain is the transmembrane domain of the α, β or ζ chain of the T cell receptor, CD3-8, CD3ζ, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137 or CD154. Preferably, the transmembrane domain is the transmembrane domain of CD8.

[0055] The transmembrane domain may be synthesized de novo, composed largely of hydrophobic residues such as leucine and valine.

[0056] In the present invention, the transmembrane domain is fused at its N-terminus to the extracellular antigen-binding domain of the CAR and at its C-terminus to the intracellular signaling domain.

[0057] In an embodiment, a short polypeptide linker may form the junction between the transmembrane domain and the intracellular signaling domain of the CAR.

[0058] CARs may further comprise a stalk, an extracellular region consisting of amino acids, between the extracellular antigen binding domain and the transmembrane domain. For example, the stalk may be a series of amino acids naturally associated with a selected transmembrane domain.

[0059] Preferably, the CAR comprises a transmembrane domain of CD8. Preferably, the CAR comprises a transmembrane domain of CD8 and a hinge domain of CD8. The hinge domain is preferably fused at its C-terminus (preferably directly) to the N-terminus of the transmembrane domain.

[0060] Preferably, the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3), which is the transmembrane domain of human CD8.

[0061] Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:2).

[0062] Intracellular signaling domains The CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, comprise an intracellular signaling domain at the C-terminus of the CAR.

[0063] Specifically, the intracellular signaling domain comprises, from the N-terminus to the C-terminus, a first intracellular signaling domain comprising the cytoplasmic terminus of CD40, and a second intracellular signaling domain comprising the intracellular domain of CD3ζ fused thereto.

[0064] The first intracellular signaling domain comprises the cytoplasmic tail of CD40. By "cytoplasmic tail of CD40" is meant the cytoplasmic domain of the CD40 molecule. CD40, also known as TNFRSF5, is a costimulatory protein present on antigen-presenting cells and is required for their activation. The sequence of human CD40 (hCD40) may be found in UniProt under accession number P25942. Human CD40 comprises 277 amino acids. The fragment comprising amino acids 216 to 277 of said sequence is the cytoplasmic portion. Said fragment corresponds to SEQ ID NO: 4.

[0065] Preferably, the first intracellular signaling domain comprises the cytoplasmic tail of CD40 that is a fragment of human CD40.

[0066] Preferably, the first intracellular signaling domain comprises the amino acid sequence KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ ID NO:4).

[0067] The first intracellular signaling domain is fused at its C-terminus to a second intracellular signaling domain comprising the intracellular domain of CD3ζ. Preferably, the fusion is direct, i.e., without any linker.

[0068] CD3ζ, also known as OKT3 or CD247, is a component of the T cell receptor (TCR) complex. In humans, in 95% of T cells, the TCR consists of α and β chains, whereas in 5% of T cells, the TCR consists of γ and δ chains. At the plasma membrane, the α and β chains of the TCR further associate with six adaptor proteins to form an octameric complex. The complex contains both the α and β chains to form the ligand binding site, but also one CD3γ chain, one CD3δ chain, two CD3ε chains, and two CD3ζ chains.

[0069] The sequence of human CD3 zeta chain (hCD3ζ) may be found in UniProt under accession number P20963. Human CD40 contains 164 amino acids. The fragment containing amino acids 52 to 164 of said sequence is the cytoplasmic part. Said fragment corresponds to SEQ ID NO:5.

[0070] Preferably, the second intracellular signaling domain comprises the intracellular domain of human CD3ζ.

[0071] Preferably, the second intracellular signaling domain comprises the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:5).

[0072] Preferably, the CAR comprises from its N-terminus to its C-terminus: an extracellular antigen-binding domain having the sequence of SEQ ID NO:1; Optionally, a hinge domain having the sequence of SEQ ID NO:2, A transmembrane domain having the sequence SEQ ID NO:3, a first intracellular signaling domain having the sequence of SEQ ID NO:4 fused, preferably directly, to a second intracellular signaling domain having the sequence of SEQ ID NO:5; Includes.

[0073] The CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, preferably exhibit targeted effector activity. By "targeted effector activity" is meant at least one effector activity selected from phagocytosis, targeted cytotoxic activity, cytokine production, reactive oxygen species (ROS) generation, myeloid activation, antigen processing and presentation to T cells, and the in vivo ability to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells. Preferably, the targeted effector activity is selected from antigen-dependent tumor cell phagocytosis, antigen-dependent tumor cell cytokine secretion, and the in vivo ability to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells. Said human antigen-dependent tumor regression will be mediated both by macrophage phagocytosis of tumor cells and by tumor-specific T cell killing of tumor cells. Antigen-dependent tumor cell phagocytosis and antigen-dependent tumor cell cytokine secretion may be measured according to methods well known in the art and are illustrated in the examples. The in vivo capacity to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells will be assessed according to the protocol described in the Examples.

[0074] The CAR myeloid cell according to the invention, or the CAR iPS or CAR HSC according to the invention preferably comprises an additional vector comprising a sequence encoding a gene of interest under the control of a cytokine-specific promoter. Preferably, the gene of interest is selected from a gene encoding IFNγ, a gene encoding IFNα, a gene encoding IFNβ, a gene encoding IFNλ, a gene encoding IL12, and a gene encoding IL10 or TGFβ.

[0075] Preferably, the gene of interest is a human gene.

[0076] The present invention further relates to a modified cell comprising a CAR, said CAR comprising: an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; A transmembrane domain; and an intracellular signaling domain comprising STING or one of its fragments; The present invention relates to a modified cell comprising: said modified cell being a myeloid cell.

[0077] With the exception of the intracellular signaling domain, the above embodiments and definitions relating to CAR myeloid cells also apply to such modified myeloid cells bearing a CAR comprising STING or one of its fragments (said CAR is referred to as "CAR-STING").

[0078] The present invention further relates to a modified cell comprising a CAR, said CAR comprising: an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; Optionally, a hinge domain, A transmembrane domain; and an intracellular signaling domain comprising STING or one of its fragments fused to (i) the cytoplasmic tail of CD40, and / or (ii) the intracellular domain of CD3ζ; Including, The modified cells are myeloid cells. Concerning modified cells.

[0079] The present invention further relates to a modified cell comprising a CAR, said CAR comprising: an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; Optionally, a hinge domain, A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused, preferably at its C-terminus, to a) a second intracellular signaling domain comprising STING or one of its fragments, or b) the intracellular domain of CD3ζ; Including, the intracellular domain of CD3ζ is fused, preferably at its C-terminus, to a second intracellular signaling domain comprising STING or a fragment thereof; The modified cells are myeloid cells. Concerning modified cells.

