JP-7855622-B2 - T cell production composition and method

Inventors

• マリト エム． ファン ビューレン
• ディヴィヤ レディ レンカラ
• ジェシカ コーラー
• フラヴィアン デュヴァル ブラウン
• クリスティーナ マーフィー ククシン
• ヨースト ヘイベルト ファン デン ベルフ
• レナーテ デ ブール
• ノール バッケル
• トン シューマッハー

Assignees

• ビオンテック ユーエス インコーポレイテッド
• スティヒティング・ヘット・ネーデルランズ・カンカー・インスティテュート－アントニ・ファン・レーウェンフック・ゼィークンホイス

Dates

Publication Date

20260508

Application Date

20240129

Priority Date

20190508

Claims (20)

1. The use of an enlarged population of cells, including tumor antigen-specific T cells, in the manufacture of a pharmaceutical product for treating cancer in a subject, The tumor antigen-specific T cells include stimulated T cells that have been augmented in vitro from a peripheral blood mononuclear cell (PBMC) sample derived from the subject, and the PBMC sample includes a first population of antigen-presenting cells (APCs) and T cells, and the first population of antigen-presenting cells (APCs) and T cells is (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, (B) Incubated for a first period in the presence of a polynucleotide encoding the polypeptide, thereby forming a population of cells including stimulated T cells, The first population of APCs and T cells is a population of immune cells derived from a sample containing APCs and T cells from which CD25+ cells and/or CD14+ cells have been depleted. The tumor antigen-specific T cells include T cells that are specific to a complex comprising the at least one tumor antigen epitope sequence and the MHC protein expressed by the human target cancer cells or APC. Here, (i) At least 0.1% of the CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells, are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells, or (ii) At least 0.1% of the CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells, are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells, (a) The cancer is an unresectable melanoma, (b) The cancer is in a subject who has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or (c) Use in which the cancer is in a subject who has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least three months and has a stable disease or an asymptomatic progressive disease.
2. The use according to claim 1, wherein the sample is washed and/or cryopreserved peripheral blood mononuclear cells (PBMCs).
3. The use according to claim 1 or 2, wherein the enlarged population of cells including tumor antigen-specific T cells comprises a total of 1 × 10⁸ to 1 × 10¹¹ cells.
4. The population of cells, including stimulated T cells, in the first period, (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) mRNA encoding a polypeptide containing at least two different tumor antigen epitope sequences expressed by cancer cells of human subjects with cancer. The use according to any one of claims 1 to 3 , comprising a first population of APCs and T cells incubated in the presence of .
5. The use according to any one of claims 2 to 4 , wherein the washed and/or cryopreserved PBMC sample directly depletes CD14+ cells and CD25+ cells.
6. The use according to claim 4 or 5 , wherein the mRNA comprises a 5' CAP and a poly-A tail.
7. The use according to claim 6 , wherein the 5'CAP is CAP-1 and the polyA tail comprises 120 to 135 nucleotide residues.
8. The use according to any one of claims 4 to 7, wherein the polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer comprises at least three, four, five, six, seven, eight , nine, ten or more different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer.
9. The use according to any one of claims 4 to 8 , wherein each of the at least two different tumor antigen epitope sequences is 8 to 12 amino acid long or 15 to 25 amino acid long.
10. The use according to any one of claims 1 to 9, wherein the percentage of CD3+ cells in the enlarged population of cells including tumor antigen-specific T cells is at least 40%, at least 50%, or at least 60 % of the total cells in the enlarged population of cells.
11. (a) The percentage of CD107a+ cells in the enlarged population of cells, including tumor antigen-specific T cells, is at least 10% of the tumor antigen-specific T cell population; (b) The percentage of TNFα+ cells in the enlarged population of cells, including tumor antigen-specific T cells, is at least 5% of the tumor antigen-specific T cell population; (c) The percentage of IFNγ+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 15% of the tumor antigen-specific T cell population; (d) The percentage of TNFα+ and IFNγ+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 2% of the tumor antigen-specific T cell population; (e) The percentage of TNFα+ and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 0.5% of the tumor antigen-specific T cell population; (f) The percentage of IFNγ+ and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 5% of the tumor antigen-specific T cell population; (g) The percentage of TNFα+, IFNγ+, and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 0.1% of the tumor antigen-specific T cell population. The use described in any one of claims 1 to 10 .
