Methods for engineering allogeneic and immunosuppressive resistant T cell for immunotherapy
Inventors
Galetto, Roman • Gouble, Agnes • Grosse, Stephanie • Mannioui, Cecile • Poirot, Laurent • Scharenberg, Andrew • Smith, Julianne
Assignees
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Publication Number
US-11891614-B2
Publication Date
2024-02-06
Expiration Date
Abstract
Methods for developing engineered T-cells for immunotherapy that are both non-alloreactive and resistant to immunosuppressive drugs. The present invention relates to methods for modifying T-cells by inactivating both genes encoding target for an immunosuppressive agent and T-cell receptor, in particular genes encoding CD52 and TCR. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.
Core Innovation
The invention relates to preparing engineered T-cells for immunotherapy by inactivating PDCD1 in the T-cells. PDCD1 inactivation is performed by introducing, by electroporation of RNAs, at least two half Transcription activator-like effector nucleases (TALE-nucleases) that are able to selectively inactivate a gene encoding PDCD1 by DNA cleavage, thereby producing engineered T-cells. The engineered T-cells are then expanded.
The method defines that the at least two half TALE-nucleases recognize and cleave a target sequence selected from SEQ ID NO:77 and SEQ ID NO:78. The at least two half TALE-nucleases are constituted by a first half-TALE nuclease and a second half-TALE nuclease. In further configurations, the first half-TALE nuclease or the second half-TALE nuclease comprises an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.
A further part of the invention specifies that introducing the RNAs encoding the half TALE-nucleases into the T-cells comprises applying an agile pulse sequence. The agile pulse sequence includes one electrical pulse with defined voltage range, pulse width, and pulse interval; an electrical pulse step with defined voltage range and pulse width and a pulse interval between steps; and four electrical pulses with defined voltage, pulse width, and pulse interval between each of the four pulses. This configuration supports the preparation of PDCD1-inactivated engineered T-cells for immunotherapy.
Claims Coverage
Two independent claims are identified. Both focus on immunotherapy preparation of engineered T-cells by PDCD1 inactivation using split half TALE-nucleases delivered by electroporation of RNAs, followed by expanding the engineered T-cells; the claims further differ in how the half-TALE components and pulse specification are defined.
PDCD1 inactivation using at least two half TALE-nucleases delivered by electroporation of RNAs
Inactivating PDCD1 in the T-cells by introducing, by electroporation of RNAs, at least two half Transcription activator-like effector nucleases (TALE-nucleases) able to selectively inactivate by DNA cleavage a gene encoding PDCD1 to produce engineered T-cells, and expanding the engineered T-cells.
Half TALE-nucleases recognizing defined PDCD1 target sequences and comprising first and second half nucleases
The at least two half TALE-nucleases recognize and cleave a target sequence selected from SEQ ID NO:77 and SEQ ID NO:78, wherein the at least two half-TALE nucleases are constituted by a first half-TALE nuclease and a second half-TALE nuclease, and wherein the first half-TALE nuclease or the second half-TALE nuclease comprises the amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.
Agile pulse sequence for electroporation of RNAs encoding half TALE-nucleases
Introducing the engineered T-cells with RNAs encoding at least two half TALE-nucleases comprises applying an agile pulse sequence consisting of one electrical pulse, one electrical pulse step, and four electrical pulses, each with defined voltage, pulse width, and pulse interval.
Overall, the independent claims cover engineered T-cells for immunotherapy prepared by PDCD1 inactivation using electroporated RNAs encoding at least two half TALE-nucleases that selectively DNA-cleave PDCD1 target sequences selected from SEQ ID NO:77 and SEQ ID NO:78, followed by expanding the engineered T-cells. The coverage includes the split into first and second half-TALE nucleases with specific sequence-defined components and, in one independent claim, an agile pulse sequence specification for the electroporation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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