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WO2019128996A1 - Lymphocyte t-car spécifique de la mésothéline exprimant un anticorps cd47, et son utilisation - Google Patents

Lymphocyte t-car spécifique de la mésothéline exprimant un anticorps cd47, et son utilisation Download PDF

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WO2019128996A1
WO2019128996A1 PCT/CN2018/123532 CN2018123532W WO2019128996A1 WO 2019128996 A1 WO2019128996 A1 WO 2019128996A1 CN 2018123532 W CN2018123532 W CN 2018123532W WO 2019128996 A1 WO2019128996 A1 WO 2019128996A1
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amino acid
seq
antibody
cancer
cell
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钱其军
金华君
江芏青
李�赫
李林芳
王超
崔连振
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Definitions

  • the present invention pertains to genetic engineering and immunology, and relates to mesothelin-specific CAR-T cells that express CD47 antibodies and uses thereof.
  • Cancer has become the number one killer of human health.
  • the fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices of cancer, making cancer's high incidence and rejuvenation trend more and more obvious.
  • the commonly used treatments are very limited, and it is still necessary to explore a more effective treatment to improve the survival rate and quality of life of cancer patients.
  • chimeric antigen receptor T cell therapy has achieved very good curative effect in malignant hematological tumors, and the complete remission rate of relapsed and refractory B cell leukemia is over 90%.
  • CAR-T cell Novartis's tissue lecleucel chimeric antigen receptor T cell
  • ALL acute lymphoblastic leukemia
  • CAR-T drug Novartis's tissue lecleucel chimeric antigen receptor T cell
  • Yescarta for the treatment of adult patients with certain types of large B-cell lymphoma.
  • the approval of CAR-T drugs has taken CAR-T treatment to a new level.
  • a chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region.
  • scFv single-chain fragment variable
  • TAA antibody-associated antigen
  • ITAM immunoreceptor tyrosine-based activation Motifs
  • the efficacy of CAR-T cells in the treatment of solid tumors is still insufficient.
  • the main reasons include: 1.
  • the solid tumor is highly heterogeneous, lacking cell surface targets suitable for CAR-T treatment; 2.
  • Solid tumors have a microenvironment that strongly inhibits immunity.
  • Mesothelin is a glycoprotein anchored to the plasma membrane by a phosphatidylinositol region (GPI), which is highly expressed in various tumor tissues and is expressed in a small amount in mesothelial cells of normal pleura, pericardium and peritoneum.
  • the mesothelin gene encodes a 69 kDa precursor protein that is processed to form a 40 kDa membrane-bound protein and a 31 kDa detached fragment called megakaryocyte promoting factor (MPF) and released out of the cell, which we usually call Peelin refers to a fragment anchored on a membrane, which can be divided into three regions, Region I, II, and III, according to its protein structure.
  • CAR-T cell research targeting this is also in full swing, targeting mesothelin.
  • CAR-T cell therapy ranking fourth among other targets except CD19, mainly for pancreatic cancer (NCT01897415, NCT02465983), mesothelioma (NCT01355965, NCT02414269), lung cancer and breast. Cancer (NCT02414269).
  • CD47 is mainly expressed on the surface of cancer cells and is generally considered to be a protective receptor for cancer cells to be protected from host immune system attack. Studies have shown that T cells and dendritic cells (DC) can exert anti-tumor effects through the CD47 blocking effect.
  • CD47 is a type of "don't eat me” signal that inhibits macrophage function by binding to SIRP- ⁇ on the surface of macrophages.
  • CD47 has unparalleled advantages as a target for cancer treatment: 1. It is widely expressed on the surface of various cancer cells, so it can be used to treat various types of cancer; 2. Normal cells lack the "eat me” signal, so Blocking CD47 alone does not trigger the phagocytic effect of macrophages on normal cells, so the side effects of CD47 blockers are also very small.
  • the present invention provides a T cell: (1) a coding sequence comprising a chimeric antigen receptor recognizing a mesothelin antigen and a coding sequence of a CD47 antibody; and/or (2) expression recognition of a mesothelin antigen Chimeric antigen receptor and CD47 antibody.
  • the expression cassette of the chimeric antigen receptor recognizing the mesothelin antigen and the expression cassette of the CD47 antibody are integrated into the genome of the T cell.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a signal peptide, a single-chain antibody against the mesothelioma proximal membrane end, a hinge region longer than 50 amino acid residues, and a cross Membrane region, intracellular costimulatory signal domain and intracellular signal domain.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably a light chain signal peptide; preferably, the light chain signal peptide
  • the amino acid sequence is shown as amino acid residues 1 to 20 of SEQ ID NO: 1.
  • the amino acid sequence of the single chain antibody is as shown in amino acid residues 21 to 270 of SEQ ID NO: 1.
  • the hinge region longer than 50 amino acid residues is selected from the group consisting of a CD8 alpha hinge region, an IgD hinge region, an IgGl Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is CD8 ⁇ Hinge region or IgG4Fc CH2CH3 hinge region; more preferably, the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is as shown in amino acid residues 271-498 of SEQ ID NO: 1.
  • the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase An intracellular domain of an inducible T cell costimulatory factor (ICOS) and a DNAX activator protein 10; preferably, the intracellular costimulatory signal domain is an intracellular domain of CD28; preferably, the amino acid sequence of the CD28 Shown as amino acid residues 527-567 of SEQ ID NO: 1.
  • the intracellular signal domain is a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. ID NO: 1 is described in amino acid residues 568-679.
  • the chimeric antigen receptor comprises a light chain signal peptide, a single chain antibody against mesothelin Region III, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, The intracellular domain of CD28 and the tyrosine activation motif of CD3 ⁇ .
  • the amino acid sequence of the CD47 antibody is set forth in amino acid residues 21 to 367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2; preferably, the CD47 antibody
  • the coding sequence is shown as bases 61-1101 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5.
  • a vector comprising an expression cassette of a CD47 antibody, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the cancer is selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer;
