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EP-4737561-A2 - PROCESSES FOR GENERATING ENGINEERED CELLS AND COMPOSITIONS THEREOF

EP4737561A2EP 4737561 A2EP4737561 A2EP 4737561A2EP-4737561-A2

Abstract

The present disclosure provides processes for genetically engineering T cells, such as primary CD4+ T cells and/or CD8+ T cells, for use in cell therapy that does not involve expanding the cells. In particular aspects, the provided processes successfully generate compositions of engineered T cells, such as containing populations of engineered T cells, that express a chimeric antigen receptor (CAR) within a shortened amount of time as compared to alternative engineering processes, such as processes that involve expanding the cells. In certain aspects, the provided processes successfully generate a composition of engineered T cells suitable for use in cell therapy within 4 days from when the process to stimulate or activate the cells is initiated. In some aspects, the resulting engineered cell compositions are composed of cell population that are less differentiated, less exhausted, and more potent than engineered T cell compositions generated by other means, such as by processes that involve expanding the cells. Also provided are compositions of T cells generated by the provided methods and their uses for treating subjects.

Inventors

  • WESTOBY, Matthew
  • CHAN, CALVIN
  • BRIGGS, ADRIAN WRANGHAM
  • Kugler, David G.
  • GERMEROTH, LOTHAR
  • STEMBERGER, Christian
  • POLTORAK, Mateusz Pawel
  • VARUN, Divya
  • BASHOUR, Keenan
  • CASPARY, Robert Guy

Assignees

  • Juno Therapeutics, Inc.

Dates

Publication Date
20260506
Application Date
20190809

Claims (10)

  1. A method for producing a composition of engineered T cells, the method comprising: (a) exposing an input composition comprising primary T cells with a stimulatory reagent comprising a bead having attached thereto (i) a primary agent that is an antibody that specifically binds to CD3, or is an antigen-binding fragment thereof and (ii) a secondary agent that is an antibody that specifically binds to CD28, or is an antigen-binding fragment thereof, wherein the ratio of beads to cells is less than 3:1 and the exposing is carried out under conditions to stimulate T cells, thereby generating a stimulated population; (b) introducing into T cells of the stimulated population, a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; (c) incubating the population of transformed cells, wherein the incubating is performed in serum free media, and wherein the cells are incubated in the presence of one or more cytokines, and wherein the one or more cytokines is or includes recombinant IL-2; (d) harvesting T cells of the transformed population, wherein the harvesting is carried out at a time between 48 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated; thereby producing a composition of engineered cells; and (e) formulating cells of the composition of engineered cells for cryopreservation and/or administration to a subject.
  2. The method of claim 1, wherein: a) the stimulation is performed in serum free media; b) the stimulating conditions include culturing the cells with and/or in the presence of one or more cytokines, optionally wherein the one or more cytokines is or includes IL-2; and/or c) the cells are stimulated under stimulating conditions in the presence of recombinant IL-2, IL-7, and IL-15.
  3. The method of claim 1 or claim 2, wherein: a) the introducing is carried out in serum free media; b) the introducing is carried out in the presence of one or more cytokines, optionally wherein the one or more cytokines is or includes IL-2; and/or c) the introducing is performed in the presence of IL-2, IL-7, and IL-15.
  4. The method of any one of claims 1-3, wherein the cells are incubated in the presence of recombinant IL-2, IL-7, and IL-15.
  5. The method of any one of claims 1-4, wherein the serum free media comprises: 0.5 mM to 5 mM of a dipeptide form of L-glutamine in a base media; 0.5 mM to 5 mM L-glutamine; and at least one protein.
  6. The method of any one of claims 1-5, wherein the recombinant protein is an anti-CD19 chimeric antigen receptor (CAR).
  7. The method of claim 6, wherein the CAR comprises an extracellular domain comprising an antigen-binding domain, a spacer and/or a hinge region, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling region and wherein the extracellular domain comprising an antigen-binding domain comprises a single chain variable fragment (scFv).
  8. The method of claim 7, wherein: a) the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3-zeta (CD3ζ) chain, or a signaling portion thereof and/or wherein the costimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof; and/or b) the intracellular region comprises: i) an intracellular costimulatory signaling region or domain of CD137 (4-1BB) or functional variant or portion thereof, wherein the intracellular region comprises the sequence of amino acids set forth in SEQ ID NO:12 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12; and/or ii) a human CD3-zeta stimulatory signaling domain or functional variant thereof, wherein the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.
  9. The method of claim 7 or claim 8, wherein: a) the scFv is derived from FMC63; b) the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or c) the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:57 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57 and/or the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.
  10. The method of any one of claims 7-9, wherein: (a) the spacer contains the hinge only spacer set forth in SEQ ID NO:1, and is encoded by the sequence set forth in SEQ ID NO: 2; and/or (b) the transmembrane domain is a transmembrane domain of human CD28 or variant thereof or is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9.

