JPWO2020102676A5

JPWO2020102676A5 -

Info

Publication number: JPWO2020102676A5
Application number: JP2021526487A
Authority: JP
Publication date: 2022-11-28

Description

１．分野
本発明は、医学分野、特にがんの治療のための細胞治療薬の製造に関する。 1. FIELD The present invention relates to the medical field , in particular to the production of cell therapeutics for the treatment of cancer.

２．背景
人工受容体、例えば、Ｔ細胞を特定の腫瘍関連抗原に標的化し、Ｔ細胞を活性化し、その増殖を増加させるための一次シグナル伝達及び一般的に共刺激シグナル伝達を提供するポリペプチドであるキメラ抗原受容体またはＴ細胞受容体（ＴＣＲ）を発現するＴ細胞に基づく免疫療法は、特に他の治療法を使い尽くした患者にとって、ますます有望な治療法となっている。しかしながら、自家ＣＡＲＴ細胞療法の場合、細胞の製造は依然として手間と時間を要する。結果として、当分野では、そのような細胞を製造する時間と費用を削減するように、ＣＡＲＴ細胞の製造方法を改善する必要がある。そのような改善された方法を、本明細書において提供する。 2. BACKGROUND Artificial receptors, e.g., polypeptides that target T cells to specific tumor-associated antigens, provide primary and generally co-stimulatory signaling to activate T cells and increase their proliferation. Immunotherapy based on T cells expressing chimeric antigen receptors or T cell receptors (TCRs) has become an increasingly promising treatment modality, especially for patients who have exhausted other therapies. However, for autologous CAR T-cell therapy, cell production remains laborious and time-consuming. Consequently, there is a need in the art to improve methods for producing CAR T cells so as to reduce the time and cost of producing such cells. Such improved methods are provided herein.

３．概要
第１の態様では、本明細書において、細胞を他の血液成分から単離するための膜濾過及び塩化アンモニウム－カリウム（ＡＣＫ）緩衝液を組み込んだ方法を使用して、血液から取得することができる細胞を製造する方法を提供する。第１の実施形態では、本明細書において、血液試料が得られる対象由来の末梢血単核球（ＰＢＭＣ）から細胞を製造する方法を提供し、方法は：（ａ）例えば、白血球除去または採血を使用して血液試料から血球を得て末梢血単核球（ＰＢＭＣ）を取得し；（ｂ）場合により血球を凍結して解凍後にＰＢＭＣを取得するか、または場合によりＰＢＭＣを取得して凍結し、その後の工程の前に解凍し；（ｃ）膜濾過系及び塩化アンモニウム－カリウム（ＡＣＫ）緩衝液を使用してＰＢＭＣを単離し；（ｄ）遠心分離によりＰＢＭＣを洗浄し；（ｅ）場合によりＰＢＭＣを凍結保存し；（ｆ）工程（ｅ）でＰＢＭＣを凍結保存している場合は、場合によりＰＢＭＣを解凍し；（ｇ）膜濾過系を使用してＰＢＭＣを洗浄し；（ｈ）ＰＢＭＣから細胞を製造し；（ｉ）膜濾過系を使用して細胞を洗浄する工程を含む。特定の実施形態では、前記方法の工程（ａ）～（ｈ）を、順番に実行する。特定の実施形態では、細胞は、キメラ抗原受容体（ＣＡＲ）を発現するＴ細胞（ＣＡＲＴ細胞）である。 3. Summary In a first aspect, herein, obtaining from blood using a method incorporating membrane filtration and an ammonium chloride-potassium (ACK) buffer to isolate cells from other blood components Provided is a method for producing cells capable of In a first embodiment, provided herein is a method of producing cells from peripheral blood mononuclear cells (PBMC) from a subject from whom a blood sample is obtained, the method comprising: (a) e.g., leukapheresis or blood collection obtaining peripheral blood mononuclear cells (PBMC) by obtaining blood cells from a blood sample using (c) isolate the PBMCs using a membrane filtration system and an ammonium chloride-potassium (ACK) buffer; (d) wash the PBMCs by centrifugation; (e) (f) optionally thawing the PBMCs if they were cryopreserved in step (e); (g) washing the PBMCs using a membrane filtration system; (h) ) producing cells from PBMCs; (i) washing the cells using a membrane filtration system. In certain embodiments, steps (a)-(h) of the method are performed in sequence. In certain embodiments, the cell is a T cell that expresses a chimeric antigen receptor (CAR) (CAR T cell).

４．詳細な説明
４．１．改善された細胞生産プロセス－概要
本明細書において、改善された細胞生産プロセスが組み込まれた、細胞の製造プロセスを提供する。製造プロセスは、患者の白血球アフェレーシスまたは採血から自己末梢血単核球（ＰＢＭＣ）を単離することから開始する。 4. Detailed Description 4.1. Improved Cell Production Processes—Overview Provided herein are processes for manufacturing cells that incorporate improved cell production processes. The manufacturing process begins with the isolation of autologous peripheral blood mononuclear cells (PBMC) from patient leukapheresis or blood draw.

