Method for proliferation of antigen-specific T cells
Inventors
Karlsson-Parra, Alex • Wallgren, Anna-Carin • Andersson, Bengt
Assignees
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Publication Number
US-9476028-B2
Publication Date
2016-10-25
Expiration Date
Abstract
The present invention relates to an in vitro method for priming genetically modified T cells suitable for administration to a patient having a tumor. The invention is also directed to the composition obtained by the method and uses thereof.
Core Innovation
The invention relates to an in vitro method for priming genetically modified antigen specific CD4+ and/or CD8+ T cells suitable for administration to a patient having a tumor. The method co-cultures tumor antigen receptor expressing target T cells from the patient to be treated with mature dendritic cells, anti-CD3 antibodies, and lymphocytes that have been sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs).
The sensitized lymphocytes are obtained by culturing non-proliferating APCs from a first healthy donor with peripheral blood mononuclear cells (PMBCs) from a second healthy donor. The described co-culture with mature dendritic cells and anti-CD3 is stated to upregulate DC CD70 and increase T-cell cytokines including IL-12, IFN-b3, and IL-2, while enriching CD27+ CD8+ T cells and reducing apoptosis, including lower Annexin-V.
The described priming strategy is positioned relative to rapid expansion protocol (REP) approaches and provides improved outcomes upon restimulation, including increased proliferation and enhanced CAR T-cell expansion and killing. The framework uses antigen presentation by mature dendritic cells together with anti-CD3 and MHC-sensitized lymphocytes derived from two healthy donors, and supports enrichment of functional tumor-reactive T-cell subsets compared with alternative approaches.
Claims Coverage
The independent claim provides a priming method with multiple inventive elements centered on co-culture of patient-derived tumor antigen receptor-expressing T cells with mature dendritic cells, anti-CD3 antibodies, and MHC-sensitized lymphocytes generated from two healthy donors using non-proliferating APCs and PMBCs. The dependent claims refine the independent claim by specifying tumor antigen receptor types, APC/dendritic-cell preparation, maturation/workflow features, culture duration, and optional restimulation and cytokine additions.
MHC-sensitized allogeneic lymphocytes generated from two healthy donors using non-proliferating APCs and PMBCs
The method uses lymphocytes sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs), where the sensitized lymphocytes are obtained by culturing non-proliferating APCs from a first healthy donor with PMBCs from a second healthy donor.
Co-culturing patient tumor antigen receptor-expressing T cells with mature dendritic cells and anti-CD3
The method primes genetically modified antigen specific CD4+ and/or CD8+ T cells by co-culturing tumor antigen receptor expressing target T cells from the patient to be treated, mature dendritic cells, and anti-CD3 antibodies together with the sensitized lymphocytes.
Using tumor antigen receptors selected as T-cell receptors (TCRs) and/or chimeric antigen receptors (CARs)
The method is characterized by selecting tumor antigen receptors from T-cell receptors (TCRs) and chimeric antigen receptors (CARs) for the tumor antigen receptor expressing target T cells.
Maturation workflow for dendritic cells using GM-CSF and IL-4 followed by maturation with a second composition
The method obtains mature dendritic cells by culturing monocytes with GM-CSF and IL-4 to form immature dendritic cells, and then culturing the immature dendritic cells with a second composition to mature them for at least about 12 hours.
Culture duration for the priming method of about 4 to 20 days
The method further includes culturing the cells for about 4 to 20 days.
Restimulation of primed antigen-specific T cells with new dendritic cells, anti-CD3 antibodies, and new sensitized allogeneic lymphocytes
The method restimulates primed antigen specific CD4+ and/or CD8+ T cells by culturing them with new dendritic cells, anti-CD3 antibodies, and new sensitized allogeneic lymphocytes, optionally adding exogenous IL-2, IL-7, IL-15, anti-IL-4, and/or IL-21.
Specifying TLR3 and TLR8 stimulatory agents for dendritic-cell maturation
The method specifies poly-1:C as the TLR3 ligand and R848 as the TLR8 agonist.
Across independent and dependent claims, the inventive core is an in vitro priming co-culture that combines patient-derived tumor antigen receptor-expressing CD4+ and/or CD8+ T cells with mature dendritic cells and anti-CD3, together with MHC-sensitized allogeneic lymphocytes generated from two healthy donors using non-proliferating APCs and PMBCs. Dependent claims narrow receptor type (TCR vs CAR), specify a GM-CSF/IL-4-based dendritic-cell maturation workflow, add culture duration, optionally include restimulation with new components, and include specific TLR ligand choices.
Stated Advantages
Upregulates DC CD70.
Increases T-cell cytokines including IL-12, IFN-b3, and IL-2.
Enriches CD27+ CD8+ T cells.
Reduces apoptosis, including lower Annexin-V.
Improves functional resistance to tumor immunosuppression.
Supports improved proliferation after restimulation.
Enhances CAR T-cell expansion and killing.
Documented Applications
Priming genetically modified antigen specific CD4+ and/or CD8+ T cells for administration to a patient having a tumor.
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