WO2020108642A1 - Immunothérapie par car-t combinée basée sur cd19 et cd30 - Google Patents

Immunothérapie par car-t combinée basée sur cd19 et cd30 Download PDF

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WO2020108642A1
WO2020108642A1 PCT/CN2019/122160 CN2019122160W WO2020108642A1 WO 2020108642 A1 WO2020108642 A1 WO 2020108642A1 CN 2019122160 W CN2019122160 W CN 2019122160W WO 2020108642 A1 WO2020108642 A1 WO 2020108642A1
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chimeric antigen
antigen receptor
domain
acid sequence
amino acid
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Yuchen Li
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Beijing Meikang Geno-Immune Biotechnology Co., Ltd.
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Definitions

  • the present application relates to the field of cellular immunotherapy for tumors, in particular to an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting CD30, and an application thereof, and specifically to a method for constructing a chimeric antigen receptor T (CAR-T) cell technology based on tumor specific targets CD19 and CD30 and its application in anti-tumor therapy.
  • CAR-T chimeric antigen receptor T
  • chimeric antigen receptor T cell CAR-T
  • the chimeric antigen receptor typically consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
  • the CAR generally comprises a single chain fragment variable (scFv) region of an antibody or a binding domain specific for a tumor-associated antigen (TAA) , which is coupled to the cytoplasmic domain of a T cell signaling molecule via hinge and transmembrane regions.
  • scFv single chain fragment variable
  • TAA tumor-associated antigen
  • the most common lymphocyte activation moieties include a T cell costimulatory domain in tandem with a T-cell effector function triggering (e.g. CD3 ⁇ ) moiety.
  • the CAR-mediated adoptive immunotherapy allows CAR-transplanted T cells to directly recognize the TAAs on target tumor cells in a non-HLA-restricted manner.
  • B-ALL B cell acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • One approach to treat these patients is to genetically modify T cells to target the antigens expressed on tumor cells through the expression of CARs.
  • CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen (HLA) -independent manner. Attempts in using genetically modified cells expressing CARs to treat these types of patients have achieved promising success.
  • CD19 molecule is a potential target for the treatment of B lymphocyte tumors, and is also a focus in CAR research.
  • the expression of CD19 is restricted to normal and malignant B cells and thus is a widely accepted CAR target for safety tests.
  • T cells genetically modified with a chimeric antigen receptor targeting the CD19 molecule have achieved great success in the treatment of multiple, refractory acute B lymphocytic leukemia, they have limited therapeutic effects in the treatment of refractory, recurrent chronic B lymphocytic leukemia and B lymphocyte lymphoma.
  • CN 104788573 A discloses a chimeric antigen receptor hCD19scFv-CD8 ⁇ -CD28-CD3 ⁇ and use thereof.
  • This second-generation chimeric antigen receptor is composed of variable regions of light and heavy chains of anti-human CD19 monoclonal antibody HI19a (hCD19scFv) , a human CD8 ⁇ hinge region, human CD28 transmembrane and intracellular regions, and a human CD3 ⁇ intracellular region in tandem arrangement.
  • hCD19scFv anti-human CD19 monoclonal antibody HI19a
  • a human CD8 ⁇ hinge region a human CD8 ⁇ hinge region
  • human CD28 transmembrane and intracellular regions and a human CD3 ⁇ intracellular region in tandem arrangement.
  • the expression level of CD19 is decreased after a single infusion of CAR-T cells, causing the tumor cells to easily escape immune mechanisms.
  • this second-generation CART causes a strong immune factor storm which has safety concerns.
  • CD30 is also a potential target for the treatment of malignant B-cell tumors.
  • CD30 is mainly expressed in Hodgkin's lymphoma and some non-Hodgkin's lymphomas including anaplastic large cell lymphoma, ALCL, mediastinal large B cell lymphoma and PMBCL.
  • CD30 is rarely expressed in normal tissues and is an ideal therapeutic target, playing a role in the diagnosis and prognosis of various lymphomas.
  • the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting CD30, and an application thereof.
  • the present application initiates combining the two tumor targets, CD19 and CD30, possesses advantages including strong specificity and high targeting ability, and it can effectively improve and prolong the therapeutic effects of CARTs, shows a better therapeutic effect on surface antigens CD19 and CD30-positive leukemia or B-cell lymphoma and can effectively avoid the off-target escape as found in single-targeted therapy.
  • the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting CD30.
  • T cells are modified with lentiviral vectors encoding antigen binding domains that bind to tumor surface antigens CD19 and CD30, thus allowing the tumor surface antigens CD19 and CD30 to specifically bind to the chimeric antigen receptors of the present application.
  • CAR-T cells eliminate both tumor cells expressing CD19 and those expressing CD30, effectively avoiding the escape of tumor cells resulting from a low antigen expression, and enhancing the long-term immune effects of CAR-T cells.
  • the two chimeric antigen receptors may be a separate chimeric antigen receptor targeting CD19 and a separate chimeric antigen receptor targeting CD30, respectively.
  • the chimeric antigen receptor targeting CD19 may be combined with the chimeric antigen receptor targeting CD30 to express as a dual chimeric antigen receptor, i.e., the antigen binding domain thereof binds to tumor surface antigens CD19 and CD30. Both cases can achieve a combination therapy of the two chimeric antigen receptors.
  • the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting CD30 each comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 ⁇ signaling domain, and an inducible suicide fusion domain in tandem arrangement.
  • the antigen-binding domain is a single chain antibody against tumor surface antigen CD19
  • the antigen-binding domain is a single chain antibody against tumor surface antigen CD30.