  • the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.
  • Figure 1 Schematic diagram of plasmid structure of Meso3CAR and CD47 antibodies.
  • Figure 3A CAR-T positive rate of ⁇ CD47-Meso3 CAR T cells constructed under different ratios of Meso3CAR and CD47 antibody plasmids.
  • Figure 4 Flow cytometry analysis of CD47 expression of Mock, Meso3CAR and ⁇ CD47-Meso3CAR T cells.
  • Figure 5 Killing of tumor cell lines by ⁇ CD47-Meso3CAR T cells.
  • FIG. 6 CD47 of the surface of tumor cells was blocked after co-culture of ⁇ CD47-Meso3CAR T cell supernatant with tumor cells.
  • FIG. 7 Blocking the surface of tumor cells CD47 can enhance the phagocytosis of macrophages.
  • Figure 8 In vivo anti-tumor effect of ⁇ CD47-Meso3CAR T cells.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region).
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • Fc fragment crystallizable (Fc) of an antibody
  • Fc fragment crystallizable
  • costimulatory molecule refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal.
  • the proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules.
  • the costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell.
  • B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.
  • linker or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (KD) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • KD Affinity
  • pharmaceutically acceptable excipient refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject or “patient” may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.
  • CAR chimeric antigen receptor
  • T cells immune cells
  • CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region.
  • a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity.
  • the polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.
  • the tumor cell which specifically targets mesothelin is simultaneously secreted, can secrete CD47 antibody, eliminate immune escape of tumor cells, and restore macrophage to tumor cells. Phagocytosis for better anti-tumor effects.
  • the Fc fragment of the CD47 antibody designed by the present invention is a mutant IgG4Fc, which avoids binding to the ⁇ -2 receptor on the surface of dendritic cells and is recognized and phagocytized by macrophages, so that the self-expressing CD47 antibody CAR-T The cells function without causing an AICD response.
  • the CD47 antibody further comprises a light chain signal peptide.
  • the CD47 antibody from the N-terminus to the C-terminus, in turn comprises a light chain signal peptide, a CD47 ligand, and an IgG4 Fc.
  • the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 2; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 5 base sequence of 1-60 is shown.
  • the amino acid sequence of the CD47 antibody is set forth in amino acid sequence 21-367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the invention also provides a host cell comprising a nucleic acid construct as described herein.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • the polynucleotide sequences described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO:4.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is typically operably linked to the coding sequence of the protein to be expressed.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered.
  • SV40 prion 40
  • MMTV mouse breast cancer virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • EB virus immediate early promoter EB virus immediate early promoter
  • Russ sarcoma virus promoter avian leukemia virus promoter
  • an inducible promoter can also be considered.
  • various promoter sequences disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1 ⁇ promoter shown in SEQ ID NO: 5 of the application.
  • the encoded amino acid sequence is as shown in SEQ ID NO: 1) and the mutant ⁇ CD47 gene (the nucleotide sequence thereof is shown as SEQ ID NO: 5; the encoded amino acid sequence is SEQ ID NO: 2 ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ RTIgt;
  • the recombinant plasmids constructed between the EcoRI and SalI cleavage sites of the pNB328 vector were designated as pNB328-Meso3CAR and pS328- ⁇ CD47, respectively.
  • the structural pattern of the constructed recombinant plasmid is shown in Figure 1.
  • Meso3CAR T cells were constructed using pNB328-Meso3CAR alone (6 ug).
  • Mock T cells were constructed using the pNB328 vector (6 ug).
  • the ⁇ CD47-Meso3CAR T cells prepared in Example 2 were collected and divided into two portions, each of which was 1 ⁇ 10 6 cells, washed twice with physiological saline, resuspended in 100 ul of physiological saline, and one ug of mesothelin antigen was added in one portion. - Biotin, the other is not added, incubate at 4 ° C for 30 minutes.
  • the saline was washed twice, and the cells were resuspended again with 100 ul of physiological saline, and 1 ul of streptomycin-PE antibody was added thereto, and incubated at 4 ° C for 30 minutes.
  • the saline was washed twice and detected by the machine, and the T cells of the empty plasmid (pNB328) were electroporated as a control. The result is shown in Figure 2A.
  • ELISA was used to detect the expression level of ⁇ CD47-Meso3 CAR T cells prepared in Example 2.
  • Blocking solution IgG Fc-HRP was diluted 1:30000, 100 ul/well, and incubated at 37 ° C for 45 minutes.
  • the OD value was measured at 450 nm on a microplate reader, a standard curve was drawn, and the CD47 antibody concentration was calculated.
  • Example 4 Comparison of the positive rate and antibody secretion of ⁇ CD47-Meso3CAR T cells under different ratios of pNB328-Meso3CAR and pS328- ⁇ CD47 plasmids
  • the amount of pNBS328-Meso3CAR and pS328- ⁇ CD47 plasmids were set to 1 ug+7 ug, 2 ug+6 ug, 3 ug+5 ug, 4 ug+4 ug, 5 ug+3 ug, 6 ug+2 ug, 7 ug +1 ug, respectively, for CAR T
  • the cell was constructed and constructed in the same manner as in Example 2.
  • Example 2 The Mock T cells, Meso3CAR T cells and ⁇ CD47-Meso3CAR T cells obtained in Example 2 were collected, and the expression of PD1 was detected using the flow antibody FITC-mouse anti-human CD47 of BD, and the flow method was the same as in Example 3.
  • the CD47 antibody secreted by ⁇ CD47-Meso3CAR T can block the expression of CD47 on the cell surface.
  • Example 6 Killing effect of Mock, Meso3CAR and ⁇ CD47-Meso3CAR T cells on tumor cells
  • Target cell plating gastric cancer cell line Hgc27, ovarian cancer cell line SKOV3, and pancreatic cancer cell line ASPC-1 (purchased from American Type Culture Collection ATCC) were plated at 10 4 cells/50 ⁇ l per well in a test electrode. Place in the plate for a few minutes, wait for the cells to stabilize, then put them into the instrument, start step 2, and culture the cells;
  • Example 7 ⁇ CD47-Meso3CAR T cell culture supernatant can block tumor cell surface CD47
  • the CD47 antibody in the supernatant of ⁇ CD47-Meso3CAR T cells blocked tumor cells from expressing CD47.