[0080] The above embodiments and definitions regarding CAR myeloid cells also apply to such modified myeloid cells having a CAR comprising STING or one of its fragments, the cytoplasmic tail of CD40 and / or the intracellular domain of CD3ζ.

[0081] Preferably, a first intracellular signalling domain comprising STING or one of its fragments is fused, preferably at its N-terminus, to a second intracellular signalling domain comprising the cytoplasmic tail of CD40; or, preferably at its N-terminus, to a third intracellular signalling domain comprising the intracellular domain of CD3ζ, which is fused, preferably at its N-terminus, to a second intracellular signalling domain comprising the cytoplasmic tail of CD40.

[0082] The Stimulator of Interferon Genes (STIMULATOR OF INTERFEON GENES) protein is an endoplasmic reticulum (ER)-resident protein that plays a central role in innate immunity. In reality, STING is an adaptor protein that orchestrates the transcriptional activation of type I interferons and proinflammatory cytokines in the presence of aberrant nucleic acid species. STING activation depends on the detection of dsDNA, ssDNA, or RNA:DNA hybrids by the pathogen recognition receptor cyclic GMP-AMP synthetase (cGAS). The association of cGAS with these nucleic acid species in the cytosol was found to lead to cGAS-dependent synthesis of cyclic GMP-AMP (cGAMP). The interaction of cGAMP with STING activates pathways that ultimately lead to the transcription of proinflammatory cytokines and type I interferons.

[0083] The sequence of human STING can be found in UniProt under accession number A0A2R3XZB7. Human CD40 contains 379 amino acids. Preferably, a sequence with several amino acid deletions at the N-terminus is used.

[0084] STING may be used in its wild-type version or, alternatively, in a mutant form that reduces its activity.

[0085] Preferably, a fragment of STING is used, which preferably contains an N-terminal deletion, preferably corresponding to amino acids 137 to 379 of A0A2R3XZB7.

[0086] Preferably, the intracellular signaling domain comprises the amino sequence KGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (SEQ ID NO: 6).

[0087] The intracellular signalling domain comprising STING or one of its fragments may further comprise the cytoplasmic tail of CD40, preferably as described above, and / or the intracellular domain of CD3ζ, preferably as described above.

[0088] The present invention also relates to a nucleic acid sequence encoding said CAR. Said nucleic acid sequence may be a DNA or RNA sequence. Said nucleic acid sequence may be used in therapy, in particular for treating cancer or inflammatory diseases. Preferably, said nucleic acid sequence is administered to a subject, preferably by injection. In this way, macrophages of said subject receive said nucleic acid sequence and subsequently express CAR, in particular CAR-STING.

[0089] Preferably, the nucleic sequence encoding the intracellular domain of CAR-STING is the sequence of SEQ ID NO:7.

[0090] therapeutic use The present invention also relates to the use of a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, as a medicament.

[0091] The present invention also relates to a pharmaceutical composition comprising a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, and a pharma- ceutical acceptable carrier.

[0092] The present invention also relates to the use of a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, or of a pharmaceutical composition as described above, in the treatment of cancer or an inflammatory disease. The inflammatory disease may be an autoimmune disease.

[0093] The present invention also relates to a product comprising a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, and a CAR-T cell as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or an inflammatory disease.

[0094] CAR-T cells are well known in the art. Preferably, the CAR-T cells are selected from tisagenlecleucel, axicabtagene ciloleucel, brexcabtagene autoleucel, lysocabtagene maraleucel, and idecabtagene biculeucel.

[0095] The present invention also relates to a product comprising a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, and an immune checkpoint inhibitor (ICI) as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or an inflammatory disease.

[0096] "Immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reducing and completely blocking the function. In particular, the immune checkpoint protein is a human immune checkpoint protein. That is, the immune checkpoint protein inhibitor is preferably an inhibitor of a human immune checkpoint protein.

[0097] Immune checkpoint proteins that may be cited as examples are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR (such as KIR3DL2, KIR2DL1 / 2 / 3, KIR2L3), TIGIT, VISTA, IDO, CEACAM-1, or A2aR.

[0098] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or preferably antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies.

[0099] Preferably, the ICI is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR (such as KIR3DL2, KIR2DL1 / 2 / 3, KIR2L3), TIGIT, VISTA, IDO, CEACAM-1, or A2aR. Preferably, the ICI is an anti-CTLA-4 antibody, more preferably tremelimumab or ipilimumab. In some embodiments, the ICI is an anti-killer cell immunoglobulin-like receptor (KIR) antibody, more preferably lirilumab and IPH4102. Preferably, the ICI is an anti-PD-1 antibody, more preferably nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335, or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475, or Keytruda), pidilizumab, AMP-514, cemiplimab (REGN2810), CT-011, BMS936559, MPDL3280A, AMP-224, tislelizumab (BGB-A317), spartalizumab (PDR001 or PDR -001), ABBV-181, JNJ-63723283, BI754091, MAG012, TSR-042, AGEN2034, and antibodies described in WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2009014708, WO2009114335, WO2013043569, and WO2014047350. Preferably, the PD-L1 inhibitor is durvalumab, atezolizumab, LY3300054, or avelumab. Preferably, the PD-L2 inhibitor is rHIgM12B7. Preferably, the LAG3 inhibitor is IMP321, BMS-986016, or an inhibitor of the LAG3 receptor described in US Patent No. 5,773,578.Preferably, the inhibitor of A2aR is PBF-509.Preferably, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, including but not limited to ipilimumab (see, e.g., U.S. Patent Nos. 6,984,720 and 8,017,114), tremelimumab (see, e.g., U.S. Patent Nos. 7,109,003 and 8,143,379), single chain anti-CTLA4 antibodies (see, e.g., WO1997020574 and WO2007123737), and antibodies described in U.S. Patent No. 8,491,895. An example of an anti-VISTA antibody is described in U.S. Patent Application Publication No. 20130177557. Preferably, the ICI is selected from tremelimumab, ipilimumab, lirilumab, nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS936559, MPDL3280A, AMP-224, durvalumab, atezolizumab, avelumab, rHIgM12B7, IMP321, BMS-986016, and PBF-509.

[0100] The present invention also relates to a product comprising a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, and an immune checkpoint therapy associated with costimulatory antibodies that positively signal through immunomodulatory receptors, including but not limited to ICOS, CD137, CD27, OX-40, and GITR, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or inflammatory diseases.

[0101] The invention also relates to a product comprising the CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, and an additional cancer therapy as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer. In particular, the product comprising the CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, may be administered in combination with targeted therapy, immunotherapy, such as immune checkpoint therapy and / or immune checkpoint inhibitors, costimulatory antibodies, chemotherapy, and / or radiation therapy.

