WO2019129146A1 - Cellule car-t spécifique de l'egfr capable de sécrétion autocrine de l'anticorps cd47 et application de la cellule car-t - Google Patents

Cellule car-t spécifique de l'egfr capable de sécrétion autocrine de l'anticorps cd47 et application de la cellule car-t Download PDF

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WO2019129146A1
WO2019129146A1 PCT/CN2018/124382 CN2018124382W WO2019129146A1 WO 2019129146 A1 WO2019129146 A1 WO 2019129146A1 CN 2018124382 W CN2018124382 W CN 2018124382W WO 2019129146 A1 WO2019129146 A1 WO 2019129146A1
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amino acid
seq
antibody
sequence
cell
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钱其军
金华君
江芏青
李�赫
刘祥箴
李林芳
王超
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上海细胞治疗研究院
上海细胞治疗集团有限公司
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    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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Definitions

  • the present invention pertains to genetic engineering and immunology, to EGFR-specific CAR-T cells that autocrine 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 solid tumor is highly heterogeneous and lacks cell surface targets suitable for CAR-T treatment.
  • Solid tumors have a microenvironment that strongly suppresses immunity.
  • EGFR (abbreviated as EGFR, ErbB-1 or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4).
  • HER1 erbB1, EGFR
  • HER2 erbB2, NEU
  • HER3 erbB3
  • HER4 erbB4
  • the HER family plays an important regulatory role in the process of cell physiology.
  • EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc.
  • EGFR signaling pathway plays an important role in the physiological processes such as cell growth, proliferation and differentiation. Studies have shown high expression or abnormal expression of EGFR in many solid tumors.
  • EGFR is involved in tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis. Overexpression of EGFR plays an important role in the progression of malignant tumors. EGFR is overexpressed in tissues such as glioma, kidney, lung, prostate, pancreatic, and breast cancer. Therefore, EGFR is a potential. Tumor treatment targets.
  • TKI small molecule tyrosine kinase inhibitors
  • Mab Monoclonal antibodies
  • cetuximab ABX-EGF
  • EMD 72000 EMD 72000
  • 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 can construct a EGFR-CAR T cell which expresses CD47 antibody, and can specifically secrete CD47 antibody while specifically targeting tumor cells with high expression of EGFR, thereby eliminating immune escape of tumor cells and restoring macrophage to tumor cells. Phagocytosis for better anti-tumor effects.
  • the CD47 antibody Fc fragment to be a mutant IgG4 Fc, which avoids binding to the ⁇ -2 receptor on the surface of dendritic cells and is recognized and phagocytized by macrophages, allowing CAR-T cells to express CD47 antibodies. Play the function without causing an AICD reaction.
  • the present invention provides a T cell comprising: (1) a coding sequence comprising a chimeric antigen receptor recognizing EGFR and a coding sequence of a CD47 antibody; and/or (2) expressing a chimeric antigen receptor recognizing EGFR And CD47 antibodies.
  • the expression cassette of the chimeric antigen receptor recognizing EGFR 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, an optional signal peptide, a single-chain antibody against EGFR, a hinge region of more than 50 amino acid residues, and a transmembrane 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 CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide As shown in amino acid residues 1-22 of SEQ ID NO: 1.
  • amino acid sequence of the single chain antibody is as shown in amino acid residues 23-263 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 IgG1 Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region Is the CD8 ⁇ hinge region or the IgG4 Fc CH2CH3 hinge region; more preferably, the amino acid sequence of the CD8 ⁇ hinge region is as shown in amino acid residues 264 to 318 of SEQ ID NO: 1.
  • the transmembrane region is a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region.
  • a CD8 transmembrane region preferably having an amino acid sequence as shown in amino acid residues 319-344 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 345-385 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 386-497.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-EGFR single-chain antibody, a CD8 alpha hinge region, a CD8 transmembrane region, a CD28 intracellular domain, and Tyrosine activation motif of CD3 ⁇ .
  • amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-497 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1.
