TW202208415A - Il-12 armored immune cell therapy and uses thereof - Google Patents

Il-12 armored immune cell therapy and uses thereof Download PDF

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TW202208415A
TW202208415A TW110117939A TW110117939A TW202208415A TW 202208415 A TW202208415 A TW 202208415A TW 110117939 A TW110117939 A TW 110117939A TW 110117939 A TW110117939 A TW 110117939A TW 202208415 A TW202208415 A TW 202208415A
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張璐
陳銳
喬瑟芬 Ａ 圖勒
里安農 史賓賽
布魯克 沃夫
趙麗霞
蘇震波
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大陸商廣東天科雅生物醫藥科技有限公司
美商天科雅生物醫藥有限公司
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Abstract

This disclosure relates to IL-12 armored immune cell therapies and the use of IL-12 in combination with immune cell therapies.

Description

IL-12裝甲的免疫細胞療法及其用途IL-12 Armored Immune Cell Therapy and Its Uses

發明領域Field of Invention

本公開涉及IL-12裝甲的免疫細胞療法和IL-12與免疫細胞療法組合的用途。The present disclosure relates to IL-12 armored immune cell therapy and the use of IL-12 in combination with immune cell therapy.

發明背景Background of the Invention

癌症是由生物學和環境因素(諸如年齡、性別、基因突變、環境暴露(諸如UV輻射)、職業危險因素、致癌物、石棉、放射性物質和病毒感染(例如，HPV、EBV、HBV、HCV、HTLV-1和KSHV))引起的最廣泛的細胞異常之一(Margaret E等人, “Viruses Associated With Human Cancer,” Biochimica et Biophysica Acta.1782:127–150 (2008))。Cancer is caused by biological and environmental factors (such as age, gender, genetic mutation, environmental exposure (such as UV radiation), occupational risk factors, carcinogens, asbestos, radioactive substances and viral infections (eg, HPV, EBV, HBV, HCV, HTLV-1 and KSHV)) (Margaret E et al., "Viruses Associated With Human Cancer," Biochimica et Biophysica Acta. 1782:127-150 (2008)).

CAR T細胞療法的最新臨床和商業成功已引起人們對免疫細胞療法的極大興趣。儘管癌症治療取得了進步，但各種治療對某些癌症的功效相對較差。因此，對於針對癌症的有效療法存在未滿足的需求。Recent clinical and commercial successes of CAR T-cell therapy have generated significant interest in immune cell therapy. Despite advances in cancer treatment, various treatments are relatively less effective for some cancers. Therefore, there is an unmet need for effective therapies against cancer.

發明概要Summary of Invention

本公開提供了經修飾(例如，膜拴系)或未修飾的IL-12，其可與免疫細胞療法(例如，TCR-T、CAR-T或TIL)組合使用以治療癌症(例如，實體瘤)。本公開還提供了與靶向腫瘤的抗體(例如，NHS76)融合的IL-12與免疫細胞療法(例如，TCR-T、CAR-T、CAR-NK或TIL)組合用於治療癌症的用途。經修飾的IL-12與免疫細胞療法組合使用可大大增加這些免疫細胞療法的功效；此外，這些方法具有改善的臨床使用的安全性。The present disclosure provides modified (eg, membrane-tethered) or unmodified IL-12 that can be used in combination with immune cell therapy (eg, TCR-T, CAR-T, or TIL) to treat cancer (eg, solid tumors) ). The present disclosure also provides the use of IL-12 fused to a tumor-targeting antibody (eg, NHS76) in combination with immune cell therapy (eg, TCR-T, CAR-T, CAR-NK, or TIL) for the treatment of cancer. The use of modified IL-12 in combination with immune cell therapies can greatly increase the efficacy of these immune cell therapies; in addition, these approaches have an improved safety profile for clinical use.

在一個方面，本文提供了一種細胞，其表現(a)外源T細胞受體(TCR)或嵌合抗原受體(CAR)；和(b)IL-12。In one aspect, provided herein is a cell expressing (a) a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); and (b) IL-12.

在一些實施方案中，IL-12是膜拴系IL-12。In some embodiments, the IL-12 is membrane-tethered IL-12.

在一些實施方案中，膜拴系IL-12包含CD4跨膜區。In some embodiments, the membrane-tethered IL-12 comprises a CD4 transmembrane domain.

在一些實施方案中，膜拴系IL-12包含免疫球蛋白CH2結構域、免疫球蛋白CH3結構域和CD4跨膜區。In some embodiments, the membrane-tethered IL-12 comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, and a CD4 transmembrane region.

在一些實施方案中，膜拴系IL-12包含兩個或多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和CD4跨膜區。In some embodiments, the membrane-tethered IL-12 comprises two or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a CD4 transmembrane region.

在一些實施方案中，免疫球蛋白CH2結構域是野生型免疫球蛋白恆定結構域。In some embodiments, the immunoglobulin CH2 domain is a wild-type immunoglobulin constant domain.

在一些實施方案中，免疫球蛋白CH2結構域的位置235處(EU編號)的胺基酸是Glu，並且免疫球蛋白CH2結構域的位置297處(EU編號)的胺基酸殘基是Gln。In some embodiments, the amino acid residue at position 235 (EU numbering) of the immunoglobulin CH2 domain is Glu, and the amino acid residue at position 297 (EU numbering) of the immunoglobulin CH2 domain is Gln .

在一些實施方案中，膜拴系IL-12還包含免疫球蛋白鉸鏈區。In some embodiments, the membrane-tethered IL-12 further comprises an immunoglobulin hinge region.

在一些實施方案中，免疫球蛋白CH2結構域是人免疫球蛋白CH2結構域，免疫球蛋白CH3結構域是人免疫球蛋白CH3結構域，並且CD4跨膜區是人CD4跨膜區。In some embodiments, the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain, and the CD4 transmembrane region is a human CD4 transmembrane region.

在一些實施方案中，IL-12是可溶的IL-12。In some embodiments, the IL-12 is soluble IL-12.

在一些實施方案中，IL-12與靶向腫瘤的抗體或其抗原結合片段連接。In some embodiments, IL-12 is linked to a tumor-targeting antibody or antigen-binding fragment thereof.

在一些實施方案中，靶向腫瘤的抗體或其抗原結合片段是單鏈可變片段(scFv)。In some embodiments, the tumor-targeting antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

在一些實施方案中，靶向腫瘤的抗體是NHS76。In some embodiments, the tumor-targeting antibody is NHS76.

在一些實施方案中，IL-12是人IL-12或小鼠IL-12。In some embodiments, the IL-12 is human IL-12 or mouse IL-12.

在一些實施方案中，TCR或CAR靶向BCMA、CD19、CD22、CD30、CD33、CD56、CD123(IL-3R)、CEA、IL13Ra2、ALPP、EBV相關抗原(例如，LMP2)、EGFR、EGFRvIII、GD2、GPC3、HER2、HPV相關抗原(例如，E6、E7)、MAGE(例如，MAGE-A3)、間皮素、MUC-1、NY-ESO-1、PSCA、PSMA、ROR1、WT1或Claudin 18.2。In some embodiments, the TCR or CAR targets BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (IL-3R), CEA, IL13Ra2, ALPP, EBV-associated antigens (eg, LMP2), EGFR, EGFRvIII, GD2 , GPC3, HER2, HPV-associated antigens (eg, E6, E7), MAGE (eg, MAGE-A3), mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.

在一些實施方案中，CAR包含細胞外結構域，In some embodiments, the CAR comprises an extracellular domain,

在一些實施方案中，細胞外結構域是單鏈可變片段(scFv)、配體(例如，受體結合配體)或抗體模擬物。In some embodiments, the extracellular domain is a single-chain variable fragment (scFv), a ligand (eg, a receptor binding ligand), or an antibody mimetic.

在一些實施方案中，細胞還表現趨化介素，例如CXCL10或XCL1。在一些實施方案中，細胞還表現Flt3L。In some embodiments, the cells also express chemokines, such as CXCL10 or XCL1. In some embodiments, the cells also express Flt3L.

在一個方面，本文提供了一種載體，其包含：a)第一核酸序列，其編碼IL-12α亞基和IL-12β亞基；b)第二核酸序列，其編碼一個或多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和跨膜區。在一些實施方案中，第一核酸序列和第二核酸序列通過第一接頭序列連接。In one aspect, provided herein is a vector comprising: a) a first nucleic acid sequence encoding an IL-12α subunit and an IL-12β subunit; b) a second nucleic acid sequence encoding one or more immunoglobulins A CH2 domain, one or more immunoglobulin CH3 domains, and a transmembrane region. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a first linker sequence.

在一些實施方案中，第一接頭序列編碼免疫球蛋白鉸鏈多肽序列。In some embodiments, the first linker sequence encodes an immunoglobulin hinge polypeptide sequence.

在一些實施方案中，跨膜區是CD4跨膜區。In some embodiments, the transmembrane domain is a CD4 transmembrane domain.

在一些實施方案中，跨膜區域是免疫球蛋白跨膜區。In some embodiments, the transmembrane region is an immunoglobulin transmembrane region.

在一些實施方案中，IL-12α亞基和IL-12β亞基通過接頭肽序列連接。In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence.

在一些實施方案中，載體還包含編碼訊息肽的序列。In some embodiments, the vector further comprises a sequence encoding a message peptide.

在一些實施方案中，IL-12α亞基是人IL-12α亞基，IL-12β亞基是人IL-12β亞基，免疫球蛋白CH2結構域是人免疫球蛋白CH2結構域，免疫球蛋白CH3結構域是人免疫球蛋白CH3結構域。In some embodiments, the IL-12α subunit is a human IL-12α subunit, the IL-12β subunit is a human IL-12β subunit, the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, the immunoglobulin The CH3 domain is a human immunoglobulin CH3 domain.

在一些實施方案中，載體還包含編碼T細胞受體(TCR)或嵌合抗原受體(CAR)的第三核酸序列。In some embodiments, the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

在一些實施方案中，第三核酸序列通過第二接頭序列與第一核酸連接。在一些實施方案中，第二接頭序列編碼P2A序列。In some embodiments, the third nucleic acid sequence is linked to the first nucleic acid through a second linker sequence. In some embodiments, the second linker sequence encodes a P2A sequence.

在一些實施方案中，第一核酸和第二核酸在調節元件(例如，啟動子)的控制下。In some embodiments, the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter).

在一些實施方案中，第一核酸、第二核酸和第三核酸在調節元件(例如，啟動子)的控制下。In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are under the control of a regulatory element (eg, a promoter).

在一些實施方案中，載體還包含編碼趨化介素例如CXCL10或XCL1的序列。在一些實施方案中，載體還包含編碼Flt3L的序列。In some embodiments, the vector further comprises a sequence encoding a chemokine such as CXCL10 or XCL1. In some embodiments, the vector further comprises a sequence encoding Flt3L.

在一個方面，本文提供了一種載體，其包含：a)第一核酸序列，其編碼靶向腫瘤的抗體的重鏈可變區(VH)和輕鏈可變區(VL)；b)第二核酸序列，其編碼IL-12α亞基和IL-12β亞基。在一些實施方案中，第一核酸序列和第二核酸序列通過第一接頭序列連接。In one aspect, provided herein is a vector comprising: a) a first nucleic acid sequence encoding the variable heavy (VH) and variable light (VL) regions of a tumor-targeting antibody; b) a second Nucleic acid sequences encoding the IL-12α subunit and the IL-12β subunit. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a first linker sequence.

在一些實施方案中，靶向腫瘤的抗體靶向腫瘤相關抗原。在一些實施方案中，靶向腫瘤的抗體是NHS76。In some embodiments, the tumor-targeting antibody targets a tumor-associated antigen. In some embodiments, the tumor-targeting antibody is NHS76.

在一些實施方案中，載體還包含編碼訊息肽的核酸。In some embodiments, the vector further comprises a nucleic acid encoding a message peptide.

在一些實施方案中，訊息肽是人訊息肽，IL-12α亞基是人IL-12α亞基，並且IL-12β亞基是人IL-12β亞基。In some embodiments, the message peptide is a human message peptide, the IL-12α subunit is a human IL-12α subunit, and the IL-12β subunit is a human IL-12β subunit.

在一些實施方案中，載體還包含編碼T細胞受體(TCR)或嵌合抗原受體(CAR)的第三核酸序列。In some embodiments, the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

在一些實施方案中，第三核酸序列通過第二接頭序列與第一核酸的5'連接。在一些實施方案中，第二接頭序列編碼P2A序列。In some embodiments, the third nucleic acid sequence is linked 5' to the first nucleic acid through a second linker sequence. In some embodiments, the second linker sequence encodes a P2A sequence.

在一些實施方案中，第一核酸和第二核酸在調節元件(例如，啟動子)的控制下。In some embodiments, the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter).

在一些實施方案中，第一核酸、第二核酸和第三核酸在調節元件(例如，啟動子)的控制下。In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are under the control of a regulatory element (eg, a promoter).

在一個方面，本文提供了一種載體，其以5'至3'方向包含：a)第一核酸序列，其編碼外源T細胞受體(TCR)或嵌合抗原受體(CAR)；b)第二核酸序列，其編碼訊息肽、IL-12α亞基和IL-12β亞基。在一些實施方案中，所述第一核酸序列和所述第二核酸序列通過接頭序列連接。In one aspect, provided herein is a vector comprising, in a 5' to 3' orientation: a) a first nucleic acid sequence encoding a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); b) A second nucleic acid sequence encoding the message peptide, the IL-12α subunit and the IL-12β subunit. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a linker sequence.

在一些實施方案中，IL-12α亞基和IL-12β亞基通過接頭肽序列連接。In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence.

在一些實施方案中，接頭序列編碼P2A序列In some embodiments, the linker sequence encodes a P2A sequence

在一個方面，本文提供了一種融合多肽，其包含：a)包含IL-12α亞基和IL-12β亞基的第一區。在一些實施方案中，IL-12α亞基和IL-12β亞基通過接頭肽序列連接。b)包含一個或多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和跨膜區的第二區。In one aspect, provided herein is a fusion polypeptide comprising: a) a first region comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence. b) a second region comprising one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region.

在一些實施方案中，第一區和第二區通過免疫球蛋白鉸鏈肽連接。In some embodiments, the first region and the second region are linked by an immunoglobulin hinge peptide.

在一個方面，本文提供了一種融合多肽，其包含：a)包含IL-12α亞基和IL-12β亞基的第一區。在一些實施方案中，IL-12α亞基和IL-12β亞基通過第一接頭肽序列連接。b)包含靶向腫瘤的抗體的重鏈可變區(VH)和輕鏈可變區(VL)的第二區。在一些實施方案中，VH和VL通過第二接頭肽序列連接。在一些實施方案中，第一區和第二區通過第三接頭肽序列連接。In one aspect, provided herein is a fusion polypeptide comprising: a) a first region comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a first linker peptide sequence. b) A second region comprising the variable heavy (VH) and light chain (VL) regions of the tumor-targeting antibody. In some embodiments, VH and VL are linked by a second linker peptide sequence. In some embodiments, the first region and the second region are linked by a third linker peptide sequence.

在一些實施方案中，第三接頭多肽序列具有與SEQ ID NO: 17至少80%相同的序列。In some embodiments, the third linker polypeptide sequence has a sequence that is at least 80% identical to SEQ ID NO: 17.

在一個方面，本文提供了編碼如本文所述的多肽的核酸。In one aspect, provided herein is a nucleic acid encoding a polypeptide as described herein.

在一個方面，本文提供了包含如本文所述的核酸的載體。In one aspect, provided herein is a vector comprising a nucleic acid as described herein.

在一個方面，本文提供了表現如本文所述的融合多肽的細胞。In one aspect, provided herein are cells expressing a fusion polypeptide as described herein.

在一個方面，本文提供了包含如本文所述的載體的細胞。In one aspect, provided herein is a cell comprising a vector as described herein.

在一些實施方案中，與不同之處在於細胞不包含如本文所述的載體的相同細胞相比，該細胞分泌更高位準的細胞介素。在一些實施方案中，細胞刺激該細胞附近的一種或多種細胞分泌細胞介素。在一些實施方案中，細胞介素是IFNγ。In some embodiments, the cell secretes a higher level of interferon than the same cell except that the cell does not comprise a vector as described herein. In some embodiments, the cell stimulates one or more cells in the vicinity of the cell to secrete interferons. In some embodiments, the interferon is IFNy.

在一些實施方案中，與不同之處在於細胞不包含本文所述的載體的相同細胞相比，該細胞表現更高位準的早期TCR活化標記物，和/或刺激該細胞附近的一種或多種細胞表現早期TCR活化標記物。In some embodiments, the cell exhibits a higher level of early TCR activation markers, and/or stimulates one or more cells in the vicinity of the cell, compared to the same cell except that the cell does not comprise a vector described herein Shows early TCR activation markers.

