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JP-2026076152-A - Modified cells and therapeutic methods

JP2026076152AJP 2026076152 AJP2026076152 AJP 2026076152AJP-2026076152-A

Abstract

[Problem] To provide a genetically modified composition for treating cancer, and a method for producing a genetically modified composition and using it in the treatment of cancer. [Solution] The compositions and methods disclosed herein can be used to identify cancer-specific T cell receptors (TCRs) that recognize unique immunogenic mutations in a patient's cancer and to treat any type of cancer in the patient. Insertion of these transgenes encoding cancer-specific TCRs into T cells, using non-viral methods (e.g., CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL), is a novel technique that opens up new opportunities to extend immunotherapy to many cancer types. [Selection Diagram] Figure 1

Inventors

  • モリアリティ， ブランデン
  • ウェバー， ビュー
  • マッキバー， アール． スコット
  • ラーガエスパーダ， デイビッド
  • チョードリー， モダシール
  • ローゼンバーグ， スティーブン エー．
  • パーマー， ダグラス シー．
  • レスティフォ， ニコラス ピー．

Assignees

  • リージェンツ オブ ザ ユニバーシティ オブ ミネソタ
  • インティマ・バイオサイエンス，インコーポレーテッド
  • ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ

Dates

Publication Date
20260511
Application Date
20251222
Priority Date
20150731

Claims (20)

  1. a. Lymphocytes derived from human subjects; b. A polynucleotide targeting polynucleotide that has been engineered to hybridize to a specific region of a target gene within the genome of the lymphocyte; c. A nuclease capable of associating with a polynucleic acid that targets the polynucleic acid to form a nuclear protein complex, wherein the nuclear protein complex is a nuclease capable of causing targeted double-strand breaks of the target gene in the genome of the lymphocyte; d. A target polynucleic acid which is genomic DNA containing a double-strand break within the target gene, wherein the double-strand break within the target gene results in the disruption of the target gene function, and when the nuclear protein complex is brought into contact with a population of primary lymphocytes, the disruption of the target gene function occurs with an efficiency of at least 60%, Expanding on this, it is possible to create a clonal population of lymphocytes in which the function of the target gene has been altered, and the clonal population of lymphocytes is a genetically modified immune cell suitable for administration to humans who need it.
  2. The endogenous CISH (cytokine-inducible SH2-containing) gene is disrupted in its sequence, and at least one further disruption occurs within the endogenous gene, wherein the endogenous gene is related to adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related protein (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), and KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains). Modified primary cells selected from the group consisting of long cytoplasmic tails, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS1), and chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5).
  3. a) with at least one exogenous T cell receptor (TCR); b) with at least one genomic disruption of programmed death ligand 1 (PD-1); c) comprising at least one genomic disruption of the TCR alpha (TCRA) chain gene and the TCR beta (TCRB) chain gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using the CRISPR endonuclease system.
  4. a. At least one exogenous T cell receptor (TCR) sequence; b. At least one nucleic acid that targets a nucleic acid nuclease complex, i. A nucleic acid that targets nucleic acids, comprising at least one sequence substantially complementary to a target genome sequence; and ii. A cell comprising nucleic acids, comprising an exogenous endonuclease.
  5. With at least one exogenous T cell receptor (TCR) sequence; At least one complex, a. A manipulated cell comprising at least one manipulated polynucleic acid having at least one genome sequence and a sequence complementary to it; and b. at least one complex comprising at least one exogenous endonuclease.
  6. a. At least one exogenous T cell receptor (TCR); b. With at least one genomic disruption of programmed death ligand 1 (PD-1); c. comprising at least one genomic disruption of at least one endogenous gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using the CRISPR system.
  7. A modified cell comprising at least one gene disruption and at least one non-viral T-cell receptor (TCR) sequence, wherein the gene is disrupted by the non-viral TCR sequence.
  8. Cells according to any of the preceding claims, which have been manipulated using CRISPR nuclease.
  9. Cells according to any of the preceding claims, which are manipulated using an AAV vector.
  10. The manipulated cell according to any one of the preceding claims, further comprising an exogenous receptor.
  11. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs).
  12. A pharmacological composition comprising the cells described in any of the preceding claims.
  13. A cell according to any of the preceding claims, which can expand more than 40 times in 12 days.
  14. A cell that is a TIL, as described in any of the preceding claims.
  15. A cell that is an immune cell, as described in any of the preceding claims.
  16. A human cell, as described in any of the preceding claims.
  17. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs).
  18. A method for treating a patient in need thereof, comprising the step of administering the cells described in any of the preceding claims.
  19. At least one guide RNA that binds to the endogenous CISH (cytokine-inducible SH2-containing) gene, and adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), A composition comprising a secondary guide RNA that binds to an endogenous gene selected from: chloroplast cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5).
  20. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs).