  • the coding sequence of the chimeric antigen receptor comprises one or more of the following features:
  • nucleotide sequence of the signal peptide is shown as bases 1-66 of SEQ ID NO: 4;
  • nucleotide sequence of the single-chain antibody is shown in nucleotide sequence 67-789 of SEQ ID NO: 4;
  • nucleotide sequence of the hinge region is shown in nucleotide sequence 490-954 of SEQ ID NO: 4;
  • the nucleotide sequence of the transmembrane region is represented by bases 955-1032 of SEQ ID NO: 4;
  • the nucleotide sequence of the intracellular costimulatory signal domain is shown as bases 1033-1155 of SEQ ID NO: 4;
  • nucleotide sequence of the intracellular signal domain is shown in pp. 1156-1491 of SEQ ID NO:4.
  • the coding sequence for the chimeric antigen receptor is set forth in bases 77-1491 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the CD47 antibody comprises a CD47 ligand and an IgG4 Fc sequence; wherein the amino acid sequence of the IgG4 Fc is represented by amino acid residues 139-367 of SEQ ID NO: 2; preferably, The amino acid sequence of the CD47 ligand is shown as amino acid residues 21 to 138 of SEQ ID NO: 2; preferably, the antibody further comprises a signal peptide sequence, preferably a light chain signal peptide, more preferably, the light The amino acid sequence of the strand signal peptide is shown as amino acid residues 1-20 of SEQ ID NO: 2.
  • 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 coding sequence for the CD47 antibody is set forth in bases 61-1101 of SEQ ID NO: 5, or as set forth in SEQ ID NO: 5.
  • the present invention also provides a CD47 antibody comprising a CD47 ligand and an IgG4 Fc sequence; wherein the amino acid sequence of the IgG4Fc is as shown in amino acid residues 139 to 367 of SEQ ID NO: 2.
  • the amino acid sequence of the CD47 ligand is as shown in amino acid residues 21-138 of SEQ ID NO:2.
  • the antibody further comprises a signal peptide sequence, preferably a light chain signal peptide, more preferably, the amino acid sequence of the light chain signal peptide is amino acids 1-20 of SEQ ID NO: 2. Residues are 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 coding sequence for a CD47 antibody described herein or a complement thereof; preferably, the coding sequence is represented by bases 61-1101 of SEQ ID NO: 5, or as set forth in SEQ ID NO: .
  • the invention also provides a composition comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor as described herein, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette for a CD47 antibody described herein, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the invention also provides a kit, the kit comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor as described herein, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette for a CD47 antibody described herein, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the invention also provides a pharmaceutical composition comprising a T cell and/or a CD47 antibody as described herein.
  • the present invention also provides the use of a T cell and/or CD47 antibody as described herein for the preparation of a medicament for treating or preventing a malignant tumor; preferably, the cancer is a cancer whose surface of the cancer cell abnormally expresses EGFR, preferably, said The cancer is selected from the group consisting of glioma, kidney cancer, 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.
  • Figure 1 Schematic diagram of the plasmid structure of EGFR-CAR and CD47 antibodies.
  • Figure 2A Flow cytometric detection of ⁇ CD47-EGFR-CAR T cell CAR positive rate.
  • Figure 2B ELISA assay for expression of CD47 antibody to ⁇ CD47-EGFR-CAR T cells.
  • Figure 3A CAR-T positive rate of ⁇ CD47-EGFR-CAR T cells constructed under different ratios of EGFR-CAR and CD47 antibody plasmids.
  • Fig. 3B The expression level of CD47 antibody of ⁇ CD47-EGFR-CAR T cells constructed under different ratios of EGFR-CAR and CD47 antibody plasmids.
  • Figure 4 Flow cytometric analysis of CD47 expression in Mock T cells, EGFR-CAR T cells, and ⁇ CD47-EGFR-CAR T cells.
  • Figure 5 Killing of different tumor cells by ⁇ CD47-EGFR-CAR T cells.
  • FIG. 6 CD47 of the ⁇ CD47-EGFR-CAR T cell supernatant blocked the surface of tumor cells after co-culture with different tumor cells.
  • FIG. 7 Blocking the surface of tumor cells CD47 can enhance the phagocytosis of macrophages.
  • Figure 8 Antitumor effect of ⁇ CD47-EGFR-CAR T cell mice in vivo.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter and a gene coding 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 CD47L.
  • 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, 10-8 M.