在一些實施方案中，活化標記物是CD69。In some embodiments, the activation marker is CD69.

在一些實施方案中，細胞是細胞系。In some embodiments, the cell is a cell line.

在一些實施方案中，細胞是從受試者(例如，人受試者)獲得的原代細胞。In some embodiments, the cells are primary cells obtained from a subject (eg, a human subject).

在一些實施方案中，細胞是免疫細胞(例如，淋巴細胞)。In some embodiments, the cells are immune cells (eg, lymphocytes).

在一些實施方案中，細胞是腫瘤浸潤性淋巴細胞(TIL)或NK細胞(例如，CAR-NK細胞)。In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs) or NK cells (eg, CAR-NK cells).

在一些實施方案中，細胞是T細胞。在一些實施方案中，T細胞是從人受試者中分離的。在一些實施方案中，T細胞是CD8+的。在一些實施方案中，T細胞是CD4+的。In some embodiments, the cells are T cells. In some embodiments, the T cells are isolated from a human subject. In some embodiments, the T cells are CD8+. In some embodiments, the T cells are CD4+.

在一些實施方案中，載體是表現載體、病毒載體、反轉錄病毒載體或慢病毒載體。在一些實施方案中，反轉錄病毒載體是pMP71。In some embodiments, the vector is an expression vector, a viral vector, a retroviral vector, or a lentiviral vector. In some embodiments, the retroviral vector is pMP71.

在一個方面，本文提供了用於產生細胞的方法，該方法包括將如本文所述的載體體外或離體引入細胞中。在一些實施方案中，通過轉導將載體引入細胞中。In one aspect, provided herein is a method for producing a cell, the method comprising introducing a vector as described herein into the cell in vitro or ex vivo. In some embodiments, the vector is introduced into the cell by transduction.

在一個方面，本文提供了治療患有癌症的受試者的方法，該方法包括向有需要的受試者施用有效量的如本文所述的細胞。In one aspect, provided herein is a method of treating a subject having cancer, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.

在一些實施方案中，癌症是異質癌症。在一些實施方案中，癌症是同質癌症。In some embodiments, the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.

在一些實施方案中，細胞從受試者的外周血單核細胞(PBMC)中分離。In some embodiments, the cells are isolated from peripheral blood mononuclear cells (PBMCs) of the subject.

在一個方面，本文提供了治療患有癌症的受試者的方法，該方法包括向有需要的受試者施用：a)有效量的表現T細胞受體(TCR)或嵌合抗原受體(CAR)的細胞；和b)有效量的包含IL-12和靶向腫瘤的抗體或其抗原結合片段的蛋白質。In one aspect, provided herein is a method of treating a subject with cancer, the method comprising administering to a subject in need thereof: a) an effective amount of an expressed T cell receptor (TCR) or a chimeric antigen receptor ( CAR); and b) an effective amount of a protein comprising IL-12 and a tumor-targeting antibody or antigen-binding fragment thereof.

在一些實施方案中，細胞從受試者的外周血單核細胞中分離。In some embodiments, the cells are isolated from peripheral blood mononuclear cells of the subject.

在一個方面，本文提供了治療患有癌症的人受試者的方法，該方法包括提供從人受試者或不同的人受試者收集的細胞；將如本文所述的載體引入細胞中；培養和擴增細胞；以及向受試者施用有效量的包含該細胞的組合物。In one aspect, provided herein is a method of treating a human subject having cancer, the method comprising providing cells collected from the human subject or a different human subject; introducing a vector as described herein into the cells; culturing and expanding the cells; and administering to the subject an effective amount of a composition comprising the cells.

在一些實施方案中，癌症是異質癌症。在一些實施方案中，癌症是同質癌症。In some embodiments, the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.

在一些實施方案中，細胞是外周血單核細胞(PBMC)。In some embodiments, the cells are peripheral blood mononuclear cells (PBMCs).

在一些實施方案中，細胞是腫瘤浸潤性淋巴細胞，並且載體包含編碼IL-12的核酸。在一些實施方案中，IL-12是膜拴系IL-12。In some embodiments, the cells are tumor-infiltrating lymphocytes and the vector comprises nucleic acid encoding IL-12. In some embodiments, the IL-12 is membrane-tethered IL-12.

在一些實施方案中，細胞是T細胞，並且載體包含編碼TCR或CAR的核酸。在一些實施方案中，載體還包含編碼IL-12的核酸。在一些實施方案中，IL-12是膜拴系IL-12。In some embodiments, the cells are T cells and the vector comprises nucleic acid encoding a TCR or CAR. In some embodiments, the vector further comprises a nucleic acid encoding IL-12. In some embodiments, the IL-12 is membrane-tethered IL-12.

如本文所用，術語“基因工程化細胞”或“遺傳修飾的細胞”是指在細胞中具有核酸序列的修飾的細胞，包括但不限於在其基因組中具有一個或多個核苷酸的***、缺失、取代、或修飾的細胞、和/或具有外源核酸序列(例如載體)的細胞，其中所述外源核酸序列不一定整合到基因組中。As used herein, the term "genetically engineered cell" or "genetically modified cell" refers to a cell having a modification of a nucleic acid sequence in the cell, including but not limited to having an insertion of one or more nucleotides in its genome, Deleted, substituted, or modified cells, and/or cells with exogenous nucleic acid sequences (eg, vectors) that are not necessarily integrated into the genome.

如本文所用，術語“膜拴系IL-12”是指呈拴系到細胞膜的修飾形式的IL-12。As used herein, the term "membrane-tethered IL-12" refers to a modified form of IL-12 that is tethered to the cell membrane.

如本文所用，術語“外周血細胞”是指通常在外周血中發現的細胞，包括但不限於嗜酸性粒細胞、嗜中性粒細胞、T細胞、單核細胞、K細胞、粒細胞和B細胞。As used herein, the term "peripheral blood cells" refers to cells commonly found in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, K cells, granulocytes, and B cells .

如本文所用，術語“癌症”或“癌細胞”是指以不受控制的方式***的細胞。此類細胞的實例包括具有以迅速增殖的細胞生長為特徵的異常狀態或狀況的細胞。所述術語意在包括癌性生長(例如腫瘤)、致癌過程、轉移性組織、以及惡性轉化的細胞、組織或器官，而不管組織病理類型或浸潤期。癌細胞可以在血液中形成實體瘤或過多的腫瘤細胞(例如血液癌)。另選地或另外地，其可以包括所有類型的癌性生長或致癌過程、轉移性組織或惡性轉化的細胞、組織或器官，而不管組織病理類型或浸潤期。實體瘤的實例包括各種器官系統的惡性腫瘤(例如肉瘤)、腺癌和癌，諸如累及肝、肺、乳腺、淋巴、胃腸道(例如結腸)、泌尿生殖道(例如腎、尿路上皮細胞)、***和咽的那些。腺癌包括惡性腫瘤，諸如大多數結腸癌、直腸癌、腎細胞癌、肝癌、肺非小細胞癌、小腸癌和食管癌。可以通過本文所述的方法治療的癌症的實例包括例如骨癌、胰腺癌、皮膚癌(例如黑色素瘤)、頭或頸癌、皮膚或眼內惡性黑色素瘤、子宮癌、卵巢癌、直腸癌、肛區癌、胃癌、睾丸癌、子宮癌、輸卵管癌、子宮內膜癌、子宮頸癌、***癌、外陰癌、霍奇金病、非霍奇金淋巴瘤、食管癌、小腸癌、內分泌系統癌、甲狀腺癌、甲狀旁腺癌、腎上腺癌、軟組織肉瘤、尿道癌、陰莖癌、慢性或急性白血病(包括急性髓樣白血病、慢性髓樣白血病、急性淋巴細胞性白血病、慢性淋巴細胞性白血病)、淋巴細胞性淋巴瘤、膀胱癌、腎或輸尿管癌、腎盂癌、中樞神經系統(CNS)贅生物、原發性CNS淋巴瘤、腫瘤血管生成、脊髓軸腫瘤、腦幹神經膠質瘤、垂體腺瘤、卡波西氏肉瘤、表皮樣癌、鱗狀細胞癌、和/或T細胞淋巴瘤。As used herein, the term "cancer" or "cancer cell" refers to a cell that divides in an uncontrolled manner. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growths (eg, tumors), oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. Cancer cells can form solid tumors or excess tumor cells in the blood (eg, blood cancers). Alternatively or additionally, it may include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasion. Examples of solid tumors include malignancies of various organ systems (eg, sarcomas), adenocarcinomas, and carcinomas, such as involving the liver, lung, breast, lymph, gastrointestinal tract (eg, colon), genitourinary tract (eg, kidney, urothelial cells) , prostate and pharynx. Adenocarcinomas include malignancies such as most colon, rectal, renal cell, liver, non-small cell lung, small bowel, and esophageal cancers. Examples of cancers that can be treated by the methods described herein include, for example, bone cancer, pancreatic cancer, skin cancer (eg, melanoma), head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia ), lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary gland Adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and/or T-cell lymphoma.

如本文所用，術語“載體”是指可以通過其將多核苷酸序列(例如外來基因)引入宿主細胞中以便獲得所引入的核苷酸序列的所需基因表現的運載體。選殖載體可以包括例如質體、噬菌體、病毒等。載體的最流行類型是“質體”，其是指可以將包含目的基因的額外DNA區段連接到其中的閉合環狀雙鏈DNA環。載體的另一種類型是將要運輸的核酸構建體連接到病毒基因組中的病毒載體。病毒載體能夠在它們被引入的宿主細胞中自主複製，或者可以將它們自身整合到宿主細胞的基因組中，從而與宿主基因組一起複製。此外，某些載體能夠指導與它們可操作地連接的基因的表現。此類載體在本文中被稱為“重組表現載體”或簡稱為“表現載體”。在一些實施方案中，載體是病毒載體(例如複製缺陷型反轉錄病毒、腺病毒和腺相關病毒)。As used herein, the term "vector" refers to a vehicle by which a polynucleotide sequence (eg, a foreign gene) can be introduced into a host cell so as to obtain the desired gene expression of the introduced nucleotide sequence. Cloning vectors can include, for example, plastids, phages, viruses, and the like. The most popular type of vector is the "plastid," which refers to a closed circular double-stranded DNA loop into which additional DNA segments containing the gene of interest can be ligated. Another type of vector is a viral vector that links the nucleic acid construct to be delivered into the viral genome. Viral vectors are capable of replicating autonomously in the host cell into which they are introduced, or they can integrate themselves into the genome of the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In some embodiments, the vector is a viral vector (eg, replication-defective retrovirus, adenovirus, and adeno-associated virus).

如本文所用，“受試者”是哺乳動物，諸如人或非人動物。在一些實施方案中，向其施用細胞、細胞群或組合物的受試者(例如患者)是哺乳動物，通常是靈長類動物，諸如人。在一些實施方案中，靈長類動物是猴子或猿。受試者可以是男性或女性，並且可以是任何合適的年齡，包括嬰兒、少年、青少年、成年和老年受試者。在一些實施方案中，受試者是非靈長類哺乳動物，諸如狗、貓、馬、齧齒動物、大鼠或小鼠。As used herein, a "subject" is a mammal, such as a human or non-human animal. In some embodiments, the subject (eg, patient) to which the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. Subjects can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a dog, cat, horse, rodent, rat or mouse.

如本文所用，術語“約”是指可測量的值，諸如量、持續時間等，並且涵蓋指定值的±20%、±10%、±5%、±1%、±0.5%或±0.1%的變化。As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and encompasses ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified value The change.

除非另有定義，否則本文所用的所有技術和科學術語均具有與本發明所屬領域的普通技術人員通常所理解的相同的含義。本文描述了用於在本發明中使用的方法和材料，也可以使用本領域已知的其他合適的方法和材料。材料、方法和實例僅僅是說明性的，而不意圖進行限制。本文提及的全部出版物、專利申請、專利、序列、資料庫條目和其他參考文獻均全文以引用方式併入。在發生衝突的情況下，以本說明書(包括定義)為准。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

根據以下具體實施方式和附圖以及根據申請專利範圍，本發明的其他特徵和優點將顯而易見。Other features and advantages of the present invention will be apparent from the following detailed description and drawings, and from the scope of the claims.

較佳實施例之詳細說明DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

人免疫系統能夠識別和消除已被感染或損傷的細胞以及已經癌變的那些細胞。免疫細胞療法利用了人免疫系統的優勢，並且正在徹底改變癌症療法。其涉及將免疫細胞轉移到患者體內。所述細胞最常見源自免疫系統，並且可以來源於患者或另一個個體。在自體癌症免疫療法中，從患者提取免疫細胞，將所述免疫細胞進行遺傳修飾和體外培養，並且再返回到同一患者。比較而言，同種異體療法涉及從供體受試者分離並擴增的細胞。The human immune system is able to recognize and eliminate cells that have been infected or damaged as well as those cells that have become cancerous. Immune cell therapy takes advantage of the human immune system and is revolutionizing cancer therapy. It involves the transfer of immune cells into a patient. The cells are most commonly derived from the immune system, and can be derived from a patient or another individual. In autologous cancer immunotherapy, immune cells are extracted from a patient, genetically modified and cultured in vitro, and returned to the same patient. In comparison, allogeneic therapy involves cells isolated and expanded from a donor subject.

免疫細胞療法中使用了許多不同種類的免疫細胞。這些細胞療法包括例如腫瘤浸潤性淋巴細胞(TIL)療法、工程化T細胞受體(TCR)療法、嵌合抗原受體(CAR)T細胞療法、和自然殺傷(NK)細胞療法。Many different kinds of immune cells are used in immune cell therapy. These cell therapies include, for example, tumor infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, and natural killer (NK) cell therapy.

癌症患者具有天然存在的T細胞，其通常能夠靶向他們的癌細胞。腫瘤浸潤性淋巴細胞(TIL)療法涉及從患者收穫這些天然存在的T細胞，然後活化和擴增它們。然後將這些活化的T細胞再輸注到患者中。相比之下，工程化T細胞受體(TCR)療法涉及從患者分離T細胞，但代替僅活化和擴增可用的抗腫瘤T細胞，還用編碼新的T細胞受體的載體轉染T細胞，所述受體允許T細胞靶向由主要組織相容性複合體(MHC)呈遞的特異性癌症抗原。嵌合抗原受體(CAR)T細胞療法類似於TCR療法。在CAR T細胞療法中，用編碼嵌合抗原受體的載體轉染所述細胞。嵌合抗原受體可以結合癌症抗原，並且不需要癌症抗原由MHC呈遞。一些其他免疫細胞也可以用於這些細胞療法。例如，天然殺傷細胞也可以用編碼嵌合抗原受體的載體轉染。Cancer patients have naturally occurring T cells that are often able to target their cancer cells. Tumor-infiltrating lymphocyte (TIL) therapy involves harvesting these naturally occurring T cells from the patient and then activating and expanding them. These activated T cells are then reinfused into the patient. In contrast, engineered T cell receptor (TCR) therapy involves isolating T cells from patients, but instead of merely activating and expanding available antitumor T cells, T cells are also transfected with vectors encoding novel T cell receptors. cells that allow T cells to target specific cancer antigens presented by the major histocompatibility complex (MHC). Chimeric antigen receptor (CAR) T cell therapy is similar to TCR therapy. In CAR T cell therapy, the cells are transfected with a vector encoding a chimeric antigen receptor. Chimeric antigen receptors can bind cancer antigens and do not require the cancer antigen to be presented by the MHC. Some other immune cells can also be used in these cell therapies. For example, natural killer cells can also be transfected with vectors encoding chimeric antigen receptors.

本公開顯示，免疫細胞療法可與白細胞介素-12(IL-12)協同組合。然而，IL-12施用通常與嚴重毒性相關，包括例如血液學毒性、貧血、淋巴細胞減少症、嗜中性粒細胞減少症、肌肉和肝毒性，甚至在一些情況下死亡。IL-12施用的毒性極大地限制了其在免疫細胞療法中的使用。The present disclosure shows that immune cell therapy can be synergistically combined with interleukin-12 (IL-12). However, IL-12 administration is often associated with severe toxicity, including, for example, hematological toxicity, anemia, lymphopenia, neutropenia, muscle and liver toxicity, and even death in some cases. The toxicity of IL-12 administration greatly limits its use in immune cell therapy.

本公開提供了一種改進的免疫細胞療法，其中工程化細胞(例如，免疫細胞、T細胞、NK細胞、腫瘤浸潤性細胞)以受控方式產生IL-12並且將IL-12直接遞送至靶位點，由此顯著提高功效和安全性。在一個方面，IL-12是膜栓系IL-12，其進一步限制IL-12對局部靶區域的作用。在另一方面，IL-12與作為靶向腫瘤壞死中心的抗體的NHS76的scFv綴合。The present disclosure provides an improved immune cell therapy in which engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) produce IL-12 in a controlled manner and deliver IL-12 directly to a target site points, thereby significantly improving efficacy and safety. In one aspect, IL-12 is membrane-tethered IL-12, which further limits the effects of IL-12 on localized target areas. In another aspect, IL-12 was conjugated to the scFv of NHS76 as an antibody targeting the tumor necrosis center.