[0102] In some embodiments, the products comprising the CAR myeloid cells of the invention or the CAR iPS or CAR HSC of the invention may be used in combination with targeted therapy. As used herein, the term "targeted therapy" refers to targeted therapeutic agents, which are drugs designed to interfere with specific molecules required for tumor growth and progression. For example, targeted therapeutic agents, such as therapeutic monoclonal antibodies, target specific antigens present on the cell surface, such as transmembrane receptors or extracellular growth factors. Small molecules can penetrate the cell membrane and interact with targets inside the cell. Small molecules are usually designed to interfere with the enzymatic activity of target proteins, such as, for example, inhibitors of proteasomes, inhibitors of tyrosine kinases or cyclin-dependent kinases, inhibitors of histone deacetylases, etc. Targeted therapy may also use cytokines.Examples of such targeted therapies include: ado-trastuzumab etansine (HER2), afatinib (EGFR (HER1 / ERBB1), HER2), aldesleukin (Proleukin), alectinib (ALK), alemtuzumab (CD52), axitinib (kit, PDGFRβ, VEGFR1 / 2 / 3), belimumab (BAFF), belinostat (HDAC), bevacizumab (VEGF ligand), blinatumomab (CD19 / CD 3), bortezomib (proteasome), brentuximab vedotin (CD30), bosutinib (ABL), brigutinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), canakinumab (IL-1β), carfilzomib (proteasome), ceritinib (ALK), cetuximab (EGFR), cofimetinib (MEK), crizotinib (ALK, MET, ROS1), dabrafenib (BR AF), daratumumab (CD38), dasatinib (ABL), denosumab (RANKL), dinutuximab (B4GALNT1(GD2)), elotuzumab (SLAMF7), enasidenib (IDH2), erlotinib (EGFR), everolimus (mTOR), gefitinib (EGFR), ibritumomab tiuxetan (CD20), sonidegib (Smoothened), sipuleucel-T, siltuximab (IL-6), sorafenib (VEGFR, PDGFR, KIT, RAF), (tocilizumab (IL-6R), temsirolimus (mTOR), tofacitinib (JAK3), trametinib (MEK), tositumomab (CD20), trastuzumab (HER2), vandetanib (EGFR), vemurafenib (BRAF), venetoclax (BCL2), vismodegib (PTCH, Smoothened), vorinostat (HDAC), Ziv-aflibercept (PIGF, VEGFA / B), olaparib (PARP inhibitor).

[0103] In some embodiments, the CAR myeloid cells of the invention, or products comprising the CAR iPS or CAR HSC of the invention, may be used in combination with chemotherapy. As used herein, the term "anti-tumor chemotherapy" or "chemotherapy" has its ordinary meaning in the art and refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several anti-neoplastic or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (synthetic analogs topoisomerase inhibitors); tecan; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and biceresin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosurea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics such as the enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gammall and calicheamicin omegall); the dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins; and the enediyne antibiotic chromophores of neocarzinostatin chromophore and related chromoproteins, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone;Aldophosphamide glycoside;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;eflornithine;elformithine acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;maytansinoids such as maytansine and ansamitocins;mitoguazone;mitoxantrone;mopidamol;nitrile Nitraerine; pentostatin; phenameth; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecines (especially T-2 toxin, verrucarin A, roridin A, and anguidin) ; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxa introne;vincristine;vinorelbine;novantrone;teniposide;edatrexate;daunomycin;aminopterin;xeloda;ibandronate;irinotecan (e.g., CPT-11);topoisomerase inhibitors RFS2000;difluoromethylomithine (DMFO);retinoids such as retinoic acid;capecitabine;anthracyclines, nitrosoureas, antimetabolites, epipodophyllotoxins, enzymes such as L-asparaginase;anthracenediones;Hormones and antagonists, including corticosteroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens such as flutamide; and pharma- ceutical acceptable salts, acids, or derivatives of any of the above.

[0104] In some embodiments, the CAR myeloid cells of the invention, or the products comprising the CAR iPS or CAR HSC of the invention, are administered to the patient in combination with radiation therapy for simultaneous, separate, or sequential use in the treatment of cancer or inflammatory diseases. Suitable examples of radiation therapy include external beam radiation therapy (superficial X-rays therapy, orthovoltage X-ray therapy, megavoltage radiation therapy, radiosurgery, stereotactic radiation therapy, fractionated stereotactic radiation therapy, cobalt beam therapy, electron beam therapy, fast neutron beam therapy, neutron capture therapy, proton beam therapy, intensity modulated radiation therapy (IMRT), three-dimensional conformal radiation therapy (3D-CRT), etc.); brachytherapy; unsealed source radiotherapy; tomotherapy, etc. Gamma rays are another form of photons used in radiation therapy. Gamma rays are naturally produced by certain elements (such as radium, uranium, and cobalt 60) releasing radiation as they decompose, i.e., break down. In some embodiments, the radiation therapy may be proton therapy or proton minibeam radiation therapy.Proton therapy is an ultra-precision form of radiation therapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun 1;104(2):266-271. doi: 10.1016 / j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1):16479. doi: 10.1038 / s41598-018-34796-8). The radiation therapy may be FLASH radiotherapy (FLASH-RT) or FLASH proton beam radiation therapy.FLASH radiotherapy involves ultrafast delivery of radiation therapy at dose rates several orders of magnitude higher than those currently used in routine clinical practice (ultra-high dose rates) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31: 121-123. DOI: 10.1051 / medsci / 20153102002); Patriarca A., Fouillade CM, Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j.ijrobp.2018.06.403. Epub (Jul 11, 2018).

[0105] Also described are methods of treating cancer or an inflammatory disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CAR myeloid cell of the invention, or a CAR iPS or CAR HSC of the invention. harvesting myeloid cells from a patient; modifying at least one of the myeloid cells by introducing into the cell a vector, preferably a lentiviral vector, comprising a nucleic sequence encoding a CAR; and reinfusing the modified myeloid cells into the patient; A method is also described that includes:

[0106] Cancer refers to tumors. Tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors. Refractory tumors include tumors that do not respond or are resistant to treatment with chemotherapy alone, antibodies alone, radiation alone, or a combination of these. Refractory tumors also include tumors that appear to be suppressed by treatment with such agents but recur within 5 years, and sometimes more than 10 years, after treatment is discontinued.