  • Affinity (KD) of 10-9 M or 10-10 M or less binds to the antigen.
  • Specific recognition has a similar meaning.
  • 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.
  • single-chain antibody refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability.
  • the single chain antibody of interest is from an antibody of interest.
  • Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies. The antibody can be secreted or membrane anchored.
  • the present invention can construct a EGFR-CAR T cell which expresses CD47 antibody, and can specifically secrete CD47 antibody while specifically targeting tumor cells with high expression of EGFR, thereby eliminating immune escape of tumor cells and restoring 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 IgG4 Fc, 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 present invention provides a CD47 antibody comprising a CD47 ligand and an IgG4Fc.
  • the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 139-367 of SEQ ID NO: 2; preferably, the coding sequence thereof is SEQ ID NO: 5 base sequence of 415-1101 Shown.
  • the amino acid sequence of the CD47 ligand is represented by amino acid residues 21 to 138 of SEQ ID NO: 2; preferably, the coding sequence thereof is at base 61-414 of SEQ ID NO: The base sequence is shown.
  • 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 encompasses a coding sequence for the CD47 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof.
  • the coding sequence of the CD47 antibody comprises the sequence set forth in bases 61-1101 of SEQ ID NO: 5, preferably the sequence set forth in SEQ ID NO: 5.
  • the present invention also encompasses a nucleic acid construct comprising the coding sequence of a CD47 antibody of the present invention or a complement thereof.
  • the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement into a host cell.
  • the invention also provides a host cell comprising a nucleic acid construct as described herein.
  • the invention also provides the use of the CD47 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignant tumor, particularly a tumor associated with CD47, including but not limited to Various malignant tumors.
  • the present invention also provides a T cell modified by the EGFR-CAR gene and capable of expressing a CD47 antibody, which can stably express the EGFR-CAR gene and the CD47 antibody at a high level.
  • the EGFR-CAR gene and the CD47 antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stably and continuously expressing in T cells.
  • the CAR of the invention typically contains an optional signal peptide sequence, an scFv that recognizes EGFR, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.
  • a signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures.
  • the signal peptide can be a secreted signal peptide or a membrane-bound signal peptide.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide or a light chain signal peptide; more preferably a CD8 signal peptide.
  • the amino acid sequence of the CD8 signal peptide can be as shown in amino acid residues 1-22 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1-66 of SEQ ID NO: 4.
  • the scFv recognizing EGFR described herein can be a single chain antibody directed against EGFR as is well known in the art.
  • An exemplary amino acid sequence of a single chain antibody recognizing EGFR is shown in amino acid residues 23 to 263 of SEQ ID NO: 1, and an exemplary coding sequence thereof is shown in nucleotide sequence 67-789 of SEQ ID NO: 4. Shown.
  • the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes.
  • a hinge region suitable for use herein may be selected from any of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4 or A variety.
  • the hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer.
  • a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein.
  • the amino acid sequence of the exemplary CD8 alpha hinge region is shown in amino acid residues 264 to 318 of SEQ ID NO: 1, and the exemplary nucleotide sequence thereof can be as shown in SEQ ID NO: 4, bases 490-954. Show.
  • the transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region
  • the amino acid sequence thereof is set forth in SEQ ID NO: 1 at 319-344; in certain embodiments, the coding sequence is set forth in SEQ ID NO: 4 at bases 955-1032.
  • the intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10).
  • the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably the amino acid sequence thereof is set forth in amino acid residues 345-385 of SEQ ID NO: 1, exemplary The coding sequence is shown as bases 1033-1155 of SEQ ID NO:4.
  • the intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be 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 As described in SEQ ID NO: 1 at amino acid residues 386-497; in certain embodiments, the coding sequence is set forth in SEQ ID NO: 4, pp. 1156-1491.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus: an optional CD8 signal peptide, an anti-EGFR scFv, a CD8 ⁇ hinge region, a CD8 transmembrane region, an intracellular domain of CD28 And a CD3 sputum intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-497 of SEQ ID NO: 1.
  • the chimeric antigen receptor further comprises a CD8 signal peptide, preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 1-22 of SEQ ID NO: 1.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • the intracellular signal domains and the like may be directly connected to each other or may be connected by a linker sequence.