IL-12的表現進一步提高免疫細胞療法的功效。抗原逃逸和下調(例如，抗原損失)已作為影響免疫細胞療法的持久性的主要問題出現。特別地，在實體瘤中，大多數抗原以較低位準和/或更異質的方式表現。由於異質性，某些腫瘤細胞可通過減少特異性抗原的表現而逃逸靶向該抗原的免疫細胞療法。免疫細胞療法的免疫壓力可通過調節癌細胞的靶抗原的表現來驅動癌細胞進化，這通過損失可檢測的抗原或將抗原的表現降低至低於免疫細胞活性所需的閾值的位準來實現。一旦大多數腫瘤細胞被殺死，那些剩餘的對免疫細胞療法有抗性的腫瘤細胞快速生長，並且整個復發性實體瘤變得對免疫細胞療法有抗性。抗原損失或抗原低逃逸已成為治療實體瘤的更大障礙。抗原損失機制和相關臨床資料的細節可見於例如Majzner和Crystal，“Tumor antigen escape from CAR T-cell therapy.” Cancer discovery 8.10 (2018): 1219-1226，該文獻全文以引用方式併入本文。在工程化免疫細胞中IL-12的表現可進一步增加局部腫瘤微環境中的免疫反應(例如，刺激周圍免疫細胞)，從而殺死不表現那些抗原或表現低位準抗原的周圍癌細胞，防止任何癌細胞逃逸免疫細胞療法。The expression of IL-12 further enhances the efficacy of immune cell therapy. Antigen escape and downregulation (eg, antigen loss) have emerged as major issues affecting the durability of immune cell therapy. In particular, in solid tumors, most antigens are expressed at lower levels and/or in a more heterogeneous manner. Due to heterogeneity, certain tumor cells can escape immune cell therapy targeting a specific antigen by reducing the expression of that antigen. Immune stress from immune cell therapy can drive cancer cell evolution by modulating the expression of target antigens in cancer cells, either through the loss of detectable antigens or by reducing the expression of antigens to levels below the threshold required for immune cell activity. . Once most tumor cells are killed, those remaining resistant to immune cell therapy grow rapidly, and the entire relapsed solid tumor becomes resistant to immune cell therapy. Antigen loss or low antigen escape has become an even greater obstacle to the treatment of solid tumors. Details of the mechanism of antigen loss and related clinical data can be found, for example, in Majzner and Crystal, "Tumor antigen escape from CAR T-cell therapy." Cancer discovery 8.10 (2018): 1219-1226, which is hereby incorporated by reference in its entirety. The expression of IL-12 in engineered immune cells can further increase the immune response in the local tumor microenvironment (eg, stimulate peripheral immune cells), thereby killing peripheral cancer cells that do not express those antigens or express low-level antigens, preventing any Cancer cells escape immune cell therapy.

在一個方面，本公開涉及IL-12裝甲的免疫細胞療法(例如，TCR-T、CAR-T、CAR-NK或TIL)。在一些實施方案中，IL-12通過融合到一個或多個(例如，1、2、3、4或5個)免疫球蛋白恆定結構域和/或膜拴系區(例如，跨膜區)來修飾，使得IL-12拴系到細胞膜。在一些實施方案中，IL-12通過將其連接到靶向腫瘤的抗體的可變區(例如，VH和VL)來修飾。這些IL-12裝甲的免疫細胞療法可增強抗腫瘤免疫力或功效、誘導宿主免疫系統活化、誘導表位擴散和/或增加人的安全性。本公開還提供了載體、包含此類載體的細胞以及製備此類載體的方法。還提供了使用本公開的IL-12裝甲的免疫細胞療法的方法，包括但不限於治療人中的各種癌症和一些其他障礙。IL-12 和免疫細胞療法 In one aspect, the present disclosure relates to IL-12 armored immune cell therapy (eg, TCR-T, CAR-T, CAR-NK, or TIL). In some embodiments, IL-12 is fused to one or more (eg, 1, 2, 3, 4, or 5) immunoglobulin constant domains and/or membrane tethered regions (eg, transmembrane regions) to be modified so that IL-12 is tethered to the cell membrane. In some embodiments, IL-12 is modified by linking it to variable regions (eg, VH and VL) of a tumor-targeting antibody. These IL-12 armored immune cell therapies may enhance anti-tumor immunity or efficacy, induce activation of the host immune system, induce epitope spread, and/or increase safety in humans. The present disclosure also provides vectors, cells comprising such vectors, and methods of making such vectors. Also provided are methods of immune cell therapy using the IL-12 armor of the present disclosure, including but not limited to the treatment of various cancers and some other disorders in humans. IL-12 and immune cell therapy

IL-12是由α鏈(p35亞基)和β鏈(p40亞基)組成的異二聚體分子，通過二硫橋共價連接以形成生物學活性的70kDa二聚體。在生物學上，IL-12是反應於感染而由免疫系統的多種細胞包括吞噬細胞、B細胞和活化的樹突狀細胞產生的炎性細胞介素。IL-12在介導免疫系統的先天性和適應性臂的相互作用、作用於T細胞和天然殺傷(NK)細胞以增強細胞毒性淋巴細胞的增殖和活性以及產生其他炎性細胞介素(尤其是干擾素-γ(IFN-γ))中起關鍵作用。IL-12的詳細描述可見於例如Colombo等人，“Interleukin-12 in anti-tumor immunity and immunotherapy.” Cytokine & growth factor reviews 13.2 (2002): 155-168；以及Hamza等人，“Interleukin 12 a key immunoregulatory cytokine in infection applications.” International journal of molecular sciences 11.3 (2010): 789-806；這些文獻全文以引用的方式併入本文。IL-12 is a heterodimeric molecule consisting of an alpha chain (p35 subunit) and a beta chain (p40 subunit) covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. Biologically, IL-12 is an inflammatory interleukin produced by various cells of the immune system, including phagocytes, B cells, and activated dendritic cells, in response to infection. IL-12 mediates the interaction of the innate and adaptive arms of the immune system, acts on T cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes, and produces other inflammatory interferons (especially It plays a key role in interferon-γ (IFN-γ). A detailed description of IL-12 can be found, for example, in Colombo et al., "Interleukin-12 in anti-tumor immunity and immunotherapy." Cytokine & growth factor reviews 13.2 (2002): 155-168; and Hamza et al., "Interleukin 12 a key immunoregulatory cytokine in infection applications." International journal of molecular sciences 11.3 (2010): 789-806; these documents are incorporated by reference in their entirety.

IL-12受體以組成型或誘導型方式在多種免疫細胞中表現，包括NK細胞以及T和B淋巴細胞。配體結合的IL-12R在酪胺酸上變得磷酸化，這為兩種激酶JAK2和TYK2提供了攜帶位點。在轉錄因子的STAT家族中，STAT4被認為是由IL-12引發的細胞反應的最特異性介質。IL-12的顯著功能是其從天然殺傷(NK)細胞以及CD4+ 和CD8+ T細胞誘導IFNγ釋放的能力。事實上，經由STAT-4的IL-12訊息傳導對於Th1分化和CD8+ T細胞的細胞溶解功能的獲得至關重要。IFNγ繼而強烈地改變腫瘤微環境。IFNγ的最佳研究有益機制是：1)增強腫瘤細胞的MHC I抗原呈遞；2)誘導CXCL9、10和11趨化介素的表現以吸引NK、Th1和CD8+ T細胞；3)將M2巨噬細胞轉化為活化的抗腫瘤M1巨噬細胞；以及4)作用於內皮細胞以CXCR3依賴性方式介導抗血管生成，同時還增強用於T細胞募集的歸巢受體的表現。The IL-12 receptor is expressed in a constitutive or inducible manner on a variety of immune cells, including NK cells and T and B lymphocytes. Ligand-bound IL-12R becomes phosphorylated on tyrosine, which provides a carrier site for two kinases, JAK2 and TYK2. Among the STAT family of transcription factors, STAT4 is considered to be the most specific mediator of cellular responses elicited by IL-12. A remarkable function of IL-12 is its ability to induce IFNy release from natural killer (NK) cells as well as CD4 + and CD8 + T cells. Indeed, IL-12 signaling via STAT-4 is critical for Th1 differentiation and acquisition of cytolytic function of CD8 + T cells. IFNγ in turn strongly alters the tumor microenvironment. The best studied beneficial mechanisms of IFNγ are: 1) enhanced MHC I antigen presentation by tumor cells; 2) induction of CXCL9, 10 and 11 chemokine expression to attract NK, Th1 and CD8 + T cells; phagocytosis into activated anti-tumor M1 macrophages; and 4) acting on endothelial cells to mediate anti-angiogenesis in a CXCR3-dependent manner, while also enhancing the expression of homing receptors for T cell recruitment.

儘管IL-12的強效抗腫瘤作用已得到充分證實，但IL-12施用也引發了嚴重的副作用。在臨床研究中與IL-12全身施用相關的這些副作用以及這種細胞介素的非常狹窄的治療指數顯著限制了IL-12作為抗癌療法的使用。IL-12的詳細描述可見於例如Lasek等人，“Interleukin 12: still a promising candidate for tumor immunotherapy?” Cancer Immunology, Immunotherapy 63.5 (2014): 419-435；Berraondo等人，“Revisiting interleukin-12 as a cancer immunotherapy agent.” Clinical Cancer Research 24.12 (2018): 2716-2718；這些文獻全文以引用的方式併入本文。Although the potent antitumor effects of IL-12 are well established, IL-12 administration also induces severe side effects. These side effects associated with systemic administration of IL-12 in clinical studies, as well as the very narrow therapeutic index of this interferon, significantly limit the use of IL-12 as an anticancer therapy. A detailed description of IL-12 can be found, for example, in Lasek et al., "Interleukin 12: still a promising candidate for tumor immunotherapy?" Cancer Immunology, Immunotherapy 63.5 (2014): 419-435; Berraondo et al., "Revisiting interleukin-12 as a cancer immunotherapy agent." Clinical Cancer Research 24.12 (2018): 2716-2718; these references are incorporated by reference in their entirety.

為了提高功效和減少毒性，本公開提供了經修飾的IL-12和/或遞送IL-12的改進方式。特別地，IL-12在靶向癌細胞的免疫細胞中表現。這些工程化細胞(例如，免疫細胞、T細胞、NK細胞、腫瘤浸潤性細胞)可以受控方式產生IL-12並將其直接遞送至靶位點。在一個方面，IL-12是膜栓系IL-12，其進一步限制IL-12對局部靶區域的作用。To increase efficacy and reduce toxicity, the present disclosure provides modified IL-12 and/or improved means of delivering IL-12. In particular, IL-12 is expressed in immune cells that target cancer cells. These engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) can produce IL-12 in a controlled manner and deliver it directly to target sites. In one aspect, IL-12 is membrane-tethered IL-12, which further limits the effects of IL-12 on localized target areas.

在一些實施方案中，經修飾的IL-12蛋白可以是本文所述的融合蛋白中的任一種。在一些實施方案中，本文提供了包含IL-12α亞基和IL-12β亞基的融合蛋白。IL-12蛋白可源自任何物種。在一些實施方案中，IL-12α亞基是人IL-12α亞基。在一些實施方案中，IL-12α亞基是小鼠IL-12α亞基。在一些實施方案中，IL-12β亞基是人IL-12β亞基。在一些實施方案中，IL-12β亞基是小鼠IL-12β亞基。在一些實施方案中，IL-12α亞基和β亞基通過多肽接頭序列連接。In some embodiments, the modified IL-12 protein can be any of the fusion proteins described herein. In some embodiments, provided herein are fusion proteins comprising an IL-12α subunit and an IL-12β subunit. IL-12 protein can be derived from any species. In some embodiments, the IL-12α subunit is human IL-12α subunit. In some embodiments, the IL-12α subunit is mouse IL-12α subunit. In some embodiments, the IL-12beta subunit is human IL-12beta subunit. In some embodiments, the IL-12beta subunit is a mouse IL-12beta subunit. In some embodiments, the IL-12 alpha and beta subunits are linked by a polypeptide linker sequence.

在一些實施方案中，IL-12亞基(例如，α或β)的胺基酸序列可被修飾。在一些實施方案中，與野生型分子例如本文所述的任何IL-12亞基的序列相比，IL-12亞基包括一個或多個胺基酸變體，例如取代、缺失、***和/或突變。示例性變體包括設計用於提高對IL-12受體的結合親和力和/或IL-12亞基的其他生物學特性(例如，蛋白質穩定性)的那些變體。IL-12亞基的胺基酸序列變體可通過將適當的修飾引入編碼IL-12α和/或β亞基的核苷酸序列中或通過肽合成來製備。此類修飾包括例如IL-12α和/或β亞基胺基酸序列內的殘基的缺失和/或***和/或取代。可進行此類缺失、***和取代的任何組合以得到最終的構建體，條件是最終的構建體具有期望的特徵，例如特異性結合IL-12受體。In some embodiments, the amino acid sequence of an IL-12 subunit (eg, alpha or beta) can be modified. In some embodiments, the IL-12 subunit includes one or more amino acid variants, such as substitutions, deletions, insertions, and/or substitutions, as compared to the sequence of a wild-type molecule, such as any of the IL-12 subunits described herein. or mutation. Exemplary variants include those designed to increase binding affinity to the IL-12 receptor and/or other biological properties of the IL-12 subunit (eg, protein stability). Amino acid sequence variants of the IL-12 subunits can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the IL-12 alpha and/or beta subunits or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the IL-12 alpha and/or beta subunit amino acid sequence. Any combination of such deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as specific binding to the IL-12 receptor.

在一些實施方案中，IL-12α亞基具有與SEQ ID NO: 15至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。在一些實施方案中，IL-12β亞基具有與SEQ ID NO: 16至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。在一些實施方案中,IL-12α和β亞基通過接頭序列GGGGSGGGGSGGGGS(SEQ ID NO: 17)連接。在一些實施方案中，本文所述的IL-12融合蛋白具有與SEQ ID NO: 8至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。In some embodiments, the IL-12α subunit has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% identical to SEQ ID NO: 15 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the IL-12 beta subunit has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% identical to SEQ ID NO: 16 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the IL-12 alpha and beta subunits are linked by a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 17). In some embodiments, the IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