  • the chimeric antigen receptor (CAR) of the genetically modified T cell and the single chain antibodies (scFv) of the antigen binding domains for CD19 and CD30 of the genetically modified T cells are exemplified below.
  • the single chain antibody against tumor surface antigen CD19 has an amino acid sequence selected from any one of the group consisting of
  • the amino acid sequence has the activity of a single chain antibody against tumor surface antigen CD19.
  • amino acid sequence (SEQ ID No. 1) of the single-chain antibody against the tumor surface antigen CD19 is listed as follows:
  • the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized CD19 single chain antibodies.
  • the amino acid mutant still functions as a CD19 single-chain antibody.
  • the single chain antibody against the tumor surface antigen CD30 has an amino acid sequence selected from any one of the group consisting of
  • the amino acid sequence has the activity of a single chain antibody against tumor surface antigen CD30.
  • amino acid sequence SEQ ID No. 2 of the single-chain antibody against tumor surface antigen CD30 is listed as follows:
  • the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized CD30 single chain antibodies.
  • the amino acid mutant still functions as a CD30 single-chain antibody.
  • T cells are genetically modified with the chimeric antigen receptor by lentiviral vectors.
  • the CD19-and CD30-based CAR-T cells bind to tumor surface antigens CD19 and CD30, exhibiting a stronger tumor-killing effect.
  • the transmembrane domain is a CD28 transmembrane domain and/or a CD8 ⁇ transmembrane domain.
  • the transmembrane domain can be selected or modified by amino acid substitution.
  • the costimulatory signaling region is any one selected from the group consisting of a CD28 signaling domain, a CD27 signaling domain or a CD137 signaling domain, or a combination of at least two thereof.
  • a person skilled in the art can adjust the arrangement of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain according to requirements. Different arrangements of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain will not affect the chimeric antigen receptor.
  • the present application employs the order of CD28-CD27.
  • the fourth-generation CAR comprises an inducible suicide fusion domain which contains a Caspase 9 domain having the amino acid sequence as shown in SEQ ID NO. 3, which is as follows:
  • the inducible suicide fusion domain is connected in tandem with the CD3 ⁇ signaling domain via a 2A sequence.
  • the 2A sequence will cause the protein expressed by the inducible suicide fusion domain to cleave off from the chimeric antigen receptor protein, thereby allowing the chimeric antigen receptor to exert its function.
  • the suicide fusion domain can be activated by injecting an activator, thereby causing the T cells expressing the chimeric antigen receptor to die to lose their functions.
  • the chimeric antigen receptor further comprises a signal peptide which is capable of directing transmembrane transfer of the chimeric antigen receptor.
  • a signal peptide which is capable of directing transmembrane transfer of the chimeric antigen receptor.
  • the signal peptide is a Secretory signal peptide, which is the signal peptide for gene GMCSFR and has the amino acid sequence as shown in SEQ ID NO. 8, which is as follows: MLLLVTSLLLCELPHPAFLLIP.
  • the Secretory signal peptide is a signal peptide for CD8a gene, and the Secretory signal peptide has the amino acid sequence as shown in SEQ ID NO. 9, which is as follows: MALPVTALLLPLALLLHAARP.
  • the chimeric antigen receptor of the present application may further comprise a hinge region.
  • the hinge region may be selected by those skilled in the art according to actual situation, and is not particularly limited herein. The presence of a hinge region will not affect the performance of the chimeric antigen receptor of the present application.
  • the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting CD30 each comprises a signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 ⁇ signaling domain, a 2A sequence and an inducible suicide fusion domain in tandem arrangement.
  • the chimeric antigen receptor is obtained by connecting a Secretory signal peptide, a CD19 antigen-binding domain and/or a CD30 antigen-binding domain, CD8 ⁇ and/or CD28 transmembrane domain (s) , a CD28 extracellular signaling domain, a CD28 intracellular signaling domain, a CD27 intracellular signaling domain, a CD3 ⁇ intracellular signaling domain, a 2A sequence and a FBKP.
  • Casp9 domain in tandem. Specifically, the arrangement is as follows:
  • Casp9 has the amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence that shares more than 90%homology therewith.
  • the amino acid sequence as shown in SEQ ID NO. 4 is as follows:
  • Casp9 has the nucleotide sequence as shown in SEQ ID NO. 5 or an nucleotide sequence that shares more than 95%homology therewith.
  • the nucleic acid sequence as shown in SEQ ID NO. 5 is as follows:
  • Casp9 has the amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence that shares more than 90%homology therewith.
  • the amino acid sequence as shown in SEQ ID NO. 6 is as follows:
  • Casp9 has the nucleotide sequence as shown in SEQ ID NO. 7 or an nucleotide sequence that shares more than 95%homology therewith.
  • the nucleic acid sequence as shown in SEQ ID NO. 7 is as follows:
  • the chimeric antigen receptor further comprises a promoter, which is any one of the group consisting of EF1a, CMV-TAR and CMV, or a combination of at least two thereof.
  • the chimeric antigen receptor is expressed by transducing the nucleic acid encoding the same into T cells.
  • the transduction is performed by transduction into T cells via any one of the group consisting of a viral vector, an eukaryotic expression plasmid and an mRNA sequence, or a combination of at least two thereof, preferably by transduction into T cells via a viral vector.
  • the viral vector is any one of the group consisting of a lentiviral vector and a retroviral vector, or a combination of at least two thereof, preferably a lentiviral vector.
  • the present application provides a recombinant lentivirus mixture comprising a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting CD19 and packaging helper plasmids pNHP and pHEF-VSVG and a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting CD30 and packaging helper plasmids pNHP and pHEF-VSVG.