５．実施例
５．１．実施例１：改善されたＣＡＲ－Ｔ細胞製造プロセス
５．１．１．ベースラインプロセス
ＢＣＭＡ指向性キメラ抗原受容体（ＣＡＲ）発現Ｔ細胞のために、ベースラインＣＡＲＴ細胞製造プロセスを開発した（Ｈｏｌｌｙｍａｎｅｔａｌ．，Ｊ．Ｉｍｍｕｎｏｔｈｅｒ．２００９，３２：１６９－１８０を参照のこと）。ＰＢＭＣの単離、Ｔ細胞の活性化、形質導入、及び増殖などの製造プロセスパラメータは、当初、小スケールのＴフラスコベースのプロセスを使用して開発され、最適化された。プロセスパラメータを確立し、最適化した後、プロセスを臨床製造のためにスケールアップした。このプロセスでは、白血球アフェレーシスから開始し、続いてＣｅｌｌ－Ｓａｖｅｒ５＋（ＣＳ５＋；Ｈａｅｍｏｎｅｔｉｃｓ、ブレーンツリー、マサチューセッツ）を使用した密度勾配遠心分離により末梢血単核球（ＰＢＭＣ）を単離し；得られたＰＢＭＣのＴＣＧＭ－ＨＡＢＳ培地（化学的に定義された造血細胞培地Ｘ－ＶＩＶＯ－１５（商標）（Ｌｏｎｚａ、バーゼル、スイス）を含み、フェノールレッドを含まず、５％ｖ／ｖヒトＡＢ血清２ｍＭＧｌｕｔａＭＡＸ（商標）（Ｇｉｂｃｏ）、１０ｍＭＨＥＰＥＳ（４－（２－ヒドロキシエチル）－１－ピペラジンエタンスルホン酸（ＴｈｅｒｍｏＦｉｓｈｅｒＳｃｉｅｎｔｉｆｉｃ）、及び３００ＩＵ／ｍＬ組換えヒトインターロイキン－２（ｒｈＩＬ２）を添加したＴ細胞増殖培地）中でのバッチ遠心分離培養を使用して前培養物を洗浄した。１％ピルビン酸ナトリウムと１％最小必須ビタミンをさらに補充した同じ培地も試験し、８日間で同数の集団倍化をもたらすことがわかった。簡略化のために、ＴＣＧＭ－ＨＡＢＳをプロセスに選択した。追加の培地と比較した後、ｒｈＩＬ２の濃度を、３００ＩＵ／ｍＬから１００ＩＵ／ｍＬに低下させた。 5. Example 5.1. Example 1: Improved CAR-T Cell Manufacturing Process 5.1.1. Baseline Process A baseline CAR T cell manufacturing process was developed for BCMA-directed chimeric antigen receptor (CAR)-expressing T cells (see Hollyman et al., J. Immunother. 2009, 32:169-180). thing). Manufacturing process parameters such as PBMC isolation, T cell activation, transduction, and expansion were initially developed and optimized using a small-scale T-flask-based process. After establishing and optimizing the process parameters, the process was scaled up for clinical manufacturing. The process started with leukoapheresis followed by isolation of peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation using Cell-Saver 5+ (CS5+; Haemonetics, Braintree, MA); of TCGM-HABS medium (chemically defined hematopoietic cell medium X-VIVO-15™ (Lonza, Basel, Switzerland) containing no phenol red, 5% v/v human AB serum 2 mM GlutaMAX ( (Gibco), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher Scientific), and 300 IU/mL recombinant human interleukin-2 (rhIL2). The pre-culture was washed using batch centrifugation cultures in medium).The same medium additionally supplemented with 1% sodium pyruvate and 1% minimum essential vitamins was also tested, yielding the same number of population doublings in 8 days. TCGM-HABS was chosen for the process for simplification, and the concentration of rhIL2 was reduced from 300 IU/mL to 100 IU/mL after comparison with supplemental media.

Claims

A method of producing chimeric antigen receptor (CAR)-expressing T cells from peripheral blood mononuclear cells (PBMC) from a blood sample from a subject, comprising:
(a) isolating PBMCs from a blood sample from said subject using a membrane filtration system and an ammonium chloride-potassium (ACK) buffer;
(b) producing said CAR T cells from said PBMC; and (c) washing said cells using a membrane filtration system.

2. The method of claim 1, wherein said PBMC are separated from said ACK buffer and washed by centrifugation after step (a).

3. The method of claim 1 or 2, wherein the T cells are isolated from isolated PBMC.

4. The method of claim 3, wherein magnetic beads coated with anti-CD3, anti-CD4, or anti-CD8 antibodies are used for isolating said T cells.

A method according to any one of claims 1 to 4, wherein said T cells are cultured after their isolation.

The method of any one of claims 1-5, wherein the isolated PBMCs are cryopreserved.

The method of any one of claims 3-5, wherein said isolated T cells are cryopreserved.