  • the linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S.
  • the linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8.
  • the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein.
  • Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino or carboxy terminus of a CAR herein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • polynucleotide sequences encoding the chimeric antigen receptor.
  • the polynucleotide sequence herein may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • 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.
  • nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the CD47 antibody, and one or more operably linked to the sequences Regulatory sequence.
  • the nucleic acid construct is an expression cassette.
  • 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.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequences for CARs herein or the coding sequences for CD47 antibodies can be cloned into a wide variety of vectors, such as, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • the vector can be an expression vector.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism.
  • the invention employs a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein or a coding sequence for a CD47 antibody And optional optional markers.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • 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.
  • an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • 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 selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo and the like.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • the coding sequence of a chimeric antigen receptor described herein and the coding sequence of a CD47 antibody are separately cloned into a vector (also referred to as an integration vector) for integration of a nucleic acid sequence of interest into the genome of a host cell.
  • a vector also referred to as an integration vector
  • the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty.
  • Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon.
  • the transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system.
  • the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art.
  • LTR is the expression cassette for the CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for the CAR or antibody, and the polyA tailing signal sequence.
  • the transposase is a transposase from a piggybac transposition system.
  • the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively.
  • the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference.
  • the transposon 3' inverted terminal repeat is as set forth in CN 201510638974.7 SEQ ID NO:4.
  • the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence.
  • the coding sequence for the piggybac transposase is set forth in CN 201510638974.7 SEQ ID NO: 5.
  • the promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence.
  • the expression of the transposase coding sequence is controlled using a CMV promoter.
  • the sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.
  • the vector of the present invention comprising a coding sequence for a chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7.
  • the coding sequence of the chimeric antigen receptor of the present invention can be prepared by a method conventional in the art and cloned into a suitable vector.
  • the vector for integrating a gene of interest into the genome of a host cell does not contain a transposase coding sequence.
  • such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector.
  • such vectors are used to integrate the coding sequence for a CD47 antibody and a signal peptide coding sequence (such as the coding sequence for a light chain signal peptide) into the genome of a host cell.
  • the amino acid sequence of an exemplary light chain signal peptide is shown in amino acid residues 1-20 of SEQ ID NO: 2, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 5 bases 1-60. Shown.
  • a T cell modified by an EGFR-CAR gene and capable of expressing a CD47 antibody described herein can be transformed into: for integration into a chimeric antigen receptor coding sequence as described herein in a T cell genome.
  • the T cell is transformed into a vector containing a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a backbone vector and constructed using a pS328 vector (with no transposase coding sequence compared to pNB328) as a backbone vector.
  • a vector containing a CD47 antibody coding sequence is set forth in SEQ ID NO: 4; the coding sequence of the CD47 antibody is as set at bases 61-1101 of SEQ ID NO: 5.
  • the signal peptide of the CD47 antibody is a light chain signal peptide.
  • amino acid sequence of an exemplary light chain signal peptide can be as shown in amino acid residues 1-20 of SEQ ID NO: 2; the coding sequence of an exemplary light chain signal peptide is nucleus 1 to 60 of SEQ ID NO: 5. The nucleotide sequence is shown.
  • the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome is at the 5' LTR, promoter, CD8 signal peptide coding sequence , the coding sequence of the scFv recognizing EGFR, the coding sequence of the CD8 ⁇ hinge region, the coding sequence of the CD8 transmembrane region, the coding sequence of the CD28 intracellular domain, the coding sequence of the CD3 intracellular signal domain, the polyA tailing signal sequence, 3' a coding sequence for LTR and a transposase and a promoter thereof; the vector containing the transposase coding sequence encoding the coding sequence of the CD47 antibody described herein in the T cell genome is at 5'LTR and 3'LTR
  • the coding sequence of the promoter, the light chain signal peptide, the coding sequence of the CD47 antibody, and the polyA tailing signal sequence are sequentially contained.
  • the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD47 antibody coding sequence is from 1 to 7:1 to 3, preferably from 1:1 to 3, more preferably from 1:1 to 2, more preferably 1:1.
  • Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • the vector is transfected into a cell of interest using electroporation.
  • the cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokines.
  • T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL).