[0107] Examples of cancers that can be treated by the CAR myeloid cells of the invention, or the CAR iPS or CAR HSC of the invention, include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may be specifically of the following histological types, but is not limited to these: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma of familial polyposis coli; solid tumors; malignant carcinoid tumors; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma carcinoma); adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystic carcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant cytoma (and roblastoma, malignant); Sertoli cell tumor; malignant Leydig cell tumor; malignant lipocytoma; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; malignant glomus tumor sarcoma);malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;malignant blue nevus;sarcoma;fibrosarcoma;malignant fibrous histiocytoma;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;Malignant mesenchymal cell tumor;Malignant Brenner's tumor;Malignant phyllodes tumor;Synovial sarcoma;Malignant mesothelioma;Dysgerminoma;Embryonal carcinoma;Malignant teratoma;Malignant ovarian thyroid tumor;Choriocarcinoma;Malignant mesonephroma;Hemangiosarcoma;Malignant hemangioendothelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma;Parosteal osteosarcoma;Chondrosarcoma;Malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblastic odontosarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal (primitive neuroectodermal);cerebellar sarcoma;ganglionioblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;malignant meningioma;neurofibrosarcoma;malignant neurilemmoma;malignant granular cell tumor;malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;lateral granuloma;small lymphocytic lymphoma;diffuse large cell lymphoma;follicular lymphoma;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia. ;

[0108] Preferably, the cancer is a solid tumor or a metastasis.

[0109] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventive treatments and therapeutic or disease-modifying treatments, including treatment of subjects at risk of or suspected of having a disease, and subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and also includes the suppression of clinical recurrence. Treatment may be administered to subjects who have a medical disorder or who may ultimately acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the survival of the subject beyond that expected in the absence of such treatment.

[0110] "Therapeutic regimen" refers to a pattern of treatment for a disease, e.g., the pattern of administration used during treatment. Therapeutic regimens include induction regimens and maintenance regimens.

[0111] The term "induction regimen" or "induction phase" refers to a treatment regimen (or a portion of a treatment regimen) used in the early stages of disease treatment. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of the treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen" that may involve administering a higher dose of drug than the physician would employ during a maintenance regimen, administering the drug more frequently than the physician would administer the drug during a maintenance regimen, or both.

[0112] The term "maintenance regimen" or "maintenance phase" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a subject during treatment of a disease, for example, to keep the subject in remission for an extended period of time (months or years). A maintenance regimen may employ continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a certain predetermined criterion (e.g., disease manifestation, etc.).

[0113] By "therapeutically effective amount" is meant a sufficient amount of the CAR myeloid cells of the present invention, or the CAR iPS or CAR HSC of the present invention, to treat a disease (e.g., cancer) at a reasonable risk / benefit ratio applicable to any medical therapy. It is understood that the total daily usage of the products of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the patient's age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the particular compound used; the duration of treatment; drugs used in combination or concomitantly with the product; and similar factors well known in the medical arts. For example, it is well within the skill of the art to begin administering an antibody at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0114] "Pharmaceutically" or "pharmaceutical acceptable" refers to molecular species and compositions that do not cause adverse, allergic or other untoward reactions when administered appropriately to mammals, particularly humans. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient can be administered to animals and humans alone or in combination with another active ingredient in unit dosage form in a mixture with conventional pharmaceutical support. Typically, the pharmaceutical composition contains a pharmaceutical acceptable vehicle for injectable formulations. These vehicles may be, in particular, isotonic sterile saline (such as monosodium or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or may be dry, in particular lyophilized, compositions that can be made into injections, optionally by adding sterile water or saline. Suitable pharmaceutical dosage forms for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage forms must be sterile and fluid to the extent that they exhibit easy syringability. The dosage forms must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the compounds of the invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The product can be formulated in a neutral or salt form composition.Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent such as sugar or sodium chloride. The sustained absorption of the injectable composition can be achieved by using an absorption retardant, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions are prepared by mixing the required amount of active polypeptide in a suitable solvent, together with some of the other ingredients as required above, followed by filtration sterilization. In general, dispersions are prepared by mixing various sterilized active ingredients in a sterile vehicle containing the basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient, plus any additional desired ingredients, from a previously sterile-filtered solution. After formulation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the above-mentioned types of injectable solutions, but drug-release capsules and the like can also be employed. For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be employed will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous fluid or injected into the intended injection site. Some dosage variation will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

[0115] In vitro methods The present invention also provides an in vitro assay for co-culturing a tumor cell line and a myeloid cell line, comprising: a) culturing at least one tumor cell line or at least one tumor cell derived from a primary tumor and at least one myeloid cell line in ultra-low attachment surface plates such that all cell lines or cells grow in spheroid form; b) Tracking the growth of 3D spheroids of co-cultured cell lines or cells by time-lapse microscopy; c) Optionally, periodically collecting samples of the three-dimensional spheroids and / or supernatants of the co-cultured cell lines or cells and analyzing their composition and performing three-dimensional imaging; The present invention relates to an in vitro assay method comprising:

[0116] Preferably, the ultra-low attachment surface plate of step a) is non-adhesive. Preferably, said plate does not contain a matrix or other solid phase, but a liquid medium. Preferably, said plate is a U-shaped plate. In such a shape, cells typically adhere to each other in a liquid medium.

[0117] The tumor cell line may be any tumor cell line known in the art, such as A549 or MDA-MB-231.

[0118] Tumor cells may be from a primary tumor. A primary tumor refers to the original, or first, tumor in the body. Cancer cells from a primary tumor may spread to other parts of the body and form new (or secondary) tumors; this is called metastasis.

[0119] Preparation method The present invention also provides a method for producing a CAR myeloid cell according to the invention, or a CAR iPS or CAR HSC according to the invention, comprising: Providing at least one cell selected from an isolated myeloid cell, an induced pluripotent stem cell (iPS), and an isolated hematopoietic stem cell (HSC); and transducing the cells with a vector, preferably a lentiviral vector, comprising a nucleic sequence encoding the CAR; The present invention relates to a method comprising the steps of:

[0120] Of course, the CAR may be an extracellular antigen-binding domain that binds to a tumor antigen or a TME antigen; optionally, a hinge domain; A transmembrane domain; and a first intracellular signaling domain comprising the cytoplasmic tail of CD40 fused to a second intracellular signaling domain comprising the intracellular domain of CD3ζ; Includes.

[0121] The first step of the preparation method is to provide at least one type of cell selected from isolated myeloid cells, induced pluripotent stem cells (iPS), and isolated hematopoietic stem cells (HSC).

[0122] The cells are then transduced with a vector, preferably a lentiviral vector, that contains a nucleic sequence encoding the CAR. This vector can be used to introduce the CAR into isolated myeloid cells, iPS or isolated HSC, preferably monocytes, macrophages or dendritic cells. The vector contains a nucleic acid sequence encoding the CAR of the present invention. In one embodiment, the vector is a plasmid vector, a viral vector, a retrotransposon (e.g., piggyback, sleeping beauty), or a vector for site-specific insertion (e.g., CRISPR, zinc finger nuclease, TALEN). Preferably, the vector is a viral vector, preferably a lentiviral vector. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools to achieve long-term gene transfer, since they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, that they can transduce non-proliferating cells. Lentiviral vectors also have the added advantage of being less immunogenic in the subject into which they are introduced.