12. (a) The percentage of CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (b) The percentage of CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+), is at most 5%; (c) The percentage of CD4+ T cells in the enlarged population of cells, including tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-), is at least 10%; (d) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+), is at most 25%; (e) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (f) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+), is at most 10%; and/or (g) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are central memory T cells (CD62L- and CD45RA-), is at least 15%. The use described in any one of claims 1 to 1 .
13. The use according to any one of claims 1 to 1 or 2 , wherein the first population of APCs and T cells is a population of immune cells derived from a biological sample in which CD11b+ cells and/or CD19+ cells have been further depleted.
14. The use according to any one of claims 4 to 13, wherein the at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain, and the first tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences is linked to the second tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences via a linker sequence.
15. The aforementioned subject is, (i) refractory to anti-checkpoint inhibitor therapy; (ii) being between 18 and 75 years of age; and/or, (iii) Having a mutation in the BRAF gene and having previously received B-raf inhibitor or B-raf/MEK combination therapy, The use described in any one of claims 1 to 14 .
16. The use according to any one of claims 1 to 15 , wherein the incubation and growth period of the first population of APCs and T cells is less than 28 days.
17. An improved ex vivo method for preparing tumor antigen-specific T cells, (a) Deplete CD14+ cells and/or CD25+ cells directly from washed and/or cryopreserved peripheral blood mononuclear cell (PBMC) samples derived from human subjects, This involves the step of forming a CD14 and/or CD25 depleted population of PBMCs, which includes a first population of APCs and T cells, (b) The first population of APCs and T cells from step (a) is subjected to a first period of time. (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, or (B) Incubating in the presence of a polynucleotide encoding the polypeptide, This involves the step of forming a population of cells including stimulated T cells, (c) augmenting the stimulated T cells from step (b) to form an augmented population of cells including tumor antigen-specific T cells, The tumor antigen-specific T cells are A method comprising (i) the at least one tumor antigen epitope sequence from step (b), and (ii) T cells specific to a complex comprising the human target cancer cells or APC expressed by step (b).
18. Step (b) above is: The method according to claim 17, comprising (i) incubating the first population of APCs and T cells from step (a) for a first period in the presence of the FLT3 L and ( ii ) the polynucleotide encoding the polypeptide.
19. A composition for treating cancer in human subjects, comprising an enlarged population of cells including tumor antigen-specific T cells, The tumor antigen-specific T cells are T cells that have been augmented in vitro from a human - derived peripheral blood mononuclear cell (PBMC) sample and stimulated with tumor antigens ex vivo, wherein the PBMC sample comprises a first population of antigen-presenting cells (APCs) and T cells, and the first population of antigen-presenting cells (APCs) and T cells is subjected to the depletion of CD25+ and/or CD14+ cells , (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, (B) A population of cells containing stimulated T cells, which is incubated for a first period in the presence of a polynucleotide encoding the polypeptide. The T cells stimulated with tumor antigens in the aforementioned ex vivo are specific to a complex comprising the at least one tumor antigen epitope sequence and the MHC protein expressed by the human target cancer cells or APCs. Here, (i) At least 0.1% of the CD8+ T cells in the enlarged population of cells including the T cells stimulated with the tumor antigen in ex vivo are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells, or (ii) In the enlarged population of cells including the T cells stimulated with the tumor antigen in ex vivo, at least 0.1% of the CD4+ T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells, (a) The cancer is an unresectable melanoma, (b) The cancer is in a subject who has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or (c) A composition wherein the cancer is in a subject who has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least three months and has a stable disease or an asymptomatic progressive disease.
20. The composition according to claim 19 , wherein the PBMC sample is washed and/or cryopreserved PBMC.