  • CIK induced killer cells
  • TIL tumor infiltrating lymphocytes
  • the T cell can be derived from a PBMC of a B cell malignancy patient.
  • the T cell is a primary cultured T cell.
  • the invention also provides a composition comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a CD47 antibody described herein.
  • Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.
  • the invention also provides a kit comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a CD47 antibody described herein, or a composition described herein.
  • a reagent or instrument for transferring the vector into a cell can also be provided in the kit.
  • the expression cassette contains at least a suitable promoter and polyA tailing signal sequence in addition to the coding sequence comprising the chimeric antigen receptor or CD47 antibody.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell as described herein and/or said CD47 antibody.
  • Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions.
  • the pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells and/or the CD47 antibody.
  • the therapeutically or prophylactically effective amount of the T cell and/or the CD47 antibody can be determined based on factors such as the condition of the patient.
  • the invention also provides the use of a T cell, or a pharmaceutical composition thereof, and/or the CD47 antibody described herein, in the manufacture of a medicament for the treatment or prevention of a malignancy (cancer).
  • the present invention also provides a method of treating or preventing a malignant tumor, which comprises administering to a subject in need thereof a therapeutically or prophylactically effective amount of the T cell of the present invention and/or the CD47 antibody.
  • a cancer suitable for treatment or prevention of a T cell and/or said CD47 antibody described herein is preferably a cancer having a surface abnormal expression of EGFR on a cancer cell surface; preferably, the cancer is selected from the group consisting of: glioma, renal cancer, adenocarcinoma , lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.
  • Example 1 Construction of recombinant plasmid PNB328-EGFR-CAR, PS328- ⁇ CD47
  • the commercial company was commissioned to synthesize EGFR-CAR (the nucleotide sequence of which is shown in SEQ ID NO: 4, the encoded amino acid sequence is shown in SEQ ID NO: 1), and the ⁇ CD47 gene (the nucleotide sequence of which is SEQ ID NO: 5). Shown, the encoded amino acid sequence is shown in SEQ ID NO: 2), and its structural pattern is shown in Figure 1.
  • the recombinant plasmid was constructed by inserting it into pNB328, pS328 vector EcoRI and SalI restriction sites, respectively. They were named pNB328-EGFR-CAR and pS328- ⁇ CD47, respectively.
  • the pS328-wt- ⁇ CD47 recombinant plasmid was constructed by the same method using the wild type ⁇ CD47 gene (the nucleotide sequence of which is shown in SEQ ID NO: 6, and the encoded amino acid sequence is shown as SEQ ID NO: 3).
  • PBMCs Peripheral blood mononuclear cells
  • the PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells.
  • the suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rmp for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rmp for 3 min to abandon the physiology.
  • EGFR-CAR T cells were constructed using the same method with 6 ug of pNB328-EGFR-CAR plasmid; Mock T cells were constructed with 6 ug of blank plasmid pNB328; 4 ug of pNB328-EGFR-CAR and 4 ug of pS328 were used.
  • -wt- ⁇ CD47 was constructed to obtain wt- ⁇ CD47-EGFR-CAR T cells.
  • the ⁇ CD47-EGFR-CAR T cells obtained in Example 2 were collected, 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 EGFR-bio The other one was not added and incubated 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 of CD47 antibody in ⁇ CD47-EGFR-CAR T cells.
  • 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 pNB328-EGFR-CAR and pS328- ⁇ CD47 plasmids to construct ⁇ CD47-EGFR-CAR T cell positive rate and antibody secretion under different ratios
  • the amount of pNBS328-EGFR-CAR and pS328- ⁇ CD47 plasmids constructed in Example 1 was set to 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively. Seven kinds of ratios were used to construct CAR T cells, and the construction method was the same as in Example 2. The positive rate of CAR-T cells and the amount of antibody secreted by the seven ratios were detected (the detection method was the same as in Example 3).
  • Example 5 Detection of CD47 expression in Mock T cells, EGFR-CAR T cells, and ⁇ CD47-EGFR-CAR T cells
  • Example 2 The Mock T cells, EGFR-CAR T cells and ⁇ CD47-EGFR-CAR T cells constructed in Example 2 were collected, and the expression of CD47 was detected by flow cytometry mouse anti-human CD47-FITC of BD, and the flow method was the same as in Example 3.