[0123] Expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. Vectors are generally capable of replicating in mammalian cells and / or integrating into the mammalian cell genome. Typical vectors contain transcriptional and translational termination regions, initiation sequences, and promoters useful for controlling expression of the desired nucleic acid sequence.

[0124] The nucleic acid sequence (nucleic acid) encoding the CAR can be cloned into a number of different types of vectors. For example, the nucleic acid can be cloned into a vector, including but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid. Vectors of particular interest include vectors for expression, vectors for replication, vectors for probe generation, and vectors for sequencing.

[0125] The expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Viruses useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Usually, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selection markers. Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Depending on the promoter, individual elements may function cooperatively or independently of each other to activate transcription.

[0126] An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, such as the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, and the like. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0127] The expression vector introduced into cells for the purpose of evaluating the expression of a polypeptide or a portion thereof can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that have been transfected or infected via a viral vector. In other embodiments, the selection marker may be carried on separate DNA fragments and used in a co-transfection method. Both selection markers and reporter genes may be flanked by appropriate control sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes such as neo. Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of control sequences. In general, reporter genes are genes that are not present or expressed in the recipient organism or tissue and that code for a polypeptide whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is evaluated at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and may be prepared using known techniques or may be obtained commercially. In general, the construct with the smallest 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0128] Preferably, the nucleic acid sequence encoding the intracellular domain of the CAR is the sequence of SEQ ID NO:7.

[0129] The above method further comprises introducing into said cell an additional vector comprising a sequence encoding a gene of interest under the control of a cytokine-specific promoter.

[0130] Preferably, the gene of interest is selected from a gene encoding IFNγ, a gene encoding IFNα, a gene encoding IFNβ, a gene encoding IFNλ, a gene encoding IL12, and a gene encoding IL10 or TGFβ.

[0131] Preferably, the gene of interest is a human gene.

[0132] The sequence of the present application is as follows: [Table 1-1] [Table 1-2]

[0133] The invention is illustrated by the following figures and examples. [Brief description of the drawings]

[0134] The drawings in this application are as follows: [Figure 1]Lentiviral transduction of monocytes allows high expression of CAR constructs on the cell surface. Figure 1A shows a schematic of the CAR constructs cloned into a lentiviral vector. scFv; single-chain antibody specific for CD19. TM: transmembrane domain. 41BB, CD3z (CD3z), and CD40 correspond to the intracellular domains of these molecules. A hinge domain is also present in each CAR construct between the scFv and TM domains, but is not shown. Figure 1B shows a representative histogram of transduced macrophages stained for CD19 expression. Freshly purified CD14+ cells were transduced with lentiviral vectors encoding the indicated CAR constructs and cultured for 10 days in the presence of M-CSF, but without selection. Cells were then harvested and analyzed by flow cytometry using rhCD19-Atto647 complex protein for staining. Note the typical high transduction efficiency of over 90%. [Diagram 2] CAR-macrophages (CAR-MΦ) capable of phagocytosing A549-CD19+ cells. FACS-based phagocytosis assay of A549-GFP or A549-GFP-CD19+ by CAR-macrophages at an E:T ratio of 1:1. Macrophages were prepared as in Figure 1 and then incubated with A549 cells expressing or not expressing CD19 for 2 hours. Figure 2A shows the flow cytometry gating strategy to track macrophages that had phagocytosed A549 cells. Figure 2B shows the quantification of the ability of macrophages expressing the indicated CAR constructs to phagocytose A549 cells expressing or not expressing CD19. The presence of phagocytosis was defined as the % of GFP+ events within the CD45+ population and plotted per macrophage population. Each dot represents one donor. Note that all CAR macrophages phagocytosed CD19+ cells but not CD19- cells, regardless of the CAR intracellular domain they possessed. [Diagram 3]CAR-MΦ can disrupt tumor spheroid growth. IncuCyte-based spheroid growth assay of A549GFP+CD19+ cells co-cultured with CAR-MΦ at an E:T ratio of 16:1. CAR-MΦ were prepared as in Figure 1. 1000 tumor cells were seeded with 16000 macrophages expressing the CAR construct in an ultra-low attachment surface 96-well plate, resulting in the formation of tumor spheroids growing in three dimensions. GFP intensity was tracked by time-lapse microscopy every 3 hours for 96 hours. Mean values ​​± SD from three donors are shown. CAR macrophages bearing the CD3z domain slowed down the growth of tumor spheroids. [Figure 4] Ability of CAR-MΦ to control tumor growth in three dimensions. IncuCyte-based spheroid growth assay of MDA-MB-231GFP+CD19+ cells (A) or A549GFP+CD19+ cells (B) co-cultured with various CAR-MΦ. CAR-MΦ were prepared as in Figure 1. On day -3, 103 A549 or 103 cells were seeded in ultra-low attachment surface 96-well plates, resulting in the formation of tumor spheroids growing in three dimensions. After 3 days, 8.103 non-transduced macrophages or CAR-MΦ were added to the established spheroids. GFP intensity was tracked by time-lapse microscopy every 3 hours for 96 hours. Mean values ​​± SD from three donors are shown. [Diagram 5] Antigen stimulation of CAR-MΦ induces secretion of inflammatory cytokines. Quantification of the indicated cytokines secreted by CAR-MΦ when co-cultured with medium alone, A549-GFP cells, or A549-GFP-CD19 cells. CAR macrophages were prepared as in Figure 1 and cultured for 24 h at an E:T ratio of 2:1. Each dot represents one donor. CAR macrophages bearing the CD40 domain secrete high levels of the proinflammatory cytokines IL-6 and IL-8 when co-cultured with CD19+ cells, and have a baseline secretion of TNFα in the absence of antigen stimulation. [Figure 6A]CAR-Monocytes can induce tumor shrinkage in vivo. Figure 6A shows NSG mice were subcutaneously injected with 5 106 MDA-MB-231 CD19+GFP+ cells. Half of these mice were injected intravenously with CD14-PBMCs (equivalent to 7. 106 cells). Mice were left untreated or injected intratumorally (it) with CAR-Mono. [Figure 6B] FIG. 6B shows body weight measured over a 35 day period. [Figure 6C] FIG. 6C: Tumor size measured with calipers over a 35-day period. [Figure 7] Flow cytometric analysis of spleens and tumors from cell-treated mice. Mice from the experiment depicted in Figure 6 were euthanized 35 days after tumor injection. Mouse spleens and residual tumors were harvested, stained for the indicated markers, and analyzed by flow cytometry to determine the percentage of human cells versus tumor cells present in these preparations. [Figure 8] Lentiviral transduction of monocytes allows high expression of CAR constructs on the cell surface. Figure 8A shows a schematic of the CAR constructs cloned into lentiviral vectors. scFv; single chain antibody specific for CD19. TM: transmembrane domain. CD3 (CD3ζ) and CD40 correspond to the intracellular domains of these molecules. STINGt corresponds to a truncation of the C-terminal domain of the STING protein (SEQ ID NO: 6). Figure 8B shows a representative histogram of transduced macrophages stained for CD19 expression. Purified CD14+ cells were transduced with lentiviral vectors encoding the indicated CAR constructs and cultured for 10 days in the presence of M-CSF without selection. Cells were then harvested and analyzed by flow cytometry using rhCD19-biotin and streptavidin-A647 for staining. [Figure 9]Antigen stimulation of CAR macrophages induces secretion of inflammatory cytokines and an interferon response. Quantification of the indicated cytokines secreted by macrophages expressing CAR constructs when co-cultured with media alone, A549 cells, or A549-CD19 cells. CAR macrophages were cultured at an E:T ratio of 1:1 for 24 hours. Each dot represents one donor. CAR macrophages carrying the STING domain secrete high levels of IP-10 and high levels of the pro-inflammatory cytokine IL-6 when co-cultured with CD19+ cells. EXAMPLES