在一些實施方案中，本文所述的融合蛋白包含膜拴系區。在一些實施方案中，膜拴系區是跨膜蛋白(例如，CD4)的跨膜區。在一些實施方案中，跨膜區是4-1BB/CD137的跨膜結構域、T細胞受體的α鏈、T細胞受體的β鏈、B7(例如，B7-1)、CD3ε、CD4、CD5、CD8、CD8α、CD9、CD16、CD19、CD22、CD28、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、或T細胞受體的ζ鏈、或它們的任何組合。在一些實施方案中，融合蛋白包含膜免疫球蛋白(mIg)的跨膜結構域。在一些實施方案中，融合蛋白包含CD28、CD3-ζ、CD3-α或CD3-β跨膜結構域。在一些實施方案中，本文所述的融合蛋白包含免疫球蛋白的0、1、2、3、4、5、6、7、8、9、10個或更多個恆定結構域。在一些實施方案中，恆定結構域是重鏈恆定結構域。在一些實施方案中，恆定結構域是輕鏈恆定結構域。在一些實施方案中，蛋白質包含2個恆定結構域。在一些實施方案中，該2個恆定結構域是CH2和CH3結構域。在一些實施方案中，該2個恆定結構域是CH1和CH3結構域。在一些實施方案中，該2個恆定結構域是CH1和CH2結構域。在一些實施方案中，該2個恆定結構域都是CH1結構域。在一些實施方案中，該2個恆定結構域都是CH2結構域。在一些實施方案中，該2個恆定結構域都是CH3結構域。在一些實施方案中，蛋白質包含免疫球蛋白的整個Fc區。在一些實施方案中，蛋白質包含3個恆定結構域。在一些實施方案中，該3個恆定結構域是兩個CH2和一個CH3。在一些實施方案中，該3個恆定結構域依次連接為CH2-CH2-CH3。在一些實施方案中，該3個恆定結構域依次連接為CH1-CH2-CH3。在一些實施方案中，蛋白質包含1、2、3、4、5個或更多個CH2結構域。在一些實施方案中，蛋白質包含1、2、3、4、5個或更多個CH3結構域。在一些實施方案中，免疫球蛋白是人免疫球蛋白。在一些實施方案中，免疫球蛋白是小鼠免疫球蛋白。在一些實施方案中，免疫球蛋白是免疫球蛋白G(IgG)、IgM、IgE、IgA或IgD分子。在一些實施方案中，免疫球蛋白是IgG1、IgG2、IgG3或IgG4。在一些實施方案中，免疫球蛋白是人IgG4。在一些實施方案中，恆定結構域來自相同的免疫球蛋白類別(例如，IgG、IgM、IgA)。在一些實施方案中，恆定結構域包含IgM的CH2、CH3和CH4。In some embodiments, the fusion proteins described herein comprise a membrane tethered region. In some embodiments, the membrane tethered region is the transmembrane region of a transmembrane protein (eg, CD4). In some embodiments, the transmembrane region is the transmembrane domain of 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, B7 (eg, B7-1), CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of the T cell receptor, or any combination thereof. In some embodiments, the fusion protein comprises the transmembrane domain of a membrane immunoglobulin (mIg). In some embodiments, the fusion protein comprises a CD28, CD3-zeta, CD3-alpha or CD3-beta transmembrane domain. In some embodiments, the fusion proteins described herein comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more constant domains of an immunoglobulin. In some embodiments, the constant domain is a heavy chain constant domain. In some embodiments, the constant domain is a light chain constant domain. In some embodiments, the protein comprises 2 constant domains. In some embodiments, the two constant domains are CH2 and CH3 domains. In some embodiments, the two constant domains are CH1 and CH3 domains. In some embodiments, the two constant domains are CH1 and CH2 domains. In some embodiments, the two constant domains are both CH1 domains. In some embodiments, the two constant domains are both CH2 domains. In some embodiments, the two constant domains are both CH3 domains. In some embodiments, the protein comprises the entire Fc region of an immunoglobulin. In some embodiments, the protein comprises 3 constant domains. In some embodiments, the three constant domains are two CH2 and one CH3. In some embodiments, the three constant domains are sequentially linked as CH2-CH2-CH3. In some embodiments, the three constant domains are linked sequentially as CH1-CH2-CH3. In some embodiments, the protein comprises 1, 2, 3, 4, 5 or more CH2 domains. In some embodiments, the protein comprises 1, 2, 3, 4, 5 or more CH3 domains. In some embodiments, the immunoglobulin is a human immunoglobulin. In some embodiments, the immunoglobulin is a mouse immunoglobulin. In some embodiments, the immunoglobulin is an immunoglobulin G (IgG), IgM, IgE, IgA, or IgD molecule. In some embodiments, the immunoglobulin is IgGl, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin is human IgG4. In some embodiments, the constant domains are from the same immunoglobulin class (eg, IgG, IgM, IgA). In some embodiments, the constant domains comprise CH2, CH3 and CH4 of IgM.

在一些實施方案中，恆定結構域是來自人的野生型恆定結構域。在一些實施方案中，恆定結構域是突變的人恆定結構域。在一些實施方案中，恆定結構域是突變的人免疫球蛋白IgG4重鏈恆定結構域。在一些實施方案中，恆定結構域是野生型人免疫球蛋白IgG4重鏈恆定結構域CH2。在一些實施方案中，恆定結構域是突變的人免疫球蛋白IgG4重鏈恆定結構域CH2。在一些實施方案中，在CH2結構域中至少1、2、3、4、5、6、7、8、9、10、11或12個胺基酸發生突變。在一些實施方案中，CH2結構域的所有胺基酸殘基的至少或約1%、2%、3%、4%、5%、6%或7%發生突變。在一些實施方案中，恆定結構域是野生型人免疫球蛋白IgG4重鏈恆定結構域CH3。在一些實施方案中，恆定結構域是突變的人免疫球蛋白IgG4重鏈恆定結構域CH3。在一些實施方案中，與對應的野生型恆定結構域相比，本文所述的突變恆定結構域(例如，hmCH2)具有對可溶性FcγR降低的結合親和力。在一些實施方案中，與對應的野生型恆定結構域相比，本文所述的突變恆定結構域(例如，hmCH2)具有增強的體內T細胞持久性。在一些實施方案中，CH2結構域中的突變是根據EU編號的L235E和/或N297Q。在一些實施方案中，突變包括以下中的一種或多種：N297A、N297Q或N297G(EU編號)。在一些實施方案中，突變是L234A/L235A(LALA)(EU編號)。在一些實施方案中，突變是F234A和或L235A。在一些實施方案中，突變包括以下中的一種或多種：H268Q、V309L、A330S和/或P331S(EU編號)。在一些實施方案中，突變包括以下中的一種或多種：V234A、G237A、P238S、H268A、V309L、A330S和/或P331S。詳細描述可見於例如Jonnalagadda等人，“Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy.” Molecular Therapy 23.4 (2015): 757-768；以及美國專利號9,914,909 B2，這些文獻中的每一者全文以引用的方式併入本文。In some embodiments, the constant domains are wild-type constant domains from humans. In some embodiments, the constant domains are mutated human constant domains. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain. In some embodiments, the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids are mutated in the CH2 domain. In some embodiments, at least or about 1%, 2%, 3%, 4%, 5%, 6%, or 7% of all amino acid residues of the CH2 domain are mutated. In some embodiments, the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the mutated constant domains described herein (eg, hmCH2) have reduced binding affinity for soluble FcyRs compared to the corresponding wild-type constant domains. In some embodiments, a mutated constant domain (eg, hmCH2) described herein has enhanced T cell persistence in vivo as compared to a corresponding wild-type constant domain. In some embodiments, the mutations in the CH2 domain are L235E and/or N297Q according to EU numbering. In some embodiments, the mutation includes one or more of the following: N297A, N297Q, or N297G (EU numbering). In some embodiments, the mutation is L234A/L235A(LALA) (EU numbering). In some embodiments, the mutation is F234A and or L235A. In some embodiments, the mutation includes one or more of the following: H268Q, V309L, A330S and/or P331S (EU numbering). In some embodiments, the mutation includes one or more of the following: V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S. A detailed description can be found in, eg, Jonnalagadda et al., "Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy." Molecular Therapy 23.4 (2015): 757-768; and US Patent No. 9,914,909 B2, Each of these documents is incorporated herein by reference in its entirety.

在一些實施方案中，本文所述的CH2結構域包含在SEQ ID NO: 41中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，本文所述的CH2結構域由與SEQ ID NO: 42至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同的胺基酸序列編碼。在一些實施方案中，本文所述的CH3結構域包含在SEQ ID NO: 43中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，本文所述的CH3結構域由與SEQ ID NO: 44至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同的胺基酸序列編碼。In some embodiments, the CH2 domain described herein comprises, or has at least 80%, 85%, 90%, 91%, 92%, of the amino acid sequence set forth in any one of SEQ ID NO: 41 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CH2 domain described herein consists of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with SEQ ID NO: 42 , 98% or 99% identical amino acid sequence encoding. In some embodiments, the CH3 domain described herein comprises, or has at least 80%, 85%, 90%, 91%, 92%, of the amino acid sequence set forth in any one of SEQ ID NO: 43 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CH3 domain described herein consists of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with SEQ ID NO: 44 , 98% or 99% identical amino acid sequence encoding.

在一些實施方案中，恆定結構域通過鉸鏈區與IL-12亞基連接。在一些實施方案中，本文所述的恆定結構域的胺基酸序列可被修飾。在一些實施方案中，與野生型分子(例如，本文所述的任何恆定結構域)的序列相比，恆定結構域(例如，CH2或CH3)包括一個或多個胺基酸變體，例如取代、缺失、***和/或突變。In some embodiments, the constant domain is linked to the IL-12 subunit through a hinge region. In some embodiments, the amino acid sequences of the constant domains described herein can be modified. In some embodiments, the constant domain (eg, CH2 or CH3) includes one or more amino acid variants, eg, substitutions, as compared to the sequence of the wild-type molecule (eg, any of the constant domains described herein). , deletions, insertions and/or mutations.

在一些實施方案中，融合蛋白是膜拴系蛋白。在一些實施方案中，膜拴系區融合至如本文所述的恆定結構域。在一些實施方案中，本文所述的膜拴系IL-12融合蛋白具有與SEQ ID NO: 10至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。在一些實施方案中，本文所述的膜拴系IL-12融合蛋白具有與SEQ ID NO: 12至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。In some embodiments, the fusion protein is a membrane tethered protein. In some embodiments, the membrane tethered region is fused to a constant domain as described herein. In some embodiments, the membrane-tethered IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the membrane-tethered IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

在一些實施方案中，融合蛋白是可溶性蛋白。在一些實施方案中，融合蛋白包含訊息肽(例如，人訊息多肽)。在一些實施方案中，訊息肽序列是分泌訊息肽。In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein comprises a message peptide (eg, a human message polypeptide). In some embodiments, the message peptide sequence is a secretion message peptide.

改善IL-12的安全性的另一策略是通過融合至靶向腫瘤的抗體來指導其遞送至腫瘤。此類抗體-細胞介素融合蛋白或“免疫細胞介素”先前在臨床前模型中已證實增強抗腫瘤免疫力的能力。為了使免疫細胞介素耐受性最大化，選擇作為媒介物的抗體通常特異性結合腫瘤相關抗原。例如，針對大量存在於腫瘤中但不存在於正常組織中的壞死相關抗原的抗體提供了有吸引力的遞送方法。在一個方面，這些抗體-細胞介素融合蛋白在靶向腫瘤的細胞(例如，免疫細胞、T細胞、NK細胞、腫瘤浸潤性細胞)中表現；因此，這些抗體-細胞介素融合蛋白在靶位點處特異性表現，這進一步提高了它們的安全性。Another strategy to improve the safety of IL-12 is to direct its delivery to tumors by fusion to tumor-targeting antibodies. Such antibody-interleukin fusion proteins or "immune interleukins" have previously demonstrated the ability to enhance anti-tumor immunity in preclinical models. To maximize immune interleukin tolerance, antibodies chosen as vehicles typically bind specifically to tumor-associated antigens. For example, antibodies directed against necrosis-associated antigens that are abundant in tumors but not in normal tissues provide an attractive delivery method. In one aspect, the antibody-interleukin fusion proteins are expressed in tumor-targeted cells (eg, immune cells, T cells, NK cells, tumor-infiltrating cells); thus, the antibody-interleukin fusion proteins are in the target site-specific performance, which further enhances their safety.

在一些實施方案中，融合蛋白包含靶向腫瘤的抗體或其抗原結合片段。靶向腫瘤的抗體或抗原結合片段可將IL-12遞送至靶位點，從而進一步減少IL-12的副作用。在一些實施方案中，融合蛋白不是膜拴系蛋白和/或不具有跨膜蛋白。靶向腫瘤的抗體或抗原結合片段可靶向腫瘤相關抗原。如本文所用，術語“腫瘤相關抗原”是指在腫瘤細胞表面上呈遞或可呈遞並且位於腫瘤細胞上或腫瘤細胞內的抗原。在一些其他實施方案中，腫瘤相關抗原可在腫瘤細胞上排他性地表現，或者與非腫瘤細胞相比可表示腫瘤特異性突變。在一些其他實施方案中，腫瘤相關抗原可在腫瘤細胞和非腫瘤細胞兩者中發現，但當與非腫瘤細胞相比時在腫瘤細胞上過表現，或由於與非腫瘤組織相比腫瘤組織的不太緊密的結構而更易於抗體在腫瘤細胞中結合。在一些實施方案中，腫瘤相關抗原位於腫瘤的脈管系統上。腫瘤相關表面抗原的例示性實例包括CD10、CD19、CD20、CD22、CD21、CD22、CD25、CD30、CD33、CD34、CD37、CD44v6、CD45、CD133、Fms樣酪胺酸激酶3(FLT-3、CD135)、硫酸軟骨素蛋白聚醣4(CSPG4、黑色素瘤相關硫酸軟骨素蛋白聚醣)、表皮生長因子受體(EGFR)、Her2neu、Her3、IGFR、IL3R、成纖維細胞活化蛋白(FAP)、CDCP1、Derlin1、生腱蛋白、frizzled 1-10、血管抗原VEGFR2(KDR/FLK1)、VEGFR3(FLT4、CD309)、PDGFR-α(CD140a)、PDGFR-β(CD140b)、內皮因子、CLEC14、Tem1-8和Tie2、A33、CAMPATH-1(CDw52)、癌胚抗原(CEA)、碳酸酐酶IX(MN/CA IX)、de2-7 EGFR、EGFRvIII、EpCAM、Ep-CAM、葉酸結合蛋白、G250、Fms樣酪胺酸激酶3(FLT-3、CD135)、c-Kit(CD117)、CSF1R(CD115)、HLA-DR、IGFR、IL-2受體、IL3R、MCSP(黑色素瘤相關細胞表面硫酸軟骨素蛋白聚醣)、Muc-1、***特異性膜抗原(PSMA)、***幹細胞抗原(PSCA)、***特異性抗原(PSA)和TAG-72。In some embodiments, the fusion protein comprises a tumor-targeting antibody or antigen-binding fragment thereof. Tumor-targeted antibodies or antigen-binding fragments can deliver IL-12 to the target site, thereby further reducing the side effects of IL-12. In some embodiments, the fusion protein is not a membrane tethered protein and/or does not have a transmembrane protein. Tumor-targeting antibodies or antigen-binding fragments can target tumor-associated antigens. As used herein, the term "tumor-associated antigen" refers to an antigen that is or can be presented on the surface of a tumor cell and that is located on or within a tumor cell. In some other embodiments, tumor-associated antigens may be expressed exclusively on tumor cells, or may express tumor-specific mutations as compared to non-tumor cells. In some other embodiments, tumor-associated antigens may be found in both tumor cells and non-tumor cells, but are overexpressed on tumor cells when compared to non-tumor cells, or due to the presence of tumor tissue compared to non-tumor tissue Less compact structure and easier antibody binding in tumor cells. In some embodiments, the tumor-associated antigen is located on the vasculature of the tumor. Illustrative examples of tumor-associated surface antigens include CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135 ), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activation protein (FAP), CDCP1 , Derlin1, tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR/FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1-8 and Tie2, A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN/CA IX), de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, folate binding protein, G250, Fms Like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate) proteoglycan), Muc-1, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA) and TAG-72.

在一些實施方案中，本文提供了包含IL-12α亞基和IL-12β亞基的融合蛋白。在一些實施方案中，蛋白質還包含靶向腫瘤的抗體或其抗原結合片段的重鏈可變區和輕鏈可變區。在一些實施方案中，靶向腫瘤的抗體或其抗原結合片段是scFv。在一些實施方案中，靶向腫瘤壞死的抗體是人抗體。在一些實施方案中，靶向腫瘤壞死的抗體是人IgG1。在一些實施方案中，靶向腫瘤壞死的抗體是NHS76。NHS76是一種完全人源、噬菌體展示衍生的IgG1抗體，因其結合壞死區並因此在體內靶向腫瘤的特異能力而選擇。詳細描述可見於例如Fallon等人，“The immunocytokine NHS-IL12 as a potential cancer therapeutic.” Oncotarget 5.7 (2014): 1869；和Sharifi等人，“Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors.” Hybridoma and hybridomics 20.5-6 (2001): 305-312；這些文獻中的每一者全文以引用的方式併入本文。因此，在一些實施方案中，抗體-細胞介素融合蛋白具有與SEQ ID NO: 14至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的胺基酸序列。T 細胞受體和結合分子 In some embodiments, provided herein are fusion proteins comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the protein further comprises the heavy and light chain variable regions of the tumor-targeted antibody or antigen-binding fragment thereof. In some embodiments, the tumor-targeting antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the antibody targeting tumor necrosis is a human antibody. In some embodiments, the antibody targeting tumor necrosis is human IgGl. In some embodiments, the antibody targeting tumor necrosis is NHS76. NHS76 is a fully human, phage-display-derived IgG1 antibody selected for its specific ability to bind necrotic regions and thus target tumors in vivo. Detailed descriptions can be found, for example, in Fallon et al., "The immunocytokine NHS-IL12 as a potential cancer therapeutic." Oncotarget 5.7 (2014): 1869; and Sharifi et al., "Characterization of a phage display-derived human monoclonal antibody counterpart (NHS76) to chimeric TNT-1 directed against necrotic regions of solid tumors." Hybridoma and hybridomics 20.5-6 (2001): 305-312; each of these references is incorporated herein by reference in its entirety. Thus, in some embodiments, the antibody-interferon fusion protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. T cell receptors and binding molecules

T細胞是通常在胸腺中發育並在適應性免疫反應中起主要作用的一種淋巴細胞類型。T細胞與其他淋巴細胞的區別在於細胞表面上存在T細胞受體。分化的T細胞在控制免疫反應中具有重要作用。CD8+ T細胞(也稱為“殺傷細胞”)具有細胞毒性。一旦它們識別出靶細胞，它們便能夠直接殺死靶細胞(例如，病毒感染的細胞或癌細胞)。CD8+ T細胞還產生細胞介素並募集其他細胞(例如，巨噬細胞和自然殺傷(NK)細胞)以啟動免疫反應。CD4+ T細胞(也稱為“輔助細胞”)可以例如通過促進B細胞成熟為漿細胞和記憶B細胞以及活化細胞毒性T細胞和巨噬細胞而間接地殺死靶細胞。當輔助T細胞被MHC II類分子與肽抗原一起呈遞時，它們變成活化的，所述MHC II類分子在抗原呈遞細胞(APC)的表面上表現。一旦活化，它們便迅速地***並分泌調節或協助免疫反應的細胞介素。調節性T細胞對於耐受性很重要，從而阻止或抑制自身免疫反應。調節性T細胞的主要作用是在免疫反應結束時關閉T細胞介導的免疫力，並抑制逃避在胸腺中的負選擇過程的自身反應性T細胞。T cells are a type of lymphocyte that normally develops in the thymus and plays a major role in the adaptive immune response. What distinguishes T cells from other lymphocytes is the presence of T cell receptors on the cell surface. Differentiated T cells have an important role in controlling the immune response. CD8+ T cells (also known as "killer cells") are cytotoxic. Once they identify the target cell, they are able to directly kill the target cell (eg, virus-infected cells or cancer cells). CD8+ T cells also produce interferons and recruit other cells (eg, macrophages and natural killer (NK) cells) to initiate immune responses. CD4+ T cells (also known as "helper cells") can kill target cells indirectly, eg, by promoting the maturation of B cells into plasma cells and memory B cells and by activating cytotoxic T cells and macrophages. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist the immune response. Regulatory T cells are important for tolerance, preventing or suppressing autoimmune responses. The main role of regulatory T cells is to shut down T cell-mediated immunity at the end of the immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus.

