Methods for selectively expanding cells expressing a TCR with a murine constant region
Inventors
DENIGER, Drew C. • Feldman, Steven A. • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-12227554-B2
Publication Date
2025-02-18
Expiration Date
2038-09-24
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Abstract
Disclosed are methods of selectively expanding a number of T cells. The methods may comprise: modifying human T cells to express a TCR, wherein the TCR comprises a murine constant region; producing a population of cells comprising a number of human T cells expressing the TCR and a number of human T cells not expressing the TCR; and culturing the population of cells in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody, or an antigen-binding portion thereof, wherein the antibody has antigenic specificity for the murine constant region of the TCR, so as to selectively expand the number of T cells expressing the TCR over the number of T cells not expressing the TCR. Also disclosed are related populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.
Core Innovation
The invention provides methods for selectively expanding a number of human T cells that have been modified to express a T cell receptor (TCR) comprising a murine constant region (mTCR). The method involves producing a mixed population comprising both T cells expressing the mTCR and those not expressing it, and culturing the population in the presence of irradiated feeder cells, one or more cytokines, and an antibody or antigen-binding portion thereof which specifically binds to the murine constant region of the TCR. This selective culture environment allows for the expansion of the T cells expressing the mTCR over those not expressing it.
The problem being solved is the low efficiency of gene delivery encoding exogenous TCRs in human T cells, which results in low numbers of T cells expressing the exogenous TCR. This limitation hinders the widespread success of cancer treatments involving adoptive transfer of T cells engineered with exogenous TCRs. The invention seeks to improve methods to produce greater numbers of T cells expressing such exogenous TCRs containing murine constant regions, thereby enhancing therapeutic applications.
The invention also encompasses producing populations of cells with a larger proportion of cells expressing the mTCR, pharmaceutical compositions comprising these populations, and methods of treating or preventing cancer by administering the selectively expanded T cells to a mammal. The methods may include multiple rounds of selective expansion with the antibody targeting the murine constant region, optionally followed by non-selective expansion using antibodies targeting the human CD3 complex to further increase the number of TCR-expressing cells.
Claims Coverage
The patent claims include one independent method claim for selectively expanding human T cells expressing a TCR with a murine constant region, and one independent method claim for treating or preventing a condition using such cells. Below are the main inventive features extracted from these independent claims.
Selective expansion of T cells expressing TCR with murine constant region
A method comprising modifying human T cells to express a TCR including a murine constant region, producing a mixed population of TCR-expressing and non-expressing cells, and culturing the population in a first culture that includes (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody specifically binding the murine constant region of the TCR, whereby the culture lacks an antibody binding to human CD3 complex, resulting in selective expansion of T cells expressing the TCR over non-expressing T cells.
Use of TCRs with antigenic specificity for cancer or viral antigens
The TCR expressed by the modified T cells has antigenic specificity for either a cancer antigen or a viral antigen.
Culture conditions including cytokines
The culture includes one or more cytokines selected from IL-2, IL-7, IL-12, IL-15, and IL-21.
Sequential culturing with selective and non-selective expansion
The method may further comprise culturing the population in a second culture containing irradiated feeder cells, cytokines, and an antibody binding the human CD3 complex to non-selectively expand the T cells after initial selective expansion.
Antibody specificity for murine constant region beta chain
The antibody used for selective expansion specifically binds to the murine constant region of the beta chain of the TCR.
Methods for modifying human T cells
Modification methods include transfection, transformation, transduction, electroporation, transposons, meganucleases, zinc finger nucleases, TALEN, or CRISPR-Cas systems.
Antibody comprising specific heavy and light chain variable regions
The antibody for selective expansion comprises amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4, representing heavy and light chain variable regions, respectively.
Population characteristics after selective expansion
The selective expansion results in a population where approximately 10% to about 75%, or 20% to about 99%, of the cells express the TCR comprising the murine constant region.
Magnitude of T cell expansion
The method increases the number of human T cells expressing the TCR with the murine constant region by about 10-fold to about 1000-fold.
Variable region origin in TCR
The TCR may comprise either a murine variable region or a human variable region.
Separation of TCR-expressing T cells
The method may further include separating T cells expressing the TCR from those that do not using the antibody specifically binding the murine constant region.
Method for treating or preventing conditions
A method of treating or preventing a condition in a mammal by preparing T cells according to the described selective expansion method and administering an effective amount to the mammal.
Applications to viral conditions and cancer
The treated or prevented condition can be a viral condition or cancer.
The claims cover methods of selectively expanding human T cells expressing exogenous TCRs containing a murine constant region using specific culture conditions including an antibody targeting the murine constant region and excluding antibodies to human CD3, methods of further expansion with human CD3-targeting antibodies, T cell modification techniques, characteristics of antibody specificity, and therapeutic applications for cancer and viral conditions.
Stated Advantages
Selective expansion increases the proportion and number of T cells expressing the murine constant region TCR compared to methods without such selective expansion.
Populations produced by the methods have improved potential for destruction of target cancer cells and cancer treatment efficacy due to higher proportions of mTCR-expressing T cells.
The inventive methods can achieve fold expansions of mTCR-expressing T cells ranging from about 5-fold up to about 4,000-fold within about 10 to 14 days.
Selective expansion allows for production of large numbers of TCR-expressing T cells suitable for clinical applications, including about 10^6 to more than 10^11 cells.
The method enables production of populations with up to approximately 99% TCR-expressing cells after multiple rounds of selection and expansion.
Documented Applications
Treatment or prevention of cancer in mammals by administration of selectively expanded T cells expressing a TCR comprising a murine constant region.
Treatment or prevention of viral conditions in mammals using selectively expanded T cells expressing the mTCR.
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