Description

Cross-reference This application claims the interests of U.S. Provisional Application No. 62/199,905 filed on 31 July 2015; No. 62/232,983 filed on 25 September 2015; No. 62/286,206 filed on 22 January 2016; No. 62/295,670 filed on 16 February 2016; No. 62/330,464 filed on 2 May 2016; and No. 62/360,245 filed on 8 July 2016, all of which are incorporated herein by reference in their entirety. Background Despite the remarkable progress in cancer treatment over the past 50 years, many tumor types still exist that are resistant to chemotherapy, radiation therapy, or biotherapy, especially in advanced stages, and cannot be treated surgically. In recent years, molecular targets on tumors have been used in Remarkable advances in the genetic engineering of vivo-recognized lymphocytes have resulted in a remarkable number of cases of targeted tumor remission. However, these successes have been largely limited to hematopoietic malignancies, and broader application to solid tumors is limited due to the lack of identifiable molecules expressed by cells within specific tumors, and the lack of molecules that can be used to specifically bind to tumor targets in order to mediate tumor destruction. Some recent advances have focused on identifying tumor-specific mutations that, in some cases, induce antitumor T cell responses. For example, these endogenous mutations can be identified using whole-exome sequencing (Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer," Science, vol. 344: pp. 641-644 (2014)). Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer," Science, Vol. 344: pp. 641-644 (2014). Figure 1 is a diagram showing an overview of some of the methods disclosed herein. Figure 2 shows several exemplary transposon constructs for the integration of the TCR transgene and the expression of TCR. Figure 3 illustrates the transcription of mRNA in vitro and its use as a template for producing homologous recombination (HR) substrates in any cell type (e.g., primary cells, cell lines, etc.). For in vitro transcription of the viral cassette, T7, T3, or other transcription start sequences can be placed upstream of the 5' LTR region of the viral genome. Yields can be improved by using mRNA encoding both the sense and antisense strands of the viral vector. Figure 4 shows the structures of four plasmids, including the Cas9 nuclease plasmid, the HPRT gRNA plasmid, the Amaxa EGFPmax plasmid, and the HPRT target vector. Figure 5 shows an exemplary HPRT target vector with a 0.5 kb targeting arm. Figure 6 illustrates three potential TCR trans gene knock-in designs targeting an exemplary gene (e.g., the HPRT gene): (1) exogenous promoter: transcribing the TCR trans gene ("TCR") by an exogenous promoter ("promoter"); (2) in-frame splice acceptor (SA) transcription: transcribing the TCR trans gene by an endogenous promoter via splicing (indicated by the arrow); and (3) in-frame fusion translation: transcribing the TCR trans gene by an endogenous promoter via in-frame translation. All three exemplary designs can knock out gene function. For example, when knocking out the HPRT gene or the PD-1 gene by insertion of a TCR trans gene, 6-thioguanine selection can be used as a selection assay. Figure 7 illustrates that co-transfection of Cas9 + gRNA + target plasmid resulted in good transfection efficiency within the bulk population. Figure 8 shows the results of the EGFP FACS analysis for CD3+ T cells, supporting the findings. Figure 9 shows two types of T cell receptors. Figure 10 shows the achievement of T cell transfection efficiency using two platforms. Figure 11 shows the efficient transfection when the number of T cells is scaled up, for example, when the number of T cells is increased. Figure 12 shows the percentage of gene modification caused by CRISPR gRNA at potential target sites. Figure 13 is a diagram that supports the CRISPR-induced DSB in stimulated T cells. Figure 14 shows the optimization of RNA delivery. Figure 15 illustrates the double-strand break at the target site. Gene targeting successfully induced double-strand breaks in T cells activated with anti-CD3 and anti-CD28 before introducing the targeted CRISPR-Cas system. For illustrative purposes, the system was validated using the immune checkpoint genes PD-1, CCR5, and CTLA4. Figure 16 shows the integration of TCR in CCR5. It is an exemplary design of a plasmid targeting vector for CCR5 with a 1 kb recombination arm. To target other target genes using homologous recombination, a 3 kb TCR expression trans gene can be inserted into a similar vector with a recombination arm to a different gene. Successful TCR integration in the gene can be confirmed by PCR analysis using primers outside the recombination arm. Figure 17 illustrates the integration of TCRs into the CCR5 gene within stimulated T cells. The positive PCR result confirms the success of homologous