[0135] Example 1: Engineering macrophages for antitumor immunity Materials and Methods cell line A549 human lung tumor adenocarcinoma cell line and MDA-MB-231 human breast adenocarcinoma cell line were maintained in RPMI complete medium (Gibco™ Roswell Park Memorial Institute 1640 supplemented with 10% fetal bovine serum and 1% GIBCO™ penicillin-streptomycin (Thermo Fisher)). HEK293 FT cells were maintained in DMEM complete medium (Gibco™ Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin). A549-GFP and A549-GFP-CD19 were obtained by lentiviral transduction of pWPXLd-GFP encoding GFP and pCDH1-CD19 encoding hCD19. Transduced cell lines were sorted by FACS to obtain homogenous cell populations.

[0136] primary cells Peripheral blood mononuclear cells (PBMCs) were isolated from the plasmapheresis residue using Ficoll-Paque (GE Healthcare). Informed consent was obtained from all donors, and samples were anonymized prior to use in the study. CD14 magnetic microbeads (Miltenyi, 130-050-201) were used to isolate CD14. + Monocytes were isolated by positive selection.

[0137] Plasmid construction and virus production CAR constructs were cloned into the pCDH1 lentiviral vector, which contains a puromycin resistance gene under the control of the EF1α promoter. All CAR constructs were expressed under the control of the CMV promoter. Lentivirus was produced in HEK293 FT cells. Lentiviral vectors were co-transfected with psPAX2 (a second generation lentiviral packaging plasmid) and pMD2.G (encoding VSV-G) using PEI MAX® (Polysciences). Vpx-VLPs were produced in HEK293FT cells by transfection of pSIV3 and pMD2.G (S. Bobadilla et al., 2013). After 18 hours, the medium was replaced with fresh medium to remove the transfection reagent. 24 hours after medium replacement, the supernatant containing the lentiviral vector was collected and filtered through a 0.45 μm filter.

[0138] Monocyte transduction and differentiation In the presence of Vpx-VLPs and 4 μg / mL protamine, CD14 + The cells were transduced with the lentiviral vector, and then the monocytes were differentiated in macrophages in macrophage medium (RPMI + 5% fetal bovine serum + 5% human serum + 1% penicillin-streptomycin) containing 50 ng / mL M-CSF in Corning® 100 mm Not TC-treated Culture Dish for 10 days.

[0139] Detection of CAR expression Human primary CAR macrophages were stained with rhCD19-Atto647 binding protein (R&D Systems, ATM9269). Cells were harvested with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher), washed with PBS, and stained with rhCD19 Atto647 binding protein for 30 minutes at 4°C. Human FcBlock™ (BD Biosciences) was added during staining. Cells were analyzed by FACS using BD Verse.

[0140] FACS-based phagocytosis assay 1x10 5 1x10 CAR macrophages 5 A549-GFP or A549-GFP-CD19 cells were co-cultured for 3 h at 37° C. Cells were harvested with Accutase, stained with anti-CD45-Alexa700 (Biolegend) antibody in the presence of FcBlock™ (BD Biosciences), and analyzed by FACS on a BioRad ZE5. The percentage of GFP+ events within the CD45+ population was plotted as the rate of phagocytosis.

[0141] IncuCyte-based spheroid growth assay 1x10 3 Tumor cells, 1.6x10 4 The cells were seeded in Corning® Costar® Ultra-Low Attachment 96-Well Plates (Merck) with 1000 macrophages. GFP fluorescence was then tracked and measured over several days with an Incucyte® S3 Live-Cell Analysis System (Essen Biosciences). GFP intensity analysis was performed with IncuCyte software. After background subtraction, green objects were defined as the threshold. Total GFP + The integrated intensity is the sum of the pixels belonging to all green objects.

[0142] Cytokine secretion assay 1.5x10 5 CAR macrophages were cultured in medium or 7.5x10 4 A549-GFP, or 7.5x10 4 A549-GFP-CD19 cells were co-cultured in Corning® Costar® Not Treated 12-well plates for 24 hours at 37° C. Supernatants were collected and clarified by centrifugation. IL-6, IL-8, and TNFα concentrations were measured by cytometric bead assay (Human Flex Set BD™ CBA, 558276, 558277, 558273) according to the manufacturer's instructions.

[0143] In vivo testing The experimental design of the xenograft model used is shown in the relevant figure panels. Cells were injected in 100 μl of PBS for all IV, IT, and SC injections. Tumor sized was measured twice weekly with a caliper. Mice were weighed weekly and underwent regular veterinary examination for any significant signs of disease. Animals were sacrificed at the end of the experiment.

[0144] result 1. Generation of CAR-expressing macrophages We developed CAR constructs for macrophages to induce their activation only when they encounter tumor-specific antigens (here CD19). That is, all CAR constructs contain an anti-CD19 single-chain antibody (scFv) fused with the hinge and transmembrane domains of CD8. Classically, CAR intracellular domains combine the CD3ζ chain with the CD28 or 41BB costimulatory domains, either separately or together. Here, we combined the intracellular domains of CD3ζ and CD40. Three different CAR constructs were constructed (see FIG. 1A). The CAR Stop does not have an intracellular domain (it is a negative control for signaling), the CAR 41BBCD3 construct is a classical CAR used in T cells but also successfully tested in macrophages, and the CAR CD40CD3 combines the cytoplasmic domain of CD40 with that of CD3ζ (and is a CAR according to the invention) since activation of CD3ζ enhances phagocytosis in macrophages.