T細胞在癌症免疫力方面起重要作用，其中來自癌細胞的抗原被吸收並呈遞到被稱為抗原呈遞細胞(APC)的特殊免疫細胞的細胞表面上，從而使其他免疫細胞可以識別所關注的抗原。在淋巴結中，APC活化T細胞並活化它們以識別腫瘤細胞。然後，活化的T細胞可以穿過血管到達腫瘤，使其浸潤，識別癌細胞並殺死它們。T cells play an important role in cancer immunity, where antigens from cancer cells are taken up and presented on the cell surface of specialized immune cells called antigen-presenting cells (APCs), allowing other immune cells to recognize the antigen. In lymph nodes, APCs activate T cells and activate them to recognize tumor cells. The activated T cells can then travel through blood vessels to the tumor, where they infiltrate, recognize cancer cells and kill them.

T細胞的活化需要T細胞受體。“T細胞受體”或“TCR”是含有可變a(或α)鏈和b(或β)鏈(分別稱為TCRα和TCRβ)或可變g(或γ)鏈和d(或δ)鏈(也分別稱為TCRγ和TCRδ)或其抗原結合部分並且能夠特異性地結合抗原(例如，與主要組織相容性複合物(MHC)分子結合的肽抗原或肽表位)的一種分子。Activation of T cells requires T cell receptors. "T cell receptors" or "TCRs" are those containing variable alpha (or alpha) chains and b (or beta) chains (referred to as TCRalpha and TCRbeta, respectively) or variable g (or gamma) chains and d (or delta) chains A molecule that chains (also referred to as TCRγ and TCRδ, respectively) or antigen-binding portions thereof and is capable of specifically binding an antigen (eg, a peptide antigen or peptide epitope bound to a major histocompatibility complex (MHC) molecule).

本公開提供了T細胞受體(TCR)或其抗原結合片段、以及衍生自TCR的結合分子。在一些實施方案中，TCR呈ab形式。以αβ和γδ形式存在的TCR通常在結構上相似，但是表現它們的T細胞可能具有獨特的解剖位置或功能。通常，TCR發現於T細胞(或T淋巴細胞)的表面，在此處其通常負責識別抗原(諸如與主要組織相容性複合物(MHC)分子結合的肽)。The present disclosure provides T cell receptors (TCRs) or antigen-binding fragments thereof, and binding molecules derived from TCRs. In some embodiments, the TCR is in the form of ab. TCRs in the αβ and γδ forms are often structurally similar, but the T cells that express them may have unique anatomical locations or functions. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are typically responsible for the recognition of antigens (such as peptides bound to major histocompatibility complex (MHC) molecules).

在一些實施方案中，TCR是完整或全長TCR，諸如含有a鏈和b鏈的TCR。在一些實施方案中，TCR是小於全長TCR但與結合在MHC分子中的特定肽結合(諸如與MHC-肽複合物結合)的抗原結合部分。在一些情況下，TCR的抗原結合部分或片段可以僅含有全長或完整TCR的結構性結構域的一部分，但仍能夠結合完整TCR所結合的肽表位(諸如MHC-肽複合物)。在一些情況下，抗原結合部分含有TCR的可變結構域(諸如TCR的可變a(Va或Vα)鏈和可變b(Vb或Vβ)鏈)、或其足以形成用於與特定的MHC-肽複合物結合的結合位點的抗原結合片段。In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing an a-chain and a b-chain. In some embodiments, the TCR is an antigen-binding portion that is smaller than a full-length TCR but binds to a specific peptide bound in an MHC molecule, such as to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may contain only a portion of the structural domain of a full-length or intact TCR, but still be capable of binding a peptide epitope (such as an MHC-peptide complex) to which the intact TCR binds. In some cases, the antigen-binding portion contains a variable domain of a TCR (such as the variable a (Va or Vα) chain and variable b (Vb or Vβ) chain of a TCR, or is sufficient to form a TCR for interaction with a particular MHC -An antigen-binding fragment of the binding site to which the peptide complex binds.

TCR的可變結構域含有互補決定區(CDR)，其通常是抗原識別以及肽、MHC和/或MHC-肽複合物的結合能力和特異性的主要貢獻者。在一些實施方案中，TCR的CDR或其組合形成給定TCR分子的全部或基本上全部抗原結合位點。在TCR鏈的可變區內的各種CDR通常被框架區(FR)分開，所述框架區與CDR相比在TCR分子之間通常顯示出較小的變異性。在一些實施方案中，CDR3是負責抗原結合或特異性的主要CDR，或者在給定TCR可變區上的三個CDR中對於抗原識別和/或對於與肽-MHC複合物的加工過的肽部分相互作用而言是最重要的。在一些情境下，α鏈的CDR1可以與某些抗原肽的N末端部分相互作用。在一些情況下，β鏈的CDR1可以與肽的C末端部分相互作用。在一些情境下，CDR2最有力地促進了與MHC-肽複合物的MHC部分的相互作用或對MHC-肽複合物的MHC部分的識別，或者是負責與MHC-肽複合物的MHC部分相互作用或識別MHC-肽複合物的MHC部分的主要CDR。The variable domains of TCRs contain complementarity determining regions (CDRs), which are generally the major contributors to antigen recognition and binding capacity and specificity of peptides, MHCs and/or MHC-peptide complexes. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen binding site of a given TCR molecule. The various CDRs within the variable regions of a TCR chain are typically separated by framework regions (FRs), which typically show less variability between TCR molecules than the CDRs. In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or the processed peptide among the three CDRs on the variable region of a given TCR for antigen recognition and/or for peptide-MHC complexes Some interactions are the most important. In some contexts, CDR1 of the alpha chain can interact with the N-terminal portion of certain antigenic peptides. In some cases, CDR1 of the beta chain can interact with the C-terminal portion of the peptide. In some contexts, CDR2 most strongly facilitates the interaction with or recognition of the MHC portion of the MHC-peptide complex, or is responsible for interacting with the MHC portion of the MHC-peptide complex Or the major CDRs that recognize the MHC portion of the MHC-peptide complex.

TCR的a鏈和/或b鏈還可以含有恆定結構域、跨膜區和/或短細胞質尾巴。在一些方面，TCR的每條鏈(例如α或β)可以擁有一個N末端免疫球蛋白可變結構域、一個免疫球蛋白恆定結構域、跨膜區和在C末端的短細胞質尾巴。在一些實施方案中，TCR例如經由細胞質尾巴與參與介導訊息轉導的CD3複合物的不變蛋白質相關。在一些情況下，所述結構允許TCR與其他分子如CD3及其亞基締合。例如，包含具有跨膜區的恆定結構域的TCR可以將蛋白質錨定在細胞膜中，並與CD3訊息傳導裝置或複合物的不變亞基締合。CD3訊息傳導亞基(例如CD3γ、CD3δ、CD3e和CD3z鏈)的細胞內尾巴含有一個或多個基於免疫受體酪胺酸的活化基序或ITAM，並且通常參與TCR複合物的訊息傳導能力。The a-chain and/or b-chain of the TCR may also contain constant domains, transmembrane regions and/or short cytoplasmic tails. In some aspects, each chain (eg, alpha or beta) of a TCR can possess an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction, eg, via a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3 and its subunits. For example, TCRs comprising constant domains with transmembrane regions can anchor proteins in the cell membrane and associate with the invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (eg, CD3γ, CD3δ, CD3e, and CD3z chains) contain one or more immunoreceptor tyrosine-based activation motifs, or ITAMs, and are typically involved in the signaling capacity of the TCR complex.

結構域或區域的確切位點可以根據用於描述特定結構域的特定結構或同源性建模或其他特徵而有所不同。應當理解，提及胺基酸(包括用於描述TCR的結構域組織的以SEQ ID NO列出的特定序列)是出於說明性目的，並不意味著限制所提供的實施方案的範圍。在一些情況下，特定結構域(例如可變或恆定)可以是更長或更短的幾個胺基酸(諸如一個、兩個、三個或四個)。在一些方面，TCR的殘基是已知的或可以根據國際免疫遺傳學資訊系統(International Immunogenetics Information System，IMGT)編號系統來鑒定(參見例如www.imgt.org；Lefranc等人，"IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77)。TCR的結構和變化是本領域已知的，並且描述於例如WO 2019 /195486中，該專利全文以引用方式併入本文。The exact location of a domain or region can vary depending on the particular structure or homology modeling or other features used to describe the particular domain. It should be understood that references to amino acids (including specific sequences listed as SEQ ID NOs used to describe the domain organization of the TCR) are for illustrative purposes and are not meant to limit the scope of the provided embodiments. In some cases, a particular domain (eg, variable or constant) may be several amino acids longer or shorter (such as one, two, three, or four). In some aspects, the residues of the TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see, eg, www.imgt.org; Lefranc et al., "IMGT unique numbering" for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77). The structures and variations of TCRs are known in the art and are described, for example, in WO 2019/195486, which is incorporated herein by reference in its entirety.

在一些實施方案中，TCR的a鏈和b鏈各自還含有恆定結構域。在一些實施方案中，a鏈恆定結構域(Ca)和b鏈恆定結構域(Cb)單獨地是哺乳動物恆定結構域，諸如是人恆定結構域或非人恆定結構域(例如，小鼠恆定結構域)。在一些實施方案中，恆定結構域與細胞膜相鄰。例如，在一些情況下，由兩條鏈形成的TCR的細胞外部分含有兩個膜近端恆定結構域和兩個膜遠端可變結構域，所述可變結構域各自含有CDR。In some embodiments, the a and b chains of the TCR each further contain a constant domain. In some embodiments, the a-chain constant domain (Ca) and b-chain constant domain (Cb) are individually mammalian constant domains, such as human constant domains or non-human constant domains (eg, mouse constant domains) domain). In some embodiments, the constant domains are adjacent to the cell membrane. For example, in some cases, the extracellular portion of a TCR formed from two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains a CDR.

在一些方面，如本文所述的TCR可以含有人恆定區，諸如含有人Ca區的α鏈和含有人Cb區的β鏈。在一些實施方案中，TCR是完全人的。在一些實施方案中，TCR的表現和/或活性，諸如當在人細胞(例如人T細胞，諸如原代人T細胞)中表現時，不受或基本上不受內源性人TCR的影響。In some aspects, a TCR as described herein can contain human constant regions, such as an alpha chain containing a human Ca region and a beta chain containing a human Cb region. In some embodiments, the TCR is fully human. In some embodiments, the expression and/or activity of the TCR, such as when expressed in human cells (eg, human T cells, such as primary human T cells), is not or substantially unaffected by endogenous human TCRs .

在一些實施方案中，與在相似的人細胞(但其中內源性TCR的表現已經被降低或被消除)中的相同TCR的表現位準、功能活性和/或抗腫瘤活性相比，當工程化TCR被含有或表現內源性人TCR的人細胞(諸如人T細胞)表現時，所述工程化TCR在細胞表面上以相似或改善的位準表現，表現出相似或更高的功能活性(例如，細胞溶解活性)和/或表現出相似或更高的抗腫瘤活性。在一些實例中，當在人T細胞中表現時，如本文所述的工程化TCR在細胞表面上以如下的位準表現，所述位準為當在相似的人T細胞(但其中內源性TCR的表現已經被降低或被消除)中表現時的相同TCR的表現位準的至少或至少約80%、85%、90%、95%、100%、105%、110%、115%或120%。In some embodiments, when engineered compared to the level of expression, functional activity and/or anti-tumor activity of the same TCR in similar human cells (but in which the expression of endogenous TCRs has been reduced or eliminated) The engineered TCRs are expressed at similar or improved levels on the cell surface and exhibit similar or greater functional activity when expressed by human cells (such as human T cells) that contain or express endogenous human TCRs (eg, cytolytic activity) and/or exhibit similar or greater antitumor activity. In some instances, when expressed in human T cells, an engineered TCR as described herein is expressed on the cell surface at levels that are comparable when expressed in similar human T cells (but where endogenous At least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or at least about the performance level of the same TCR at the time when the performance of the sexual TCR has been reduced or eliminated) 120%.

在一些實施方案中，Ca結構域和Cb結構域中的每一者都是人的。在一些實施方案中，Ca由TRAC基因(IMGT命名法)編碼或為其變體。在一些實施方案中，Ca的變體含有至少一個非天然半胱胺酸的替代。In some embodiments, each of the Ca domain and the Cb domain is human. In some embodiments, Ca is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, the variant of Ca contains at least one substitution of unnatural cysteine.

在一些實施方案中，TCR可以是諸如通過一個或多個二硫鍵連接的兩條鏈a和b的異二聚體。在一些實施方案中，TCR的恆定結構域可以含有短連接序列，其中半胱胺酸殘基形成二硫鍵，從而連接TCR的兩條鏈。在一些實施方案中，TCR可以在a鏈和b鏈的每一者中具有另外的半胱胺酸殘基，使得TCR在恆定結構域中含有兩個二硫鍵。在一些實施方案中，恆定結構域和可變結構域中的每一者都含有由半胱胺酸殘基形成的二硫鍵。In some embodiments, the TCR may be a heterodimer such as two chains a and b linked by one or more disulfide bonds. In some embodiments, the constant domains of the TCR may contain short linking sequences in which the cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine residues in each of the a and b chains, such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, each of the constant and variable domains contain disulfide bonds formed by cysteine residues.

在一些實施方案中，TCR包含CDR、Va和/或Vb以及如本文所述的恆定區序列。In some embodiments, the TCR comprises CDRs, Va and/or Vb and constant region sequences as described herein.

在一些實施方案中，TCR是二聚體TCR(dTCR)。在一些實施方案中，dTCR含有其中與所提供的TCR a鏈可變區序列對應的序列融合至與TCR a鏈恆定區細胞外序列對應的序列的N末端的第一多肽、以及其中與所提供的TCR b鏈可變區序列對應的序列融合至與TCR b鏈恆定區細胞外序列對應的序列的N末端的第二多肽，所述第一多肽和所述第二多肽通過二硫鍵連接。In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the dTCR comprises a first polypeptide in which the sequence corresponding to the provided TCR alpha chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR alpha chain constant region extracellular sequence, and in which the provided sequence corresponds to the TCR alpha chain constant region extracellular sequence. The sequence corresponding to the provided sequence of the variable region of the TCR b chain is fused to a second polypeptide at the N-terminus of the sequence corresponding to the extracellular sequence of the constant region of the TCR b chain, the first polypeptide and the second polypeptide passing through two Sulfur bond.

在一些實施方案中，TCR可以是細胞結合的或呈可溶的形式。在一些實施方案中，TCR呈在細胞表面上表現的細胞結合形式。In some embodiments, the TCR may be cell-bound or in a soluble form. In some embodiments, the TCR is in a cell-bound form expressed on the cell surface.

