Enhanced generation of cytotoxic T-lymphocytes by IL-21 mediated FOXP3 suppression
Inventors
Assignees
National Institutes of Health NIH • Fred Hutchinson Cancer Center
Publication Number
US-9809797-B2
Publication Date
2017-11-07
Expiration Date
2028-09-25
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Abstract
A method of carrying out adoptive immunotherapy by administering a subject an antigen-specific cytotoxic T lymphocytes (CTL) preparation in a treatment-effective amount is described. In the method, the CTL preparation is preferably administered as a preparation of an in vitro antigen-stimulated and expanded primate CTL population, the CTL population: (i) depleted of FoxP3+ T lymphocytes prior to antigen stimulation; (ii) antigen-stimulated in vitro in the presence of interleukin-21; or (iii) both depleted of FoxP3+ T lymphocytes prior to antigen stimulation and then antigen-stimulated in vitro in the presence of interleukin-21. Methods of preparing such compositions, and compositions useful for carrying out the adoptive immunotherapy, are also described.
Core Innovation
The invention describes an improved method for adoptive immunotherapy by administering an antigen-specific cytotoxic T lymphocyte (CTL) preparation that has been in vitro antigen-stimulated and expanded. This CTL population is characterized by depletion of FoxP3+ T lymphocytes (or CD25+ cells) prior to antigen stimulation or being antigen-stimulated in the presence of interleukin-21 (IL-21), or a combination of both depleting FoxP3+ T lymphocytes prior to stimulation and stimulating in the presence of IL-21.
The problem being addressed is the difficulty in isolating tumor-reactive T cells due to their low frequency and low target avidity caused by central and peripheral tolerance mechanisms. Additionally, regulatory T cells (Treg), expressing FoxP3 and CD25, suppress potentially autoreactive T cells and impair proliferative response to antigenic stimulation. This suppressive influence presents a significant barrier in generating effective anti-tumor immunity and limits the capacity to generate antigen-specific T cells both in vivo and in vitro.
The present invention overcomes these barriers by depleting FoxP3+ T regulatory cells and introducing IL-21 during antigen stimulation, which enhances the generation, expansion, and avidity of tumor-associated antigen-specific CTLs. IL-21 uniquely suppresses the outgrowth of FoxP3+ Tregs during stimulation and expansion, partially reverses Treg-mediated inhibition of CD8+ T cell proliferation, and enriches for a memory CTL phenotype with superior functions. This combined approach results in a dramatic increase (150- to over 450-fold) in frequency and absolute numbers of antigen-specific CTLs in vitro, surpassing effects of other gamma-chain cytokines alone or Treg depletion alone.
Claims Coverage
The patent includes one independent claim focused on a pharmaceutical formulation comprising antigen-specific CD8+CD25− human cytotoxic T lymphocyte cells prepared by a process involving depletion of CD25+ cells and in vitro culturing with antigen and IL-21. The inventive features relate to cell sorting, antigen stimulation with IL-21, and formulation characteristics.
Pharmaceutical formulation of antigen-specific CD8+CD25− CTL cells
A formulation comprising a single T cell population of antigen-specific CD8+CD25− human CTLs specific for an antigen, enriched at least 100-fold compared to populations not subjected to CD25 depletion or IL-21 culture, with at least 109 CTL cells in the formulation.
Depletion of CD25+ cells by antibody-mediated sorting
The process includes sorting lymphocytes by depleting CD25+ cells through contacting the population with anti-CD25 antibodies, thereby enriching the starting population for CD8+CD25− cells prior to culture.
In vitro promotion of antigen-specific CTL production with interleukin-21
Antigen-specific CTLs are produced by culturing the sorted CD8+CD25− subpopulation with antigen in a medium containing interleukin-21, at concentrations ranging from 1 to 1000 ng/ml, which uniquely enhances CTL expansion.
Use of antigen-presenting cells including dendritic cells in culture
The in vitro culture involves antigen presentation by cells such as dendritic cells (DCs) pulsed with antigen peptides to stimulate CTL expansion.
Optionally, transgene expression in CTL cells
The antigen-specific CTL cells in the formulation may comprise a transgene introduced to improve therapeutic efficacy or for tracking purposes.
The claims collectively cover a method and pharmaceutical formulation for enhancing the generation of antigen-specific cytotoxic T lymphocytes by combining depletion of regulatory CD25+ cells with IL-21 mediated in vitro antigen stimulation and expansion, resulting in a highly enriched and therapeutically potent CTL population.
Stated Advantages
Markedly enhanced generation and expansion of antigen-specific cytotoxic T lymphocytes—up to over 450-fold increase in frequency compared to untreated controls.
Combined use of CD25 depletion and IL-21 results in profound (>99.5%) depletion of FoxP3+ regulatory T cells, alleviating suppressive effects.
IL-21 uniquely enriches for antigen-specific CTL with a memory phenotype (CD28+, CCR7−) associated with improved function and helper independence.
IL-21 partially reverses regulatory T cell-mediated suppression of CD8+ T cell proliferation without inducing Treg proliferation.
The method enables generation of tumor-reactive T cells with enhanced avidity and function, potentially improving adoptive cellular therapy outcomes.
Documented Applications
Adoptive immunotherapy for cancer treatment by administering antigen-specific CTL preparations to primate subjects.
Treatment of infectious diseases in primates, including viral, retroviral, bacterial, and protozoal infections, by enhancing CTL responses.
Treatment of immunodeficient patients with viral infections such as Cytomegalovirus, Epstein-Barr virus, adenovirus, and BK polyomavirus, particularly in transplant patients.
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