[0145] These three CAR constructs were cloned into lentiviral vectors and used to transduce human primary monocytes together with pseudoparticles carrying the Vpx SIV accessory protein. Cells were then differentiated for 10 days in culture medium containing M-CSF without antibiotic selection. The resulting three transduced macrophages (designated CAR-MΦ) showed high cell surface expression of CAR as quantified by FACS using recombinant CD19 ectodomain labeled with a fluorophore. Importantly, i) for each donor tested, at least 90% of CAR-MΦ expressed the CAR construct on their surface (Figure 1B), and ii) these high transduction rates were obtained without any antibiotic selection.

[0146] 2. Phagocytic Ability of CAR-MΦ in Two Dimensions To evaluate the tumor cell phagocytosis ability of CAR-MΦ, we needed to generate suitable target cells. Starting from the A549 lung adenocarcinoma cell line, we established cells stably expressing CD19 and GFP by lentiviral transduction. Three different CAR-expressing MΦs were transfected with A549GFP + CD19 + Incubated with cells at 37°C for 2 hours (except for A549GFP + CD19- but not incubated with GFP when viewed by flow cytometry + And CD45 + In contrast, no double-positive cells were detected when non-transduced MΦs were used instead of CAR-MΦs. These results arguably demonstrate that primary human anti-CD19 CAR-MΦs express A549GFP-A ... + CD19 + These results suggest that the phagocytosis of cells is antigen-specific (Figure 2).

[0147] 3. CAR-MΦ can disrupt the growth of tumor spheroids Next, we tested the ability of CAR-MΦs to disrupt the three-dimensional growth of A549 cell spheroids over a 4-day period. CAR-MΦs with CD3z domains, combined with either 41BB or CD40 domains, were used to express A549GFP. + CD19 + CAR-MΦs slowed the growth of tumor spheroids (Figure 3). Importantly, CAR-MΦs had no antitumor activity against CD19- tumor spheroids. These data suggest that CAR-MΦs carrying the CD3z intracellular domain can mediate the uptake of CD19 cells that can then be phagocytosed. + It was suggested that it was efficiently activated by target cells.

[0148] 4. CAR-MΦ regulates tumor growth in three dimensions We tested the antitumor potential of CAR-macrophages under more stringent conditions. We added CAR MΦ to established tumor cell spheroids. We formed spheroids from 1000 A549-GFP-CD19 cells or 1000 MDA-MB-231-GFP-CD19 cells, and after 3 days, added 8000 non-transduced macrophages or 8000 CAR MΦ. CAR MΦ containing only the CD3ζ domain achieved very limited control of the growth of existing spheroids in both tumor models tested, suggesting that activation of phagocytosis is not sufficient for efficient control of tumor growth. Addition of the CD40 domain to the CD3ζ domain at the CAR cytoplasmic end increased the ability to control tumor growth in both tumor models.

[0149] 5. Cytokine production by CAR-MΦ We next examined the polarization of CAR-MΦ when co-cultured with target cells that either express or do not express CD19. CAR-MΦs bearing the CD40 domain were polarized in both types of cells, A549CD19. + When co-cultured with IL-10 cells, they secreted high levels of the proinflammatory cytokines IL-6 and IL-8, and in the absence of antigenic stimulation, they secreted baseline levels of TNFα. Classical CARs (containing CD3z and 41BB domains), CAR-Stop MΦs, and non-transduced MΦs produced very little of any cytokine in all conditions (Figure 5). + Exposure to the cells induced a transition to a proinflammatory phenotype.

[0150] 6. Antitumor activity of CAR-MΦ in vivo To evaluate the antitumor activity of CAR-MΦ in vivo, we used the human breast cancer MDA-MB-231 cell line to generate cells expressing CD19 and GFP by lentiviral transduction, i.e., MDA-MB-231 GFP. + CD19 +The cells were injected subcutaneously into immunodeficient NSG mice. Half of these mice were injected intravenously (iv) with monocyte-depleted PBMCs (CD14- cells) 10 days later. CD3 + The proportion of T cells was assessed by flow cytometry, and 6 CD3 + The number of CD14- cells injected was adjusted to contain T cells. One day later, mice were injected with 10.10 6 Intratumoral (it) injection of CAR CD40CD3-monocytes (CAR-Mono) of the invention was performed. Body weight and subcutaneous tumor growth were monitored periodically over a period of 35 days (Figure 6A). The treatment did not affect the body weight of the mice compared to untreated control mice, suggesting the absence of widespread toxic effects (Figure 6B). The present inventors confirmed that CD14-cells cannot control tumor growth by themselves. Only 1 out of 6 mice in the group administered CAR-Mono alone showed a reduction in tumor volume, and only on day 35. In contrast, 5 out of 6 mice in the group administered both CD14-cells and CAR-Mono showed a significant reduction in tumor burden. These data suggest that the injected CAR-Mono of the present invention needs to cooperate with CD14-cells (possibly T cells) to control tumor growth (Figure 6C). The inventors concluded that transducing monocytes with a vector encoding the CAR CD3zCD40 (invention) yields cells capable of inducing tumor regression in immunodeficient mice partially reconstituted with T cells.

[0151] 7. Flow cytometric analysis of cell populations present in treated mice Mice from the experiment described in Figure 6 were sacrificed 35 days after tumor injection, and the spleens and residual tumors were removed, stained, and analyzed by flow cytometry. + These analyses demonstrated that these monocyte preparations transduced with CAR CD3CD40 (invention) contained human CD3 +Contaminating T cells were confirmed. However, we could not detect the presence of human myeloid cells using CD64-specific antibodies 35 days after tumor injection. Further studies are required to measure the presence of injected cells in tumors and lymphoid organs at various time points.

[0152] Thus, this study is the first to demonstrate cooperation between engineered myeloid cells and T cells in vivo. For the first time, the efficiency of a CAR in monocytes containing the cytoplasmic domain of CD40 combined with the cytoplasmic domain of CD3ζ, its effect on cytokine expression, and its effect on tumor regression are shown.

[0153] Example 2: Engineering macrophages with STING-containing CAR for anti-tumor immunity We developed CAR constructs for macrophages to induce activation only when the resulting CAR macrophages encounter the appropriate tumor-specific antigen (here CD19). That is, all CAR constructs contain an anti-CD19 single-chain antibody (scFv) fused to the hinge and transmembrane domains of CD8. Here, we combined the intracellular domain of STINGt (SEQ ID NO: 6), the intracellular domain of CD40, and the intracellular domain of CD3ζ (CD3 zeta). Four different CAR constructs were constructed (see FIG. 8A). CAR Stop has no intracellular domain (and is a negative control for signaling), the CAR STING construct contains a truncation of the C-terminal part of STING; CAR CD40STING combines the CD40 and STING domains (i.e., the cytoplasmic tail of CD40 is fused at its C-terminus to the STING domain), and CAR CD40CD3STING combines the cytoplasmic domains of CD40, CD3ζ, and STINGt (i.e., the cytoplasmic tail of CD40 is fused at its C-terminus to the intracellular domain of CD3ζ, which is fused at its C-terminus to the STING domain).