在一些實施方案中，TCR是單鏈TCR(scTCR)。scTCR是含有能夠與MHC-肽複合物結合的a鏈和b鏈的一條單胺基酸鏈。通常，可以使用本領域技術人員已知的方法來產生scTCR。這些方法描述於例如WO 96/13593、WO 96/18105、W099/18129、WO 04/033685、W02006/037960、WO2011/044186；WO 2019 /195486；美國專利號7,569,664中；這些專利中的每一者全文以引用方式併入本文。In some embodiments, the TCR is a single-chain TCR (scTCR). The scTCR is a monoamino acid chain containing a and b chains capable of binding to the MHC-peptide complex. In general, scTCRs can be generated using methods known to those skilled in the art. These methods are described, for example, in WO 96/13593, WO 96/18105, WO 99/18129, WO 04/033685, WO2006/037960, WO2011/044186; WO 2019/195486; US Patent No. 7,569,664; each of these patents The entire contents are incorporated herein by reference.

TCR、其抗原結合片段和TCR衍生的結合分子可以結合或識別與所關注的抗原(例如，腫瘤相關抗原)相關的肽表位。在一些實施方案中，抗原可以是在癌細胞和/或感染病毒的細胞的表面上表現的肽表位。在一些實施方案中，在MHC分子的背景下呈遞抗原。此類結合分子包括例如T細胞受體(TCR)及其抗原結合片段、抗體及其抗原結合片段、和TCR樣CAR。它們表現出結合或識別此類肽表位的抗原特異性。在一些方面，表現所提供的結合分子(例如TCR或抗原結合片段)的工程化細胞對表現肽表位的靶細胞(諸如癌細胞或感染病毒(例如，HPV或EBV)的細胞)表現出細胞毒性活性。TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules can bind or recognize peptide epitopes associated with an antigen of interest (eg, a tumor-associated antigen). In some embodiments, the antigen may be a peptide epitope expressed on the surface of cancer cells and/or virus-infected cells. In some embodiments, the antigen is presented in the context of an MHC molecule. Such binding molecules include, for example, T cell receptors (TCRs) and antigen-binding fragments thereof, antibodies and antigen-binding fragments thereof, and TCR-like CARs. They exhibit antigen specificity for binding or recognizing such peptide epitopes. In some aspects, engineered cells expressing a provided binding molecule (eg, TCR or antigen-binding fragment) express cellular Toxic activity.

在一些方面，TCR、其抗原結合片段和TCR衍生的結合分子識別或結合在MHC分子(諸如MHC I類分子或MHC II類分子)的背景下的表位。MHC I類分子或MHC II類分子兩者都是人白細胞抗原(HLA)。它們扮演著適應性免疫系統的重要組分。HLA表現受位於6號染色體上的基因的控制。其編碼專門向T細胞上的T細胞受體呈遞抗原肽的細胞表面分子。In some aspects, TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules recognize or bind epitopes in the context of MHC molecules, such as MHC class I molecules or MHC class II molecules. Both MHC class I molecules or MHC class II molecules are human leukocyte antigens (HLA). They act as an important component of the adaptive immune system. HLA expression is controlled by genes located on chromosome 6. It encodes a cell surface molecule that specifically presents antigenic peptides to T cell receptors on T cells.

在一些實施方案中，TCR、其抗原結合片段和TCR衍生的結合分子識別或結合在MHC I類分子的背景下的表位。MHC I類分子是人白細胞抗原(HLA)-A2分子，包括其任何一種或多種亞型，例如HLA-A*020l、*0202、*0203、*0206或*0207。人白細胞抗原A2(HLA-A2)是最常見的人血清型之一。在一些情況下，不同群體之間亞型的頻率可能有所不同。例如，HLA-A2陽性的白種人群中超過95%是HLA-A*020l，然而據報導在中國人群中，HLA-A*020l的頻率為大約23%、HLA-A*0207的頻率為45%，HLA-A*0206的頻率為8%、並且HLA-A*0203的頻率為23%。在一些實施方案中，MHC分子是HLA-A*020l。In some embodiments, TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules recognize or bind epitopes in the context of MHC class I molecules. MHC class I molecules are human leukocyte antigen (HLA)-A2 molecules, including any one or more subtypes thereof, such as HLA-A *0201, *0202, *0203, *0206, or *0207. Human leukocyte antigen A2 (HLA-A2) is one of the most common human serotypes. In some cases, the frequency of subtypes may vary between different groups. For example, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency of HLA-A*0201 has been reported to be approximately 23% and the frequency of HLA-A*0207 to be 45% , the frequency of HLA-A*0206 was 8%, and the frequency of HLA-A*0203 was 23%. In some embodiments, the MHC molecule is HLA-A*0201.

在一些實施方案中，結合分子(例如TCR或其抗原結合片段或TCR衍生的結合分子)是分離的或純化的，或者是重組的。在一些方面，結合分子(例如TCR或其抗原結合片段或TCR衍生的結合分子)是完全人的。在一些實施方案中，結合分子是單株的。在一些方面，結合分子是單鏈。在其他實施方案中，結合分子含有兩條鏈。在一些實施方案中，結合分子(例如TCR、其抗原結合片段或TCR衍生的結合分子)在細胞表面上表現。In some embodiments, a binding molecule (eg, a TCR or antigen-binding fragment or TCR-derived binding molecule) is isolated or purified, or recombinant. In some aspects, the binding molecule (eg, TCR or antigen-binding fragment or TCR-derived binding molecule) is fully human. In some embodiments, the binding molecule is monoclonal. In some aspects, the binding molecule is single-stranded. In other embodiments, the binding molecule contains two chains. In some embodiments, the binding molecule (eg, TCR, antigen-binding fragment thereof, or TCR-derived binding molecule) is expressed on the cell surface.

在一些實施方案中，TCR、其抗原結合片段或TCR衍生的結合分子特異性地結合腫瘤相關抗原，例如BCMA、CD19、CD22、CD30、CD33、CD56、CD123(也稱為IL-3R)、CEA、IL13Ra2、ALPP、EBV相關抗原(例如，LMP2)、EGFR、EGFRvIII、GD2、GPC3、HER2、HPV相關抗原(例如，E6或E7)、MAGE抗原、間皮素、MUC-1、NY-ESO-1、PSCA、PSMA、ROR1、WT1或Claudin 18.2。In some embodiments, the TCR, antigen-binding fragment thereof, or TCR-derived binding molecule specifically binds a tumor-associated antigen, eg, BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R), CEA , IL13Ra2, ALPP, EBV-associated antigen (eg, LMP2), EGFR, EGFRvIII, GD2, GPC3, HER2, HPV-associated antigen (eg, E6 or E7), MAGE antigen, mesothelin, MUC-1, NY-ESO- 1. PSCA, PSMA, ROR1, WT1 or Claudin 18.2.

TCR、其抗原結合片段或TCR衍生的結合分子可以具有Va和Vb、或與Va相似的區域和與Vb相似的區域。在一些實施方案中，TCR結合愛潑斯坦巴爾病毒(EBV)的潛在膜蛋白2(LMP2)。在一些實施方案中，Va區包含在SEQ ID NO: 22中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 23中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Va區包含在SEQ ID NO: 24中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 25中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Va區包含在SEQ ID NO: 26中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 27中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。A TCR, antigen-binding fragment thereof, or TCR-derived binding molecule can have Va and Vb, or a region similar to Va and a region similar to Vb. In some embodiments, the TCR binds latent membrane protein 2 (LMP2) of Epstein Barr virus (EBV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

在一些實施方案中，TCR結合人乳頭狀瘤病毒(HPV)的腫瘤抗原E6。在一些實施方案中，Va區包含在SEQ ID NO: 28中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 29中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Va區包含在SEQ ID NO: 32中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 33中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。In some embodiments, the TCR binds the tumor antigen E6 of human papillomavirus (HPV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

在一些實施方案中，TCR結合人乳頭狀瘤病毒(HPV)的腫瘤抗原E7。在一些實施方案中，Va區包含在SEQ ID NO: 30中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 31中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。In some embodiments, the TCR binds the tumor antigen E7 of human papillomavirus (HPV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

在一些實施方案中，TCR結合NY-ESO-1(癌症/睾丸抗原1，也稱為LAGE2)。在一些實施方案中，Va區包含在SEQ ID NO: 34中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，Vb區包含在SEQ ID NO: 35中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。In some embodiments, the TCR binds NY-ESO-1 (cancer/testis antigen 1, also known as LAGE2). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

在一些實施方案中，Va區包含一個或多個如本文所述的Va CDR序列。在一些實施方案中，Vb區包含一個或多個如本文所述的Vb CDR序列。In some embodiments, the Va region comprises one or more Va CDR sequences as described herein. In some embodiments, the Vb region comprises one or more Vb CDR sequences as described herein.

在一些實施方案中，編碼α鏈的核酸和編碼β鏈的核酸可以通過接頭(諸如本文其他地方所描述的任何接頭)連接。In some embodiments, the nucleic acid encoding the alpha chain and the nucleic acid encoding the beta chain can be linked by a linker, such as any of the linkers described elsewhere herein.

在一些實施方案中，通過與所關注的抗原結合，TCR或其抗原結合片段或TCR衍生的結合分子可以活化T細胞(例如，通過活化TCR訊息傳導途徑)。在一些實施方案中，活化可以上調免疫反應，增加細胞介素(例如IFNγ)和/或CD107a的表現，促進T細胞增殖和T細胞介導的殺傷。In some embodiments, the TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule can activate T cells by binding to the antigen of interest (eg, by activating the TCR signaling pathway). In some embodiments, activation can upregulate the immune response, increase the expression of cytokines (eg, IFNy) and/or CD107a, promote T cell proliferation and T cell mediated killing.

在一些實施方案中，如本文所述的TCR或其抗原結合片段或TCR衍生的結合分子可以使T細胞的免疫反應、活性或數量增加至少10%、20%、30%、40%、50%、60%、70%、80%、90%、100%、2倍、3倍、5倍、10倍或20倍。在一些實施方案中，當存在所關注的抗原時，TCR或其抗原結合片段或TCR衍生的結合分子可以增加IFN-γ的血清濃度。在一些實施方案中，活化可以誘導IFN-γ的血清濃度增加至少10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、5倍、10倍、100倍或1000倍。在一些實施方案中，活化可以誘導對靶細胞的特異性殺傷增加至少10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、3倍、4倍或5倍。In some embodiments, a TCR or antigen-binding fragment thereof or TCR-derived binding molecule as described herein can increase the immune response, activity or number of T cells by at least 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, 100%, 2X, 3X, 5X, 10X or 20X. In some embodiments, the TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule can increase the serum concentration of IFN-γ in the presence of the antigen of interest. In some embodiments, activation can induce at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5x, 10x, 100x or 1000x. In some embodiments, activation can induce at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold increase in specific killing of target cells , 3 times, 4 times or 5 times.

在一些方面，提供的重組TCR包括至少部分地獨立於CD8的TCR。在一些方面，提供的重組TCR包括至少部分地依賴於CD8的TCR。In some aspects, provided recombinant TCRs include TCRs that are at least partially independent of CD8. In some aspects, provided recombinant TCRs include TCRs that are at least partially dependent on CD8.

在一些實施方案中，TCR或其抗原結合片段或TCR衍生的結合分子具有相對高的表現效率。例如，在相同條件下，本文所述的TCR或其抗原結合片段或TCR衍生的結合分子的表現效率可以比參考分子(例如，內源性TCR)高至少10%、20%、30%、40%、50%或100%。In some embodiments, the TCR or antigen-binding fragment thereof or TCR-derived binding molecule has a relatively high expression efficiency. For example, a TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule described herein can perform at least 10%, 20%, 30%, 40% higher than a reference molecule (eg, an endogenous TCR) under the same conditions %, 50% or 100%.

在一些實施方案中，結合分子(例如TCR)不表現出交叉反應性或脫靶結合，諸如不期望的脫靶結合，例如與健康或正常組織或細胞中存在的抗原的脫靶結合。嵌合抗原受體和結合分子 In some embodiments, the binding molecules (eg, TCRs) do not exhibit cross-reactivity or off-target binding, such as undesired off-target binding, eg, off-target binding to antigens present in healthy or normal tissues or cells. Chimeric Antigen Receptors and Binding Molecules

嵌合抗原受體(CAR)將正常T細胞活化的許多方面特徵組合成單個蛋白質。它們將細胞外抗原識別結構域連接至細胞內訊息傳導結構域，所述細胞內訊息傳導結構域當結合抗原時活化T細胞。CAR通常由四個區域構成：抗原識別結構域、細胞外鉸鏈區、跨膜結構域和細胞內T細胞訊息傳導結構域。Chimeric antigen receptors (CARs) combine many aspects of normal T cell activation into a single protein. They link extracellular antigen recognition domains to intracellular signaling domains that activate T cells when bound to antigen. CAR is usually composed of four regions: antigen recognition domain, extracellular hinge region, transmembrane domain and intracellular T cell signaling domain.

抗原識別結構域在受體的胞外結構域部分中暴露於細胞外部。其與潛在的靶分子相互作用，並且負責將CAR-T細胞靶向表現匹配分子的任何細胞。抗原識別結構域通常源自作為單鏈可變片段(scFv)連接在一起的單株抗體的可變區。scFv是由用短接頭肽連接的免疫球蛋白的輕鏈(VL)和重鏈(VH)組成的嵌合蛋白。兩條鏈之間的接頭由親水性殘基組成，其中的一段甘胺酸和絲胺酸用於柔性，以及一段麩胺酸和離胺酸用於增加溶解度。在一些實施方案中，抗原結合結構域特異性地結合腫瘤相關抗原，例如BCMA、CD19、CD22、CD30、CD33、CD56、CD123(也稱為IL-3R)、CEA、EBV相關抗原(例如LMP2)、EGFR、GD2、GPC3、HER2、HPV相關抗原(例如E6)、MAGE抗原、間皮素、MUC-1、NY-ESO-1、PSCA、PSMA、ROR1、WT1、或Claudin 18.2。The antigen recognition domain is exposed outside the cell in the extracellular domain portion of the receptor. It interacts with potential target molecules and is responsible for targeting CAR-T cells to any cell that expresses the matching molecule. Antigen recognition domains are typically derived from the variable regions of monoclonal antibodies linked together as single-chain variable fragments (scFvs). scFvs are chimeric proteins consisting of the light (VL) and heavy (VH) chains of immunoglobulins linked with a short linker peptide. The linker between the two chains consists of hydrophilic residues, with a stretch of glycine and serine for flexibility and a stretch of glutamic and lysine for solubility. In some embodiments, the antigen binding domain specifically binds a tumor-associated antigen, eg, BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R), CEA, EBV-associated antigen (eg, LMP2) , EGFR, GD2, GPC3, HER2, HPV-associated antigen (eg, E6), MAGE antigen, mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.

鉸鏈，也稱為間隔子，是位於抗原結識別區與細胞外膜之間的小結構的結構域。理想的鉸鏈增強scFv受體頭部的柔性，從而減少CAR與其靶抗原之間的空間限制。這促進了CAR-T細胞與靶細胞之間的抗原結合和突觸形成。鉸鏈序列通常是基於來自其他免疫分子(包括IgG、CD8和CD28)的膜近端區。A hinge, also known as a spacer, is a domain of a small structure located between the antigen-knot recognition region and the outer cell membrane. An ideal hinge enhances the flexibility of the scFv receptor head, thereby reducing the steric confinement between the CAR and its target antigen. This promotes antigen binding and synapse formation between CAR-T cells and target cells. Hinge sequences are often based on membrane-proximal regions from other immune molecules, including IgG, CD8, and CD28.

跨膜結構域是一種結構組分，由橫跨細胞膜的疏水性α螺旋組成。它將CAR錨定在質膜上，將細胞外鉸鏈和抗原識別結構域與細胞內訊息傳導區橋接。此結構域對於整個受體的穩定性至關重要。通常，使用來自胞內結構域的最接近膜的組分的跨膜結構域，但是不同的跨膜結構域導致不同的受體穩定性。已知CD28跨膜結構域導致高度表現的穩定受體。在一些實施方案中，跨膜結構域是4-1BB/CD137的跨膜結構域、T細胞受體的α鏈、T細胞受體的β鏈、CD3ε、CD4、CD5、CD8α、CD9、CD16、CD19、CD22、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、或T細胞受體的ζ鏈、或它們的任何組合。The transmembrane domain is a structural component consisting of hydrophobic alpha helices that span the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen recognition domain with the intracellular signaling region. This domain is critical for the stability of the entire receptor. Typically, the transmembrane domain from the closest membrane component of the intracellular domain is used, but different transmembrane domains result in different receptor stability. The CD28 transmembrane domain is known to result in a highly expressed stable receptor. In some embodiments, the transmembrane domain is the transmembrane domain of 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of the T cell receptor, or any combination thereof.