  • the recombinant lentivirus can efficiently immunize cells including T cells to prepare targeting T cells.
  • the mammalian cell is any one of the group consisting of a 293 cell, a 293T cell and a TE671 cell, or a combination of at least two thereof.
  • the present application provides a pharmaceutical composition comprising the immune cell mixture as described in the first aspect and/or the recombinant lentivirus mixture as described in the second aspect.
  • the present application provides use of the immune cell mixture as described in the first aspect, the recombinant lentivirus mixture as described in the second aspect or the pharmaceutical composition as described in the third aspect for the preparation of chimeric antigen receptor T cells, immune competent cells or tumor therapeutics.
  • the antigen receptor T cells have a good targeting effect and are capable of releasing low dose of immune factors, having a property of low toxic reaction.
  • the tumor is a blood-associated neoplastic disease and/or a solid tumor.
  • the neoplastic disease is selected from, but not limited to, leukemia.
  • the present application provides a method for treating a tumor comprising administrating to a subject in need thereof a therapeutically effective amount of
  • the CD19-and CD30-based CAR-T cells obtained by genetically modifying T cells with the chimeric antigen receptors of the present application bind to tumor surface antigens CD19 and CD30, and kill tumors with a stronger effect, achieving a more significant tumor reduction effect;
  • the two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and CD30, which are highly expressed in leukemia and lymphoma, and thus have a safer and more significant effect than other chimeric antigen receptors and other tumor antigens.
  • Figure 1 is a diagram showing the synthetic gene sequence map of the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting CD30 according to the present application;
  • Figure 2 is a diagram showing the mechanism for using chimeric antigen receptors targeting CD19 in combination with chimeric antigen receptors targeting CD30 according to the present application;
  • Figure 3 shows a flow chart of the clinical trial using CD19-targeted chimeric antigen receptor T cells and CD30-targeted chimeric antigen receptor T cells;
  • Figure 4 is a diagram showing the expression level and ratio of antigens CD19 and CD30 in tumor tissues of lymphoma patients as detected by IHC staining;
  • Figure 5 is a diagram showing the imaging comparison before and after infusion, when a combination of CD19-targeted and CD30-targeted chimeric antigen receptor T cells is administered to a PMBCL patient in clinical trial;
  • Figures 6 is a graph showing the copy numbers of CAR genes detected in vivo in a B cell malignancy patient (Patient 1) after infusion with a combination of CD19-targeted and CD30-targeted chimeric antigen receptor T cells in clinical trial;
  • Figures 7 is a graph showing the copy numbers of CAR genes detected in vivo in a B cell malignancy patient (Patient 2) after infusion with a combination of CD19-targeted and CD30-targeted chimeric antigen receptor T cells in clinical trial.
  • reagents or instruments used herein which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.
  • Casp9 had the nucleotide sequence as shown in SEQ ID NO. 5, and
  • Casp9 had the nucleotide sequence as shown in SEQ ID NO. 7.
  • virus supernatant was filtered with a 0.45 ⁇ m low protein-binding filter, and the virus was divided into small portions and stored at -80 °C;
  • lentiviral vectors at a titer of 10 6 to 10 7 transducing units can be produced by transduced cells per ml media.
  • the virus supernatant was added to the Centricon filter tube or the like, then centrifuged at 2500g for 30 minutes;
  • the activated T cells were seeded into a culture dish, and concentrated lentiviruses containing target genes were added, centrifuged at a centrifugal force of 100 g for 100 minutes, then cultured at 37 °C for 24 hours, and AIM-V media containing cell culture factors were added, after 2-3 days of culture, the cells were harvested and counted to produce available CD19 CAR-T cells and CD30 CAR-T cells.
  • the two chimeric antigen receptors as used in the present application can achieve significant therapeutic effects in treating tumors, especially tumors with weak CD19 expression, and effectively prevent CD19 escape by combining CAR-T cells expressing CD19-targeted chimeric antigen receptors and CD30-targeted chimeric antigen receptors.
  • Figure 4 is a graph showing the intensity and ratio of CD19 and CD30 expression in tumor tissues of a lymphoma patient as detected by IHC staining.
  • the expression intensity of CD19 in patient tumor tissues was 2.5+, and the expression area was about 60%; and the expression intensity of CD30 was 1+, and the expression area was about 60%. It shows that CD30 has a broader expression area in lymphoma tissues and is a good auxiliary target.
  • Sample a PMBCL patient, male, 31 years old. The lesion involved chest and pre-cardiac lymph nodes.
  • Figure 5 is a graph showing the imaging comparison in a PMBCL patient before and after infusion of a combination of CD19-targeted and CD30-targeted chimeric antigen receptor T cells in clinical.
  • Patchy soft tissue densities which had a size of about 6.3 cm ⁇ 2.2 cm and enhanced in enhanced scanning were found in the superoanterior mediastinum of patient before infusion.
  • 20 days after infusion patchy soft tissue densities which had a largest layer size of 4.7 cm ⁇ 1.6 cm and enhanced in enhanced scanning were found in the superoanterior mediastinum of patient. The lesion was reduced compared to that before infusion.
  • Figures 6 and 7 are graphs showing the copy numbers of CAR genes detected in the peripheral blood of two B cell malignancy patients after infusion of a combination of CD19-targeted and CD30-targeted chimeric antigen receptor T cells in clinical.
  • the two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and CD30.
  • the expression level of CD19 is decreased after a CAR-T cell infusion, causing the tumor cells to escape immune mechanisms.
  • CAR-T cells By using a combination of two types of CAR-T cells, better therapeutic effects are achieved for CD19-negative relapse and B cell tumors with low CD19 expression, and the disease is easier to be relieved.