[0154] These four CAR constructs were cloned into lentiviral vectors. The corresponding viral particles were used to transduce primary human monocytes together with pseudoparticles carrying the Vpx SIV accessory protein. The cells were then differentiated for 10 days in culture medium containing M-CSF without antibiotic selection. The resulting four transduced macrophages (designated CAR-MΦ) showed high cell surface expression of CAR as quantified by FACS using recombinant CD19 ectodomain labeled with a fluorophore. Importantly, i) for each donor tested, at least 50% of CAR-MΦ expressed the CAR construct on their surface (Figure 8B), and ii) these high transduction rates were obtained without any antibiotic selection. We next examined the polarization of CAR-MΦ when co-cultured with target cells expressing or not expressing CD19. CAR-MΦ carrying the STING domain polarized A549CD19 + When co-cultured with A549CD19 cells, CAR-MΦs secreted high levels of the ISG (interferon-stimulated gene) IP-10, indicating activation of the interferon pathway. + When co-cultured with CAR-Stop MΦs, they secreted higher levels of the proinflammatory cytokine IL-6. CAR-Stop MΦs and non-transduced MΦs produced very little of any cytokine in all conditions (Figure 9).

Claims

1. Modified cells containing a chimeric antigen receptor (CAR), wherein the CAR is An extracellular antigen-binding domain that binds to tumor antigens or antigens present on cells in the tumor microenvironment (TME); Hinged domain, upon request; Transmembrane domain; and, A first intracellular signaling domain, including the cytoplasmic end of CD40, is fused to a second intracellular signaling domain, which includes (i) STING or one of its fragments, and / or (ii) the intracellular domain of CD3ζ; Includes, The modified cells are myeloid cells. Modified cells.

2. The modified cell according to claim 1, wherein the cell is a monocyte, macrophage, or dendritic cell.

3. Modified induced pluripotent stem cells (iPS) or hematopoietic stem cells (HSC) containing CAR, wherein the CAR is Extracellular antigen-binding domain that binds to tumor antigen or TME antigen; Hinged domain, upon request; Transmembrane domain; and, A first intracellular signaling domain containing the cytoplasmic end of CD40 is fused to a second intracellular signaling domain containing the intracellular domain of CD3ζ; Modified iPS cells or HSCs, including those containing iPS cells.

4. The modified cell according to claim 1, wherein the extracellular antigen-binding domain is an anti-CD19-binding domain, preferably an anti-CD19 scFV domain.

5. The aforementioned CAR extends from its N-terminus toward its C-terminus, An extracellular antigen-binding domain having the sequence of Sequence ID No. 1, If desired, a hinge domain having the sequence of sequence number 2, A transmembrane domain having the sequence of Sequence ID No. 3, A first intracellular signaling domain having the sequence of Sequence ID No. 4 is fused to a second intracellular signaling domain having the sequence of Sequence ID No.

5. A modified cell according to claim 1, comprising:

6. The aforementioned CAR, Extracellular antigen-binding domain that binds to tumor antigen or TME antigen; Hinged domain, upon request; Transmembrane domain; and, A first intracellular signaling domain of CD40, including its cytoplasmic end, is preferably fused at its C-terminus to either a) a second intracellular signaling domain containing STING or one of its fragments, or b) an intracellular domain of CD3ζ. Includes, The intracellular domain of CD3ζ is preferably fused at its C-terminus to a second intracellular signaling domain containing STING or one of its fragments. The modified cell according to claim 1.

7. The modified cell according to claim 1, wherein the cell comprises an additional vector, the vector comprising a sequence encoding a target gene under the control of a cytokine-specific promoter.

8. A pharmaceutical composition comprising the modified cells described in claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, for use in the treatment of cancer or inflammatory diseases.

10. The pharmaceutical composition for use according to claim 9, wherein the cancer is a solid tumor.

11. A product comprising the modified cells described in claim 1 and CAR-T cells as a combination formulation for simultaneous, individual, or sequential use in the treatment of cancer or inflammatory diseases.

12. A product comprising the modified cells described in claim 1 and an immune checkpoint inhibitor as a combination formulation for simultaneous, individual, or sequential use in the treatment of cancer or inflammatory diseases.

13. A method for producing modified cells containing the CAR described in claim 1, Prepare at least one cell selected from isolated myeloid cells, iPS cells, and isolated HSCs; The cells are transduced using a vector containing a nucleic acid sequence encoding the CAR, preferably a lentiviral vector. Methods that include...

14. The method according to claim 13, further comprising introducing an additional vector into the cells, wherein the vector comprises a sequence encoding a target gene under the control of a cytokine-specific promoter.

15. An in vitro assay method for co-culturing tumor cell lines and myeloid cell lines, a) Culturing at least one tumor cell line or at least one primary tumor-derived tumor cell line together with at least one myeloid cell line in an ultra-low adhesion surface plate so that all cell lines or cells proliferate in a spheroidal manner; b) Tracking the growth of the co-cultured cell line or three-dimensional spheroid of cells by time-lapse microscopy observation; c) If desired, periodically collect samples of the three-dimensional spheroids and / or supernatants of the co-cultured cell lines or cells, analyze their composition, and perform three-dimensional imaging. In vitro assay methods, including those mentioned above.

16. Modified cells containing CAR, wherein the CAR is Extracellular antigen-binding domains having antigen specificity for tumor antigens or TME antigens; Upon request, hinge domain, Transmembrane domain; and, An intracellular signaling domain containing STING or one of its fragments; Includes, The modified cells are myeloid cells. Modified cells.

17. Modified cells containing CAR, wherein the CAR is Extracellular antigen-binding domains having antigen specificity for tumor antigens or TME antigens; Upon request, hinge domain, Transmembrane domain; and, An intracellular signaling domain containing STING or one of its fragments is fused to (i) the cytoplasmic terminal of CD40 and / or (ii) the intracellular domain of CD3ζ; Includes, The modified cells are myeloid cells, Preferably, the first intracellular signaling domain containing STING or one of its fragments is fused at its N-terminus to a second intracellular signaling domain containing the cytoplasmic end of CD40; or, preferably, at its N-terminus, to a third intracellular signaling domain containing the intracellular domain of CD3ζ, and the intracellular domain of CD3ζ is fused at its N-terminus to the second intracellular signaling domain containing the cytoplasmic end of CD40. Modified cells.

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