MX2021005372A - Composiciones de linfocitos t con propiedades fenotípicas mejoradas. - Google Patents

Composiciones de linfocitos t con propiedades fenotípicas mejoradas.

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Publication number
MX2021005372A
MX2021005372A MX2021005372A MX2021005372A MX2021005372A MX 2021005372 A MX2021005372 A MX 2021005372A MX 2021005372 A MX2021005372 A MX 2021005372A MX 2021005372 A MX2021005372 A MX 2021005372A MX 2021005372 A MX2021005372 A MX 2021005372A
Authority
MX
Mexico
Prior art keywords
cell compositions
cell
composition
cells
phenotypic properties
Prior art date
Application number
MX2021005372A
Other languages
English (en)
Inventor
Mathias Oelke
Sojung Kim
Kristi Jones
Lauren Suarez
Ken Carter
Scott Carmer
Dan Bednarik
Vineetha Edavana
Emily Lu
Original Assignee
Neximmune Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neximmune Inc filed Critical Neximmune Inc
Publication of MX2021005372A publication Critical patent/MX2021005372A/es

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/461Cellular immunotherapy characterised by the cell type used
    • A61K39/4611T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/464Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643Vertebrate antigens
    • A61K39/4644Cancer antigens
    • A61K39/464448Regulators of development
    • A61K39/464449Cell cycle regulated proteins, e.g. cyclin, CDC, CDK or INK-CCR
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/464Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643Vertebrate antigens
    • A61K39/4644Cancer antigens
    • A61K39/464452Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453Wilms tumor 1 [WT1]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/464Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643Vertebrate antigens
    • A61K39/4644Cancer antigens
    • A61K39/464484Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464489PRAME
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/464Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643Vertebrate antigens
    • A61K39/4644Cancer antigens
    • A61K39/46449Melanoma antigens
    • A61K39/464491Melan-A/MART
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/7051T-cell receptor (TcR)-CD3 complex
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70514CD4
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70517CD8
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/57Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48Blood cells, e.g. leukemia or lymphoma

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La presente invención brinda una composición celular aislada que, en algunas modalidades, es adecuada para inmunoterapia adoptiva, métodos para producir las composiciones de linfocitos y métodos de tratamiento con las composiciones de linfocitos. En un vehículo farmacéuticamente aceptable, la composición comprende al menos alrededor de 106 linfocitos T CD8+específicos para actuar sobre antígenos peptídicos, los cuales comprenden linfocitos T memoria troncales (TSCM). En varias modalidades, la composición es de alrededor de 1 % a alrededor de 100 % linfocitos T memoria troncales, lo que genera una terapia adoptiva fuerte y duradera, y con lo cual son posibles genomodificaciones de linfocitos T.
MX2021005372A 2018-11-08 2019-11-08 Composiciones de linfocitos t con propiedades fenotípicas mejoradas. MX2021005372A (es)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862757467P 2018-11-08 2018-11-08
US201962821031P 2019-03-20 2019-03-20
US201962867499P 2019-06-27 2019-06-27
PCT/US2019/060477 WO2020097466A1 (en) 2018-11-08 2019-11-08 T cell compositions with improved phenotypic properties

Publications (1)

Publication Number Publication Date
MX2021005372A true MX2021005372A (es) 2021-09-14

Family

ID=70612526

Family Applications (1)

Application Number Title Priority Date Filing Date
MX2021005372A MX2021005372A (es) 2018-11-08 2019-11-08 Composiciones de linfocitos t con propiedades fenotípicas mejoradas.

Country Status (12)

Country Link
US (2) US20200188435A1 (es)
EP (1) EP3876979A4 (es)
JP (1) JP2022512948A (es)
KR (1) KR20210095157A (es)
CN (1) CN113226359B (es)
AU (1) AU2019375997A1 (es)
BR (1) BR112021008963A2 (es)
CA (1) CA3118757A1 (es)
IL (1) IL282914A (es)
MX (1) MX2021005372A (es)
SG (1) SG11202104611YA (es)
WO (1) WO2020097466A1 (es)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113880953A (zh) * 2020-07-01 2022-01-04 华夏英泰(北京)生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用
IL308257A (en) 2021-05-05 2024-01-01 Immatics Biotechnologies Gmbh Antigen binding proteins that uniquely bind PRAME
EP4321613A1 (en) * 2021-05-07 2024-02-14 Yasuhito Tokumoto Method for producing memory t cells
CN113388612A (zh) * 2021-06-18 2021-09-14 重庆天科雅生物科技有限公司 一种针对表位点为iyvlvmlvl的tcr所设计的引物及其应用