8. The method of claim 7, wherein said cryopreserved T cells are thawed, washed and resuspended in a growth medium suitable for growth of such cells.

9. The method of claim 8, wherein prior to cell culturing of the thawed T cells, the thawed T cells are washed using the membrane filtration system or centrifugation.

10. The method of claim 8 or 9, wherein the growth medium suitable for expanding T cells comprises 5% (v/v) human AB serum and 100 or 300 IU/mL IL-2.

11. The method of any one of claims 3-10, wherein producing said CAR T cells comprises activating said T cells and transducing said T cells with a vector encoding a CAR. .

A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from peripheral blood mononuclear cells (PBMC) from a subject from whom a blood sample is obtained, comprising:
a. obtaining PBMCs from said blood sample;
b. isolating the PBMCs obtained from the blood sample using a membrane filtration system and an ammonium chloride-potassium (ACK) buffer;
c. washing the PBMCs by centrifugation;
d. producing CAR T cells from said PBMC of step (c); and e. The above method, comprising washing the CAR T cells of step (d) using a membrane filtration system.

13. The method of claim 12, wherein said method further comprises cryopreserving said PBMC of step (c).

14. The method of claim 13, wherein said method further comprises thawing said cryopreserved PBMC.

15. The method of claim 14, wherein said method further comprises washing thawed PBMCs using said membrane filtration system prior to producing said CAR T cells from said PBMCs.

A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from peripheral blood mononuclear cells (PBMC) from a leukodepleted blood sample from a subject, comprising:
a. isolating the PBMCs from the leukodepleted blood sample using a membrane filtration system and an ammonium chloride-potassium (ACK) buffer;
b. cryopreserving the PBMC of step (a);
c. Thawing the PBMC of step (b);
d. washing the thawed PBMC of step (c) using a membrane filtration system;
e. producing CAR T cells from said PBMC of step (d); and f. The above method, comprising washing the CAR T cells of step (e) using a membrane filtration system.

A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from peripheral blood mononuclear cells (PBMC) derived from a blood sample from a subject, comprising:
a. isolating said PBMC from said blood sample using a membrane filtration system and an ammonium chloride-potassium (ACK) buffer;
b. cryopreserving and thawing the PBMC of step (a);
c. washing the thawed PBMC of step (b) using a membrane filtration system;
d. producing CAR T cells from said PBMC; and e. Said method, comprising washing said CAR T cells using a membrane filtration system.

18. The method of any one of claims 1-17, wherein producing said CAR T cells from said PBMCs comprises transducing said cells with a vector expressing said CAR.

The method of any one of claims 1-18, wherein the ACK buffer comprises 50-300 mM ammonium chloride, 5-25 mM potassium carbonate, and 0.05-0.25 mM sodium EDTA.

The method of any one of claims 1-19, wherein the ACK buffer comprises 150 mM ammonium chloride, 10 mM potassium carbonate, and 0.1 mM sodium EDTA.

said method reduces platelet and red blood cell depletion from said PBMC by 10% to 50%, 15% to 45% compared to the same method using density gradient centrifugation instead of each use of said membrane filtration system; 20% to 40%, 20% to 35%, 20% to 30%, 25% to 35%, 25% to 30%, 25% to 40%, or 30 to 35% improvement, of claims 1 to 20 A method according to any one of the preceding claims.

Claims 1-21, wherein the method improves platelet and red blood cell depletion from the PBMC by 18% to 36% compared to the same method using density gradient centrifugation instead of each use of the membrane filtration system. A method according to any one of

said method yields about 10% to 50% recovery of said cells, e.g., CAR T cells, after T cell production compared to the same method using density gradient centrifugation in place of each use of said membrane filtration system; improve by 15%-45%, 20%-40%, 20%-35%, 20%-30%, 25%-35%, 25%-30%, 25%-40%, or 30-35%; A method according to any one of claims 1-22.

4, wherein said method improves CAR T cell recovery after CAR T cell production by 17% to 36% compared to the same method using density gradient centrifugation in place of each use of said membrane filtration system. 24. The method according to any one of 1 to 23.

The method of any one of claims 1-24, wherein any or all of said membrane filtration systems are spinning membrane filtration systems.

26. The method of claim 25, wherein the spinning membrane filtration system is a 3.6 [mu]m pore size system.

27. The method of claim 25 or 26, wherein said spinning membrane filtration system is a LOVO automated cell processing system.

19. The method of claim 11 or 18, wherein said vector is a lentiviral vector.

29. The method of any one of claims 11, 18 or 28, wherein prior to said transducing step said PBMCs or T cells are activated by incubating in the presence of a T cell activating reagent.

30. The method of claim 29, wherein said T cell activating reagent comprises antibodies against CD3 and CD28.

31. The method of any one of claims 11, 18 or 28-30, further comprising the step of incubating and growing said cells after said transducing step.

32. The method of any one of claims 1-31, wherein the CAR is an anti-BCMA CAR.