Abstract

L'invention concerne un mélange de cellules immunitaires comprenant une cellule immunitaire génétiquement modifiée avec un récepteur antigénique chimérique ciblant CD19 et une cellule immunitaire génétiquement modifiée avec un récepteur antigénique chimérique ciblant CD30, et une application correspondante. Le récepteur antigénique chimérique ciblant CD19 et le récepteur antigénique chimérique ciblant CD30 comprennent chacun un domaine de liaison à l'antigène, un domaine transmembranaire, une région de signalisation costimulatrice, un domaine de signalisation de CD3ζ, et un domaine de fusion suicide inductible en agencement en tandem. Les récepteurs antigéniques chimériques reconnaissent spécifiquement des antigènes de surface tumorale CD19 et CD30. En utilisant la combinaison de cellules CAR-T ciblant les deux antigènes, de meilleurs effets thérapeutiques sont obtenus quant aux rechutes CD19 négatives et aux lymphomes de type B avec une faible expression de CD19, de plus, la maladie est plus facile à soulager par comparaison avec l'utilisation d'autres lymphocytes T avec récepteur d'antigène chimérique unique.
PCT/CN2019/122160 2018-11-30 2019-11-29 Immunothérapie par car-t combinée basée sur cd19 et cd30 WO2020108642A1 (fr)

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CN111875710A (zh) * 2020-07-31 2020-11-03 广东昭泰体内生物医药科技有限公司 异质性肿瘤治疗用免疫细胞及其应用
WO2022147444A2 (fr) 2020-12-30 2022-07-07 Alaunos Therapeutics, Inc. Vecteurs recombinants comprenant des cassettes d'expression polycistronique et leurs procédés d'utilisation
CN116143943A (zh) * 2022-10-31 2023-05-23 济南泰和医药科技有限公司 一种靶向baffr嵌合抗原受体、car-t细胞及应用
CN117384968A (zh) * 2023-12-07 2024-01-12 广东赛尔生物科技有限公司 一种嵌合抗原受体（car）及其用于治疗白血病的用途

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CN116143943A (zh) * 2022-10-31 2023-05-23 济南泰和医药科技有限公司 一种靶向baffr嵌合抗原受体、car-t细胞及应用
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