  • the CD47 antibody secreted by ⁇ CD47-EGFR-CAR T can block the CD47 expression of the cells themselves.
  • Example 6 Killing effect of Mock T cells, EGFR-CAR T cells and ⁇ CD47-EGFR-CAR T cells on tumor cells
  • EGFR-positive cells of lung cancer cell line H23, ovarian cancer cell line SKOV3 and pancreatic cancer cell line ASPC-1 were selected as target cells, and the Mock obtained in Example 2 was detected by using Essen's real-time label-free cell function analyzer (RTCA).
  • RTCA real-time label-free cell function analyzer
  • lung cancer cell line H23, ovarian cancer cell line SKOV3, and pancreatic cancer cell line ASPC-1 purchased from American Type Culture Collection ATCC
  • ASPC-1 purchased from American Type Culture Collection ATCC
  • Example 7 ⁇ CD47-EGFR-CAR T cell culture supernatant can block tumor cell surface CD47
  • the culture supernatant of ⁇ CD47-EGFR-CAR T cells obtained in Example 2 was co-cultured with lung cancer cell line H23, ovarian cancer cell line SKOV3, and pancreatic cancer cell line ASPC-1, respectively. After 24 hours, tumor cells were collected and CD47 was detected. The expression was not compared with the co-culture of ⁇ CD47-EGFR-CAR T cell supernatant.
  • the flow detection method is the same as in the third embodiment.
  • the CD47 antibody in the ⁇ CD47-EGFR-CAR T cell supernatant blocked the tumor cell surface CD47.
  • Example 8 Blocking the surface of tumor cells CD47 can enhance the phagocytosis of macrophages
  • PBMC Peripheral blood mononuclear cells
  • Adherent cells were added to AIM-V medium and rhGM-CSF (final concentration 1000 U/ml). The medium was changed by half a day and cultured for 7 days to obtain adherent cells, which were macrophages.
  • Phagocytosis of tumor cells by macrophages Tumor cells were stained blue with Hoechst dye, and macrophages were stained red with CM-Dil. For specific staining methods, see the dye instructions. The stained two cells were mixed and divided into two, one was added to the culture supernatant of the EGFR-CAR T cells constructed in Example 2 as a control, and the other was added to the ⁇ CD47-EGFR-CAR constructed in Example 2. The culture supernatant of T cells was observed by confocal microscopy, and the phagocytosis efficiency was counted by the number of cells.
  • Example 9 In vivo anti-tumor effect of ⁇ CD47-EGFR-CAR T cells
  • mice Twenty NSC mice aged 4-6 weeks were randomly divided into 5 groups, 4 rats in each group, inoculated with lung cancer cell line H23, each 1 ⁇ 10 7 cells. After 10 days of tumor formation, PB (100ul) was injected into the tail vein respectively. S and Mock T cells obtained in Example 2, EGFR-CAR T cells, wt- ⁇ CD47-EGFR-CAR T cells, and ⁇ CD47-EGFR-CAR T cells (1 ⁇ 10 7 cells/cell) were observed for tumor volume recording. .

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Abstract

La présente invention concerne une cellule CAR-T spécifique de l'EGFR capable de sécrétion autocrine d'un anticorps CD47, ainsi que l'application de la cellule CAR-T. La cellule T fournie par la présente invention contient une séquence codante d'un récepteur d'antigène chimère reconnaissant l'EGFR et d'une séquence codante d'un anticorps CD47, et/ou exprime un récepteur d'antigène chimère reconnaissant l'EGFR et un anticorps CD47. La cellule T fournie par la présente invention peut spécifiquement cibler les cellules tumorales avec une expression élevée de l'EGFR, et peut également sécréter un anticorps CD47, empêcher l'échappement immunitaire des cellules tumorales, récupérer la phagocytose des cellules tumorales par les macrophages, atteignant ainsi un meilleur effet anti-tumoral.
PCT/CN2018/124382 2017-12-28 2018-12-27 Cellule car-t spécifique de l'egfr capable de sécrétion autocrine de l'anticorps cd47 et application de la cellule car-t WO2019129146A1 (fr)

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