Family Cites Families (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
JP3680219B2 (ja) * 1994-06-28 2005-08-10 独立行政法人理化学研究所 癌細胞障害性tリンパ球の誘導培養方法
US6458354B1 (en) 1996-03-28 2002-10-01 The Johns Hopkins University Molecular complexes which modify immune responses
JPH11507843A (ja) 1996-03-28 1999-07-13 ザ・ジョンズ・ホプキンス・ユニバーシティー タンパク質の可溶性二価および多価ヘテロ二量体類縁体
US6140113A (en) 1996-03-28 2000-10-31 The Johns Hopkins University Polynucleotides encoding molecular complexes which modify immune responses
US7973137B1 (en) 1996-03-28 2011-07-05 Johns Hopkins University Cell compositions comprising molecular complexes that modify immune responses
US6268411B1 (en) 1997-09-11 2001-07-31 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US20050169898A1 (en) 1997-04-15 2005-08-04 Jianlin Gong Cell fusions and methods of making and using the same
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells
AU2001243288B2 (en) 2000-02-24 2005-11-24 Life Technologies Corporation Simultaneous stimulation and concentration of cells
US6867041B2 (en) 2000-02-24 2005-03-15 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US6797514B2 (en) 2000-02-24 2004-09-28 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US20020131960A1 (en) 2000-06-02 2002-09-19 Michel Sadelain Artificial antigen presenting cells and methods of use thereof
US7638325B2 (en) 2002-01-03 2009-12-29 The Trustees Of The University Of Pennsylvania Activation and expansion of T-cells using an engineered multivalent signaling platform
CA2800113C (en) 2002-07-12 2015-02-03 The Johns Hopkins University Reagents and methods for engaging unique clonotypic lymphocyte receptors
DK1623017T3 (da) 2003-05-08 2011-01-10 Life Technologies Corp Frembringelse og isolering af antigenspecifikke T-celler
SI1956080T1 (sl) 2005-08-08 2012-02-29 San Raffaele Centro Fond Uporaba IL-7 in IL-15 za genetsko modifikacijo spominskih limfocitov T
WO2009094273A2 (en) 2008-01-15 2009-07-30 Yale University Compositions and methods for adoptive and active immunotherapy
WO2010065544A2 (en) 2008-12-01 2010-06-10 The Johns Hopkins University Diagnostic and treatment methods for cancer based on immune inhibitors
US8415150B2 (en) 2009-02-24 2013-04-09 The Trustees Of The University Of Pennsylvania Methods for treating progressive multifocal leukoencephalopathy (PML)
US8075895B2 (en) * 2009-09-22 2011-12-13 Janssen Pharmaceutica N.V. Identification of antigenic peptides from multiple myeloma cells
KR102315754B1 (ko) 2010-05-14 2021-10-22 더 제너럴 하스피톨 코포레이션 종양 특이적 신생항원을 확인하는 조성물 및 방법
EP2404618A1 (en) 2010-07-07 2012-01-11 Stichting Katholieke Universiteit meer in het bijzonder Radboud Universiteit Nijmegen Immunomodulatory protein constructs with a helical polymeric backbone.
EP2532740A1 (en) 2011-06-11 2012-12-12 Michael Schmück Antigen-specific CD4+ and CD8+ central-memory T cell preparations for adoptive T cell therapy
WO2013086500A1 (en) 2011-12-09 2013-06-13 The Johns Hopkins University Artificial antigen presenting cells having a defined and dynamic shape
WO2013088114A1 (en) 2011-12-12 2013-06-20 Cell Medica Limited Process of expanding t cells
GB201121308D0 (en) 2011-12-12 2012-01-25 Cell Medica Ltd Process
WO2014127220A1 (en) 2013-02-15 2014-08-21 The Johns Hopkins University Antigen-specific t cell redirectors
SG10201707377XA (en) 2013-03-14 2017-10-30 Univ Johns Hopkins Nanoscale artificial antigen presenting cells
US11807675B2 (en) 2013-10-03 2023-11-07 The University Of Maryland, Baltimore Nanoparticle based artificial antigen presenting cell mediated activation of NKT cells
US20170065690A1 (en) * 2014-02-21 2017-03-09 Argos Therapeutics, Inc. Tscm CELLS AND METHODS FOR USE
US10066265B2 (en) 2014-04-01 2018-09-04 Adaptive Biotechnologies Corp. Determining antigen-specific t-cells
CN106062185A (zh) 2014-04-24 2016-10-26 美天旎生物技术有限公司 用于自动生成遗传修饰的t细胞的方法
NZ725201A (en) 2014-04-25 2018-05-25 Bluebird Bio Inc Improved methods for manufacturing adoptive cell therapies
RU2739770C2 (ru) 2014-06-11 2020-12-28 полибайосепт ГмбХ Экспансия лимфоцитов с использованием композиции цитокинов для активной клеточной иммунотерапии
AU2015317712B2 (en) * 2014-09-17 2019-01-17 The Johns Hopkins University Reagents and methods for identifying, enriching, and/or expanding antigen-specific T cells
MA45488A (fr) 2015-10-22 2018-08-29 Juno Therapeutics Gmbh Procédés, kits et appareil de culture de cellules
AU2016344745A1 (en) 2015-10-28 2018-05-17 Life Technologies As Selective expansion of different subpopulations of T cells by the alteration of cell surfacing signals and signal ratio
MA44314A (fr) * 2015-11-05 2018-09-12 Juno Therapeutics Inc Récepteurs chimériques contenant des domaines induisant traf, et compositions et méthodes associées
EP3384012A1 (en) * 2015-12-04 2018-10-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Method of amplifying a population of antigen-specific memory cd4+ t cells using artificial presenting cells expressing hla class ii molecules
RU2021107053A (ru) * 2016-03-16 2021-08-24 Нексиммьюн, Инк. Получение антигенспецифических t-клеток
BR112020005552A2 (pt) * 2017-09-20 2020-10-27 Neximmune, Inc. composições de células compreendendo células t específicas de antígeno para terapia adotiva
ES2971659T3 (es) * 2017-11-01 2024-06-06 Juno Therapeutics Inc Procedimiento para la producción de una composición de células T

Also Published As

Publication number Publication date
AU2019375997A1 (en) 2021-06-03
EP3876979A4 (en) 2022-08-24
JP2022512948A (ja) 2022-02-07
KR20210095157A (ko) 2021-07-30
WO2020097466A1 (en) 2020-05-14
CN113226359B (zh) 2024-05-24
BR112021008963A2 (pt) 2021-09-14
CN113226359A (zh) 2021-08-06
US20200215115A1 (en) 2020-07-09
CA3118757A1 (en) 2020-05-14
IL282914A (en) 2021-06-30
US11007222B2 (en) 2021-05-18
SG11202104611YA (en) 2021-06-29
US20200188435A1 (en) 2020-06-18
EP3876979A1 (en) 2021-09-15

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