細胞內T細胞訊息傳導結構域位於細胞內部的受體胞內結構域中。在抗原結合外部抗原識別結構域之後，CAR受體聚集在一起並傳輸活化訊息。然後，受體的內部細胞質末端使T細胞內部的訊息傳導永久化。正常的T細胞活化依賴於CD3-ζ細胞質結構域中存在的基於免疫受體酪胺酸的活化基序(ITAM)的磷酸化。為了模擬此過程，通常將CD3-ζ的細胞質結構域用作主要的CAR胞內結構域組分。還嘗試了其他含ITAM的結構域，但不是那麼有效。T細胞除CD3訊息傳導外還需要共刺激分子，以便在活化之後持續存在。由於這個原因，CAR受體的胞內結構域通常還包括來自共刺激蛋白的一個或多個嵌合結構域。已經成功地測試了來自多種共刺激分子的訊息傳導結構域，包括CD28、CD27、CD134(OX40)和CD137(4-1BB)。The intracellular T cell signaling domain is located in the receptor intracellular domain inside the cell. After antigen binding to the external antigen recognition domain, CAR receptors come together and transmit activation messages. The inner cytoplasmic end of the receptor then perpetuates the signaling inside the T cell. Normal T cell activation relies on phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) present in the cytoplasmic domain of CD3-ζ. To mimic this process, the cytoplasmic domain of CD3-ζ is typically used as the main CAR intracellular domain component. Other ITAM-containing domains were also tried, but not as effective. T cells require co-stimulatory molecules in addition to CD3 signaling in order to persist after activation. For this reason, the intracellular domains of CAR receptors often also include one or more chimeric domains from co-stimulatory proteins. Messaging domains from a variety of costimulatory molecules have been successfully tested, including CD28, CD27, CD134 (OX40) and CD137 (4-1BB).

多種CAR分子和表現這些CAR分子的載體可以用於本文所述的方法。在一些實施方案中，CAR分子特異性地結合腫瘤相關抗原，例如BCMA。在一些實施方案中，CAR包含在SEQ ID NO: 37、38或39中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，CAR分子特異性地結合CD19。在一些實施方案中，CAR包含在SEQ ID NO: 36中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。在一些實施方案中，CAR分子特異性地靶向IL13Ra2。在一些實施方案中，CAR包含在SEQ ID NO: 40中任一者中列出的胺基酸序列、或者與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的胺基酸序列。例如WO2018200496A1、WO2019241686A1、WO2018085690A1、WO2018028647A1和WO2018052828A1描述了這些CAR分子或其靶結合結構域中的一些，這些專利中的每一者全文以引用方式併入本文。A variety of CAR molecules and vectors expressing these CAR molecules can be used in the methods described herein. In some embodiments, the CAR molecule specifically binds a tumor-associated antigen, such as BCMA. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, Amino acid sequences of 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the CAR molecule specifically binds CD19. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, of the amino acid sequence set forth in any one of SEQ ID NO: 36 %, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CAR molecule specifically targets IL13Ra2. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, of the amino acid sequence set forth in any one of SEQ ID NO: 40 %, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. Some of these CAR molecules or target binding domains thereof are described, for example, in WO2018200496A1, WO2019241686A1, WO2018085690A1, WO2018028647A1, and WO2018052828A1, each of which is incorporated herein by reference in its entirety.

示例性抗原受體(包括CAR)以及將此類受體工程化並引入細胞中的方法包括描述於例如以下文獻中的那些：Chandran等人，“T cell receptor‐based cancer immunotherapy: Emerging efficacy and pathways of resistance.” Immunological reviews 290.1 (2019): 127-147；Cartellieri, Marc等人，“Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.” BioMed Research International 2010 (2010)；以及PCT公開號WO2017173256A1；US2002/131960、US2013/287748、US2013/0149337、U.S. 6,451,995、U.S. 7,446,190、U.S. 8,252,592；這些文獻中的每一者全文以引用方式併入本文。Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells include those described, for example, in Chandran et al., "T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.” Immunological reviews 290.1 (2019): 127-147; Cartellieri, Marc et al., “Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.” BioMed Research International 2010 (2010); and PCT Publication No. WO2017173256A1; US2002 /131960, US2013/287748, US2013/0149337, U.S. 6,451,995, U.S. 7,446,190, U.S. 8,252,592; each of these documents are incorporated herein by reference in their entirety.

在一些實施方案中，本文所述的CAR包含抗體模擬物。抗體模擬物是以類似於抗體的方式表現並且結合特定抗原但與抗體(例如，抗體片段)無關的化合物。在一些實施方案中，抗體模擬物是肽、核酸、小分子或它們的組合。在一些實施方案中，抗體模擬物是親和體分子(例如，蛋白A的Z結構域)、adnectin、單體(例如，纖連蛋白的第10類III結構域)、肽適體、黏合素(affimer)(例如，胱抑素)、affilin(例如，γ-B晶體蛋白或泛素)、affitin(例如，Sac7d)、α體(例如，三重螺旋捲曲螺旋)、抗運載蛋白(例如，脂質運載蛋白)、avimer(例如，各種膜受體的A結構域)、fynomer(例如，Fyn的SH3結構域)、犰狳重複蛋白(armadillo repeat protein)、DARPin(例如，錨蛋白重複基序)、Kunitz結構域肽(例如，各種蛋白酶抑制劑的Kunitz結構域)、設計的錨蛋白重複蛋白、nanoCLAMP(例如，碳水化合物結合模組32-2)、半胱胺酸結肽(knottin)或它們的組合。詳細描述可見於例如Yu等人 ，“Beyond antibodies as binding partners: the role of antibody mimetics in bioanalysis.” Annual Review of Analytical Chemistry 10 (2017): 293-320；以及Ta和Brian，“Antibody and antibody mimetic immunotherapeutics.” (2017): 1301-1304；這些文獻中的每一者全文以引用的方式併入本文。工程化細胞 In some embodiments, the CARs described herein comprise antibody mimetics. Antibody mimetics are compounds that behave in a similar manner to antibodies and bind a specific antigen but are unrelated to antibodies (eg, antibody fragments). In some embodiments, the antibody mimetic is a peptide, nucleic acid, small molecule, or a combination thereof. In some embodiments, the antibody mimetic is an avidin molecule (eg, the Z domain of protein A), adnectin, a monomer (eg, the class 10 III domain of fibronectin), a peptide aptamer, a cohesin ( affimer) (eg, cystatin), affilin (eg, gamma-B crystallin or ubiquitin), affitin (eg, Sac7d), alpha body (eg, triple helix coiled-coil), anticalin (eg, lipocalin) protein), avimer (eg, A domain of various membrane receptors), fynomer (eg, SH3 domain of Fyn), armadillo repeat protein, DARPin (eg, ankyrin repeat motif), Kunitz Domain peptides (eg, Kunitz domains of various protease inhibitors), designed ankyrin repeat proteins, nanoCLAMPs (eg, carbohydrate binding module 32-2), cysteine knot peptides (knottins), or combinations thereof . Detailed descriptions can be found, for example, in Yu et al ., "Beyond antibodies as binding partners: the role of mimetics in bioanalysis." Annual Review of Analytical Chemistry 10 (2017): 293-320; and Ta and Brian, "Antibody and antibody mimetic antibody immunotherapeutics ." (2017): 1301-1304; each of these documents is incorporated by reference in its entirety. engineered cells

本公開提供了表現如本文所述的IL-12(例如，膜拴系IL-12)、TCR、CAR和/或各種蛋白質的工程化細胞(例如，免疫細胞、T細胞、NK細胞、腫瘤浸潤性淋巴細胞)。這些工程化細胞可以用於治療如本文所述的各種障礙或疾病(例如，病毒感染、癌症、病毒誘導的障礙)。The present disclosure provides engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) expressing IL-12 (eg, membrane-tethered IL-12), TCR, CAR, and/or various proteins as described herein lymphocytes). These engineered cells can be used to treat various disorders or diseases as described herein (eg, viral infections, cancer, virus-induced disorders).

在各種實施方案中，被工程化的細胞可以從例如人和非人動物中獲得。在各種實施方案中，被工程化的細胞可以從細菌、真菌、人、大鼠、小鼠、兔、猴、豬或任何其他物種獲得。優選地，所述細胞來自人、大鼠或小鼠。在一些實施方案中，所述細胞是小鼠淋巴細胞，並且被工程化(例如，轉導)以表現TCR、CAR或它們的抗原結合片段。在一些實施方案中，所述細胞從人獲得。在各種實施方案中，被工程化的細胞是血細胞。優選地，所述細胞是白細胞(例如T細胞)、淋巴細胞或任何其他合適的血細胞類型。在一些實施方案中，所述細胞是外周血細胞。在一些實施方案中，所述細胞是腫瘤浸潤性淋巴細胞(TIL)。在一些實施方案中，所述細胞是T細胞、B細胞或NK細胞。在一些實施方案中，所述細胞是人外周血單核細胞(PBMC)。在一些實施方案中，人PBMC是CD3+細胞。在一些實施方案中，人PBMC是CD8+細胞。In various embodiments, the engineered cells can be obtained from, for example, humans and non-human animals. In various embodiments, the engineered cells can be obtained from bacteria, fungi, human, rat, mouse, rabbit, monkey, pig, or any other species. Preferably, the cells are from human, rat or mouse. In some embodiments, the cells are mouse lymphocytes and are engineered (eg, transduced) to express TCRs, CARs, or antigen-binding fragments thereof. In some embodiments, the cells are obtained from a human. In various embodiments, the engineered cells are blood cells. Preferably, the cells are leukocytes (eg T cells), lymphocytes or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are T cells, B cells, or NK cells. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs). In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells.

在一些實施方案中，細胞是T細胞。在一些實施方案中，T細胞可以表現識別靶細胞表面上的特異性抗原部分的細胞表面受體。細胞表面受體可以是野生型或重組T細胞受體(TCR)、嵌合抗原受體(CAR)或能夠識別與靶細胞相關的抗原部分的任何其他表面受體。T細胞可以通過本領域已知的各種方法獲得，例如，通過從患者中分離的T細胞(例如，腫瘤浸潤性淋巴細胞)的體外培養獲得。可以通過用病毒載體轉導T細胞(例如，從患者外周血中分離的)來獲得遺傳修飾的T細胞。在一些實施方案中，T細胞是TCR基因修飾或CAR修飾的T細胞。在一些實施方案中，T細胞是CD4+ T細胞、CD8+ T細胞或調節性T細胞。在一些實施方案中，T細胞是1型T輔助細胞和2型T輔助細胞。在一些實施方案中，表現此受體的T細胞是αβ-T細胞。在替代性實施方案中，表現此受體的T細胞是γδ-T細胞。在一些實施方案中，T細胞是中樞記憶T細胞。在一些實施方案中，T細胞是效應記憶T細胞。在一些實施方案中，T細胞是幼稚T細胞。In some embodiments, the cells are T cells. In some embodiments, T cells may express cell surface receptors that recognize specific antigenic moieties on the surface of target cells. Cell surface receptors can be wild-type or recombinant T cell receptors (TCRs), chimeric antigen receptors (CARs), or any other surface receptors capable of recognizing antigenic moieties associated with target cells. T cells can be obtained by various methods known in the art, eg, by in vitro culture of T cells (eg, tumor-infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (eg, isolated from the peripheral blood of a patient) with a viral vector. In some embodiments, the T cells are TCR gene-modified or CAR-modified T cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are type 1 T helper cells and type 2 T helper cells. In some embodiments, the T cells expressing this receptor are αβ-T cells. In an alternative embodiment, the T cells expressing this receptor are γδ-T cells. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are naive T cells.

在一些實施方案中，所述細胞是NK細胞。在一些實施方案中，工程化細胞的製備包括一個或多個培養和/或製備步驟。可以從樣品(諸如生物樣品，例如從受試者獲得或來源的生物樣品)中分離用於引入結合分子(例如，TCR或CAR)的細胞。在一些實施方案中，從其分離細胞的受試者是患有疾病或病症或需要細胞療法、或將向其施用細胞療法的受試者。在一些實施方案中，受試者是需要特定治療干預(諸如針對其分離、處理細胞和/或對細胞進行工程化的過繼細胞療法)的人。In some embodiments, the cells are NK cells. In some embodiments, the preparation of engineered cells includes one or more culturing and/or preparation steps. The cells used to introduce the binding molecule (eg, TCR or CAR) can be isolated from a sample, such as a biological sample, eg, obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of, or to which cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

在一些實施方案中，所述細胞是幹細胞，諸如多潛能和多能幹細胞，包括誘導的多能幹細胞(iPSC)。所述細胞可以是原代細胞，諸如直接從受試者中分離和/或從受試者中分離並冷凍的那些細胞。在一些實施方案中，將幹細胞與另外的分化因子一起培養以獲得所需的細胞類型(例如T細胞)。In some embodiments, the cells are stem cells, such as pluripotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells may be primary cells, such as those cells isolated directly from the subject and/or isolated from the subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain the desired cell type (eg, T cells).

可以從適當的分離方法中獲得不同的細胞類型。所述分離方法包括基於一種或多種特定分子(諸如表面標記，例如表面蛋白、細胞內標記、或核酸)在細胞中的表現或存在來分離不同的細胞類型。在一些實施方案中，可以使用基於此類標記的任何已知的分離方法。在一些實施方案中，所述分離是基於親和力或免疫親和力的分離。例如，在一些方面，所述分離包括例如基於一種或多種標記(通常是細胞表面標記)的細胞表現或表現位準，通過與特異性地結合此類標記的抗體或結合伴侶一起孵育，隨後通常是洗滌步驟和將已經結合抗體或結合伴侶的細胞從未結合抗體或結合伴侶的細胞分開而分離細胞和細胞群。Different cell types can be obtained from appropriate isolation methods. The methods of separation include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, eg, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is an affinity or immunoaffinity-based separation. For example, in some aspects, the isolating comprises, for example, the expression or level of expression of cells based on, for example, one or more markers (usually cell surface markers), by incubating with antibodies or binding partners that specifically bind such markers, followed by usually It is the washing steps and separation of cells and cell populations that separate cells that have bound the antibody or binding partner from cells that have not bound the antibody or binding partner.

此類分離步驟可以是基於陽性選擇，其中已經結合試劑的細胞被保留以供進一步使用，和/或基於陰性選擇，其中未結合抗體或結合伴侶的細胞被保留。在一些實例中，兩個級分均被保留供進一步使用。在一些方面，在沒有可用的抗體特異性地鑒定異源群體中的細胞類型，使得最好基於由除所需群體以外的細胞表現的標記來進行分離的情況下，陰性選擇可能特別有用。Such isolation steps may be based on positive selection, in which cells that have bound the reagent are retained for further use, and/or based on negative selection, in which cells not bound to the antibody or binding partner are retained. In some instances, both fractions are reserved for further use. In some aspects, negative selection may be particularly useful where no antibodies are available that specifically identify cell types in a heterologous population such that separation is best performed based on markers expressed by cells other than the desired population.

還提供了用於表現結合分子並用於生產表現此類結合分子的基因工程化細胞的方法、核酸、組合物和試劑盒。基因工程化通常涉及諸如通過反轉錄病毒轉導、轉染或轉化將編碼治療分子(例如TCR、CAR(例如TCR樣CAR)、IL-12(例如，膜拴系IL-12)、多肽、融合蛋白)的核酸引入細胞中。在一些實施方案中，通過以下方式來實現基因轉移：首先刺激細胞，諸如通過將其與誘導反應(諸如增殖、存活和/或活化，例如通過細胞介素或活化標記的表現所測量的)的刺激物組合，隨後轉導活化的細胞並在培養物中擴增至足以用於臨床應用的數量。Also provided are methods, nucleic acids, compositions and kits for expressing binding molecules and for producing genetically engineered cells expressing such binding molecules. Genetic engineering typically involves transduction, transfection, or transformation, such as by retroviral transduction, encoding a therapeutic molecule (eg, TCR, CAR (eg, TCR-like CAR), IL-12 (eg, membrane-tethered IL-12), polypeptide, fusion protein) nucleic acid into cells. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with an induction response (such as proliferation, survival, and/or activation, eg, as measured by the expression of interleukins or activation markers) The combination of stimuli, activated cells are then transduced and expanded in culture to a sufficient number for clinical use.