Description

Cross-Reference to Related Applications This application claims priority from U.S. provisional application No. 62/716,981 filed August 9, 2018, entitled "Processes for Generating Engineered Cells and Compositions Thereof," U.S. provisional application No. 62/742,249 filed October 5, 2018, entitled "Processes for Generating Engineered Cells and Compositions Thereof," U.S. provisional application No. 62/798,433 filed January 29, 2019, entitled "Processes for Generating Engineered Cells and Compositions Thereof," U.S. provisional application No. 62/842,402 filed May 2, 2019, entitled "Processes for Generating Engineered Cells and Compositions Thereof," and U.S. provisional application No. 62/861,308 filed June 13, 2019, entitled "Processes for Generating Engineered Cells and Compositions Thereof," the contents of which are incorporated by reference in their entirety. Incorporation by Reference of Sequence Listing The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042017440SeqList.txt, created on August 5, 2019, which is 93,105 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety. Field The present disclosure provides processes for genetically engineering T cells, such as primary CD4+ T cells and/or CD8+ T cells, for use in cell therapy that does not involve expanding the cells. In particular aspects, the provided processes successfully generate compositions of engineered T cells, such as populations of engineered T cells, which express a chimeric antigen receptor (CAR) within a shortened amount of time as compared to alternative engineering processes, such as processes that involve expanding the cells. In certain aspects, the provided processes successfully generate a composition of engineered T cells suitable for use in cell therapy within 4 days from when the process to stimulate or activate the cells is initiated. In some aspects, the resulting engineered cell compositions are composed of cell population that are less differentiated, less exhausted, and more potent than engineered T cell compositions generated by other means, such as by processes that involve expanding the cells. Also provided are compositions of T cells generated by the provided methods and their uses for treating subjects. Background Various cell therapy methods are available for treating diseases and conditions. Among cell therapy methods are methods involving immune cells, such as T cells, genetically engineered with a recombinant receptor, such as chimeric antigen receptors. However, in some cases, some of the existing processes for generating genetically engineered cell compositions may be time consuming or may vary in the amount of time required for successful completion. In certain cases, some of the existing processes may result in a composition in which the cell population has a low potency or persistence in vivo. Improved methods for manufacturing and/or engineering such cell therapies are needed, including to provide for a more efficient process and/or an improved cell composition product. Summary Provided herein is a method for producing a composition of engineered T cells, the method comprising: (a) exposing an input composition comprising primary T cells with a stimulatory reagent under conditions to stimulate T cells, thereby generating a stimulated population; (b) introducing into T cells of the stimulated population, a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting T cells of the transformed population, wherein the harvesting is carried out: (i) at a time between 24 and 120 hours, or between 1 day and 5 days, or between about 1 day and about 5 days, inclusive, after the exposing to the stimulatory reagent is initiated; (ii) at a time when integrated vector is detected in the genome but prior to achieving a stable integrated vector copy number (iVCN) per diploid genome; (iii) at a time before the total number of viable cells at the harvesting is more than or more than about three times, two times, or the same or about the same as the number of total viable cells of the stimulated population; and/or (iv) at a time when the percentage of CD27+CCR7+ is greater than or greater than about 60% among total T cells in the population, total CD3+ T cells in the population, total CD4+ T cells in the population, or total CD8+ T cells, or of recombinant protein-expressing cells thereof, in the population; thereby producing a composition of engineered cells. In some embodiments, the input composition comprises at least 300 x 106 viable primary T cells. In certain embodiments, the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more co