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Abstract

L'invention concerne un lymphocyte T-CAR spécifique de la mésothéline exprimant un anticorps CD47. Le lymphocyte T : (1) comprend une séquence codante pour un récepteur d'antigène chimérique qui reconnaît un antigène de mésothéline et une séquence codante pour un anticorps CD47, et/ou (2) exprime un récepteur d'antigène chimérique reconnaissant l'antigène de mésothéline, et un anticorps CD47. Tout en ciblant spécifiquement des cellules tumorales exprimant fortement la mésothéline, le lymphocyte T de la présente invention peut sécréter un anticorps CD47, empêcher une évasion immunitaire de cellules tumorales, et rétablir la phagocytose de cellules tumorales par des macrophages, ce qui permet d'obtenir un meilleur effet antitumoral.
PCT/CN2018/123532 2017-12-28 2018-12-25 Lymphocyte t-car spécifique de la mésothéline exprimant un anticorps cd47, et son utilisation Ceased WO2019128996A1 (fr)

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CN116410332B (zh) * 2021-12-29 2025-04-15 上海细胞治疗集团股份有限公司 抗间皮素纳米抗体嵌合抗原受体及其应用

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EP3690033A4 (fr) * 2017-09-27 2021-07-14 Gracell Biotechnologies (Shanghai) Co., Ltd. Cellule immunitaire modifiée capable d'induire la sécrétion d'anticorps anti-cd47

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