Description

Cross-reference This application claims the interests of U.S. Provisional Patent Application No. 62/845,251, filed on 8 May 2019, which is incorporated herein by reference in its entirety. Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines contain either a mixture of co-existing and patient-specific antigens, either in the form of a co-existing tissue-restricted tumor antigen or a whole-tumor cell preparation. Co-existing tissue-restricted tumor antigens are, ideally, immunogenic proteins that are selectively expressed in tumors across many individuals and are generally delivered to patients as synthetic peptides or recombinant proteins. In contrast, whole-tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations, or whole mRNA. Since whole-tumor cells are isolated from the patient's own cells, the cells can contain patient-specific tumor antigens along with co-existing tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, which have rarely been used in vaccines, consisting of proteins with tumor-specific mutations (which may be patient-specific or well-shared) resulting in altered amino acid sequences. Such mutated proteins (a) are specific to tumor cells (because the mutation and its corresponding protein are found only in tumors); (b) evade central tolerance and are therefore more likely to be immunogenic; and (c) provide excellent targets for immune recognition, including both humoral and cellular immunity. Adoptive immunotherapy, or adoptive cell therapy (ACT), is the transfer of lymphocytes into a target for the treatment of disease. It is yet to be seen how adoptive immunotherapy can realize its potential to treat a wide variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency. However, most, if not all, adoptive immunotherapy strategies require T cell activation and augmentation steps to generate clinically effective, therapeutically administered T cells. Due to the inherent complexities of live cell culture and patient-to-patient variability, current techniques for generating therapeutically administered T cells, including engineered T cells, remain limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are neither easily scalable, repeatable, reliable, nor efficient, and often produce inferior T cell products that may be prone to depletion and loss of effector immune cell function. To date, engineered T cell adoptive immunotherapy has achieved only very limited success and routinely exhibits variable clinical activity. Therefore, such therapies are not suitable for broad clinical use. Therefore, there is still a need to develop compositions and methods for the enlargement and induction of antigen-specific T cells with desirable phenotypes and functions. Figure 1A shows an illustrative schematic diagram of an antigen-specific T cell production protocol. Figure 1B shows an illustrative schematic diagram of an antigen-specific T cell production protocol. Figure 1C shows an illustrative alternative schematic diagram of an antigen-specific T cell production protocol. Figure 2 shows illustrative results illustrating the percentage of antigen-specific CD8 + memory T cells induced by long or short peptides. "Bulk" indicates that the sample containing the T cells used for induction is the entire peripheral blood mononuclear cell (PBMC). "Treg - " indicates that the sample containing the T cells used for induction is PBMC with depleted CD25-expressing cells. Figure 3 shows an exemplary flow cytometry analysis illustrating the percentage of antigen-specific CD8 + naive T cells induced by the GAS7 peptide. Figure 4 shows exemplary results illustrating antigen-specific CD8 + T cell responses to peptide pools of short HIV peptides, short previously identified neoantigens (PINs), or long PINs. “Whole PBMC” indicates that the sample containing the T cells used for induction is the entire PBMC. “CD25 - PBMC” indicates that CD25 + cells have been depleted from the sample containing the T cells used for induction. Short, short peptide, or shortmer; long, long peptide, or longmer. Figure 5A shows an exemplary flow cytometry analysis of antigen-specific CD8 + naive T cell responses to a single previously identified neoantigen (PIN) under the conditions shown. Figure 5B shows an exemplary flow cytometry analysis of antigen-specific CD8 + naive T cell responses to a single previously identified neoantigen (PIN) under the conditions shown. Figure 6 shows exemplary results illustrating antigen-specific CD8 + T cell responses to the indicated peptides using PBMC samples from two human donors. Figure 7 shows exempla