在一些實施方案中，使用重組感染性病毒顆粒(諸如，例如源自猿猴病毒40(SV40)、腺病毒、腺相關病毒(AAV)的載體)，將重組核酸轉移到細胞中。在一些實施方案中，使用重組慢病毒載體或反轉錄病毒載體(諸如γ-反轉錄病毒載體)，將重組核酸轉移到T細胞中。在一些實施方案中，反轉錄病毒載體具有長末端重複序列(LTR)，例如衍生自莫洛尼鼠白血病病毒(MoMLV)、骨髓增生性肉瘤病毒(MPSV)、鼠胚胎幹細胞病毒(MESV)、鼠幹細胞病毒(MSCV)或脾病灶形成病毒(SFFV)的反轉錄病毒載體。大多數反轉錄病毒載體衍生自鼠反轉錄病毒。在一些實施方案中，所述反轉錄病毒包括衍生自任何禽類或哺乳動物細胞來源的那些。所述反轉錄病毒通常是雙嗜性的，這意味著它們能夠感染包括人在內的若干個物種的宿主細胞。在一些實施方案中，載體是慢病毒載體。在一些實施方案中，重組核酸通過電穿孔轉移到T細胞中。在一些實施方案中，重組核酸通過轉座轉移到T細胞中。在免疫細胞中引入和表現遺傳物質的其他方法包括磷酸鈣轉染、原生質體融合、陽離子脂質體介導的轉染；鎢粒子促進的微粒轟擊和磷酸鍶DNA共沉澱。例如在WO2019195486中描述了這些方法中的許多，該專利全文以引用方式併入本文。In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant lentiviral or retroviral vectors (such as gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells. In some embodiments, the retroviral vector has long terminal repeats (LTR), eg, derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Retroviral vector for stem cell virus (MSCV) or spleen foci forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses are generally amphiphilic, which means that they are capable of infecting host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, recombinant nucleic acids are transferred into T cells by electroporation. In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment and strontium phosphate DNA coprecipitation. Many of these methods are described, for example, in WO2019195486, which is incorporated herein by reference in its entirety.

在一些方面，人源化和/或完全人源重組TCR的開發提出了技術挑戰。例如，在一些方面，人源化和/或全人源重組TCR受體當被工程化為人T細胞時可以與內源性TCR複合物競爭和/或可以與內源性TCRa鏈和/或TCRb鏈形成錯誤配對，這在某些方面可能降低重組TCR訊息傳導、活性和/或表現，並最終導致工程化細胞的活性降低。可以對工程化細胞進行基因修飾。在一些實施方案中，工程化細胞可以包含T細胞受體α恆定(TRAC)基因和/或T細胞受體β恆定(TRBC)基因的遺傳破壞。在一些實施方案中，TRBC基因是T細胞受體β恆定1(TRBCJ)或T細胞受體β恆定2(TRBC2)基因中的一者或兩者。在一些實施方案中，工程化細胞不表現內源性TCR a鏈和/或TRC b鏈。在一些其他方面，使用非人恆定結構域，例如齧齒動物(例如小鼠)恆定結構域。使用非人恆定結構域可以有效地降低配對錯誤的可能性。In some aspects, the development of humanized and/or fully human recombinant TCRs presents technical challenges. For example, in some aspects, humanized and/or fully human recombinant TCR receptors, when engineered into human T cells, can compete with endogenous TCR complexes and/or can compete with endogenous TCRα chains and/or The TCRb chains form mispairings, which in some ways may reduce recombinant TCR signaling, activity, and/or expression, and ultimately lead to reduced activity in the engineered cells. The engineered cells can be genetically modified. In some embodiments, the engineered cells may comprise genetic disruption of the T cell receptor alpha constant (TRAC) gene and/or the T cell receptor beta constant (TRBC) gene. In some embodiments, the TRBC gene is one or both of the T cell receptor beta constant 1 (TRBCJ) or T cell receptor beta constant 2 (TRBC2) genes. In some embodiments, the engineered cells do not express endogenous TCR alpha chains and/or TRC beta chains. In some other aspects, non-human constant domains are used, eg, rodent (eg, mouse) constant domains. The use of non-human constant domains can effectively reduce the possibility of pairing errors.

還提供了工程化細胞群、含有此類細胞和/或富集此類細胞(諸如其中表現結合分子的細胞組成至少15%、20%、25%、30%、35%、40%、50%、60%、70%、80%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高百分比的在組合物中的總細胞或某種類型的細胞(諸如T細胞、CD8+或CD4+細胞))的組合物。Also provided are populations of engineered cells, containing such cells and/or enriched for such cells (such as in which cells expressing the binding molecule constitute at least 15%, 20%, 25%, 30%, 35%, 40%, 50%) , 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentages of the total in the composition A composition of cells or cells of a certain type, such as T cells, CD8+ or CD4+ cells.

在一些實施方案中，將工程化細胞(例如TCR-T細胞)與靶細胞(例如，抗原呈遞細胞)一起共培養至少或約1小時、2小時、3小時、4小時、5小時、6小時、12小時、18小時、1天、2天、3天或更長時間，使得可以活化工程化細胞(例如，TCR-T細胞)。在一些實施方案中，靶細胞是Jurkat細胞。在一些實施方案中，靶細胞是LLC-HLA-A2-Peplinker(LLW)黑色素瘤細胞。在一些實施方案中，靶細胞是抗原呈遞細胞。在一些實施方案中，靶細胞表現主要組織相容性複合物(MHC)(例如，I類、II類和/或III類MHC)。在一些實施方案中，靶細胞包含人白細胞抗原(HLA)系統。In some embodiments, engineered cells (eg, TCR-T cells) are co-cultured with target cells (eg, antigen-presenting cells) for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours , 12 hours, 18 hours, 1 day, 2 days, 3 days, or longer, so that engineered cells (eg, TCR-T cells) can be activated. In some embodiments, the target cells are Jurkat cells. In some embodiments, the target cells are LLC-HLA-A2-Peplinker (LLW) melanoma cells. In some embodiments, the target cells are antigen presenting cells. In some embodiments, the target cell expresses a major histocompatibility complex (MHC) (eg, class I, class II, and/or class III MHC). In some embodiments, the target cells comprise the human leukocyte antigen (HLA) system.

在一些實施方案中，工程化細胞可以表現IL-12和經修飾的IL-12。例如，例如當融合蛋白是膜拴系蛋白時，包含本文所述的經修飾的IL-12的融合蛋白可以在工程化細胞的細胞表面上表現。在一些情形下，例如當融合蛋白是可溶蛋白時，可以表現和分泌包含本文所述的經修飾的IL-12的融合蛋白。In some embodiments, the engineered cells can express IL-12 and modified IL-12. For example, a fusion protein comprising modified IL-12 described herein can be expressed on the cell surface of an engineered cell, eg, when the fusion protein is a membrane-tethered protein. In some cases, such as when the fusion protein is a soluble protein, a fusion protein comprising the modified IL-12 described herein can be expressed and secreted.

IL-12在工程化細胞中的表現提供了一些另外的益處。例如，它可以增加從NK和T細胞的IFN-γ(其是IL-12作用的最強介體)產生，刺激活化的NK細胞、CD8+和CD4+ T細胞的生長和細胞毒性，將CD4+ Th0細胞的分化朝向Th1表型改變，增加針對腫瘤細胞的抗體依賴性細胞毒性(ADCC)，並誘導IgG和將從B細胞的IgE產生抑制例如至少或約1倍、2倍、3倍、4倍、5倍、10倍或20倍。The expression of IL-12 in engineered cells provides several additional benefits. For example, it can increase the production of IFN-γ (which is the strongest mediator of IL-12 action) from NK and T cells, stimulate the growth and cytotoxicity of activated NK cells, CD8+ and CD4+ T cells, reduce the production of CD4+ Th0 cells Differentiation changes towards a Th1 phenotype, increases antibody-dependent cytotoxicity (ADCC) against tumor cells, and induces IgG and inhibition of IgE production from B cells, eg, at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold times, 10 times or 20 times.

在一些實施方案中，如與沒有共培養的工程化細胞的細胞介素分泌位準相比，與靶細胞一起共培養可以使工程化細胞的細胞介素(例如IFNγ)分泌增加至少或約1倍、2倍、5倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍、200倍、500倍、1000倍、2000倍、5000倍、10000倍或更多。In some embodiments, co-cultivation with target cells can increase the secretion of interleukins (eg, IFNγ) by the engineered cells by at least or about 1 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times or more.

在一些實施方案中，與不表現經修飾的IL-12的工程化細胞的細胞介素表現或分泌位準相比，經修飾的IL-12表現可使工程化細胞(例如，TCR-T細胞)的細胞介素(例如，IFNγ)表現或分泌增加至少或約1倍、2倍、5倍、10倍、20倍、50倍、100倍、200倍、500倍、1000倍、2000倍、5000倍、10000倍或更多。在一些實施方案中，與不表現經修飾的IL-12的工程化細胞附近的細胞的細胞介素表現或分泌位準相比，工程化細胞(例如，TCR-T細胞)中經修飾的IL-12表現可刺激工程化細胞附近的免疫細胞的細胞介素(例如，IFNγ)表現或分泌增加至少或約1倍、2倍、5倍、10倍、20倍、50倍、100倍、200倍、500倍、1000倍、2000倍、5000倍、10000倍或更多。在一些實施方案中，與不表現經修飾的IL-12的工程化細胞或不表現經修飾的IL-12的工程化細胞附近的免疫細胞的一種或多種早期TCR活化標記物(例如，CD69)的表現位準相比，經修飾的IL-12表現可使工程化細胞(例如，TCR-T細胞)或工程化細胞附近的免疫細胞的一種或多種早期TCR活化標記物的表現增加至少或約1倍、2倍、5倍、10倍、20倍、50倍、100倍、200倍、500倍、1000倍或更多倍。In some embodiments, the expression of modified IL-12 may allow engineered cells (eg, TCR-T cells) to compare to the level of interferon expression or secretion of engineered cells that do not express modified IL-12 ) at least or about a 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000 times, 10000 times or more. In some embodiments, the modified IL in an engineered cell (eg, a TCR-T cell) is compared to the level of cytokine expression or secretion in cells adjacent to the engineered cell that does not express the modified IL-12 -12 expression can stimulate at least or about a 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold increase in interferon (eg, IFNγ) expression or secretion by immune cells adjacent to the engineered cells times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times or more. In some embodiments, one or more early TCR activation markers (eg, CD69) of immune cells in the vicinity of engineered cells that do not express modified IL-12 or engineered cells that do not express modified IL-12 Modified IL-12 expression may increase the expression of one or more early TCR activation markers by engineered cells (e.g., TCR-T cells) or immune cells adjacent to the engineered cells by at least or about the expression level of 1x, 2x, 5x, 10x, 20x, 50x, 100x, 200x, 500x, 1000x or more.

在一些實施方案中，細胞是人PBMC，並且被工程化(例如，轉導)以表現IL-12、TCR、CAR或它們的抗原結合片段。在一些實施方案中，工程化細胞還可表現如本文所述的經修飾的IL-12。在一些實施方案中，經修飾的IL-12拴系至工程化細胞的膜。在一些實施方案中，當工程化細胞(例如，PBMC、TCRb+PBMC或TCRb-PBMC)與靶細胞(例如，抗原呈遞細胞)共培養時，與不表現膜拴系IL-12的工程化細胞的細胞介素表現或分泌位準相比，膜拴系IL-12可使工程化細胞的細胞介素(例如，IFNγ)表現或分泌增加至少或約10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、3倍、4倍、5倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍或更多。在一些實施方案中，當工程化細胞(例如，PBMC或TCRb-PBMC)與靶細胞(例如，抗原呈遞細胞)共培養時，與不表現膜拴系IL-12的工程化細胞中的活化T細胞群相比，膜拴系IL-12可使活化T細胞群增加至少或約10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、3倍、4倍、5倍、10倍、20倍、50倍、100倍或更多。在一些實施方案中，可以通過CD69表現位準來測量T細胞活化狀態。In some embodiments, the cells are human PBMCs and are engineered (eg, transduced) to express IL-12, TCR, CAR, or antigen-binding fragments thereof. In some embodiments, the engineered cells may also express modified IL-12 as described herein. In some embodiments, the modified IL-12 is tethered to the membrane of the engineered cell. In some embodiments, when engineered cells (eg, PBMCs, TCRb+PBMCs, or TCRb-PBMCs) are co-cultured with target cells (eg, antigen-presenting cells), engineered cells that do not express membrane-tethered IL-12 Membrane-tethered IL-12 can increase the expression or secretion of interferons (e.g., IFNγ) by at least or about 10%, 20%, 30%, 40% in engineered cells compared to the level of interleukin expression or secretion , 50%, 60%, 70%, 80%, 90%, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times times, 80 times, 90 times, 100 times or more. In some embodiments, when engineered cells (eg, PBMCs or TCRb-PBMCs) are co-cultured with target cells (eg, antigen-presenting cells), activated T cells in engineered cells that do not express membrane-tethered IL-12 Membrane-tethered IL-12 can increase activated T cell population by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold compared to cell population , 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or more. In some embodiments, T cell activation status can be measured by the level of CD69 expression.

在一些實施方案中，與不表現膜拴系IL-12的工程化細胞中的CD4+ T細胞群相比，膜拴系IL-12可使工程化細胞(例如，PBMC)中的CD4+ T細胞群增加至少或約10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、3倍、4倍、5倍、10倍、20倍、50倍、100倍或更多。在一些實施方案中，與不表現膜拴系IL-12的工程化細胞中的CD8+ T細胞群相比，膜拴系IL-12可使工程化細胞(例如，PBMC)中的CD8+ T細胞群減少至小於或約90%、80%、70%、60%、50%、40%、30%、20%或更少。In some embodiments, membrane-tethered IL-12 enables a population of CD4+ T cells in engineered cells (eg, PBMCs) as compared to a population of CD4+ T cells in engineered cells that do not express membrane-tethered IL-12 Increase at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20 times, 50 times, 100 times or more. In some embodiments, membrane-tethered IL-12 enables a population of CD8+ T cells in engineered cells (eg, PBMCs) as compared to a population of CD8+ T cells in engineered cells that do not express membrane-tethered IL-12 Reduce to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less.

在一些實施方案中，與不表現膜拴系IL-12的工程化細胞中的效應記憶細胞群相比，膜拴系IL-12可使工程化細胞(例如，CD4+ PBMC或CD8+ PBMC)中的效應記憶細胞群增加至少或約10%、20%、30%、40%、50%、60%、70%、80%、90%、1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、20倍、50倍、100倍或更多。在一些實施方案中，與不表現膜拴系IL-12的工程化細胞中的中樞記憶細胞群相比，膜拴系IL-12可使工程化細胞(例如，CD4+ PBMC或CD8+ PBMC)中的中樞記憶細胞群減少至小於或約90%、80%、70%、60%、50%、40%、30%、20%或更少。In some embodiments, membrane-tethered IL-12 can increase IL-12 in engineered cells (eg, CD4+ PBMC or CD8+ PBMC) as compared to a population of effector memory cells in engineered cells that do not express membrane-tethered IL-12 Increase in effector memory cell population by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6x, 7x, 8x, 9x, 10x, 20x, 50x, 100x or more. In some embodiments, membrane-tethered IL-12 can increase the number of cells in an engineered cell (eg, CD4+ PBMC or CD8+ PBMC) compared to a population of central memory cells in engineered cells that do not express membrane-tethered IL-12 The central memory cell population is reduced to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less.

在一些實施方案中，細胞(例如，PBMC)被工程化為表現包含可溶性部分的膜拴系IL-12融合蛋白。在一些實施方案中，可溶性部分包含IL-12A和/或IL-12B。在一些實施方案中，可溶性部分包含與SEQ ID NO: 8至少或約50%、60%、70%、80%、90%或100%相同的序列。在一些實施方案中，膜拴系IL-12融合蛋白不能從細胞中釋放。在一些實施方案中，與工程化以表現可溶性IL-12的細胞相比，在培養基中來自表現膜拴系IL-12融合蛋白的細胞的IL-12位準小於10%、5%、4%、3%、2%、1%或更小。在一些實施方案中，如通過ELISA檢測到的，培養基中IL-12的濃度小於300pg/ml、200pg/ml或100pg/ml。In some embodiments, cells (eg, PBMCs) are engineered to express a membrane-tethered IL-12 fusion protein comprising a soluble moiety. In some embodiments, the soluble fraction comprises IL-12A and/or IL-12B. In some embodiments, the soluble portion comprises a sequence at least or about 50%, 60%, 70%, 80%, 90%, or 100% identical to SEQ ID NO: 8. In some embodiments, the membrane-tethered IL-12 fusion protein cannot be released from the cell. In some embodiments, the level of IL-12 from cells expressing a membrane-tethered IL-12 fusion protein is less than 10%, 5%, 4% in culture medium compared to cells engineered to express soluble IL-12 , 3%, 2%, 1% or less. In some embodiments, the concentration of IL-12 in the culture medium is less than 300 pg/ml, 200 pg/ml or 100 pg/ml as detected by ELISA.

在一些實施方案中，工程化細胞可表現趨化介素，例如CXCL-8、CCL2、CCL3、CCL4、CCL5、CCL11、CXCL10、XCL1或XCL2。在一些實施方案中，趨化介素是CXCL10或XCL1。在一些實施方案中，工程化細胞可表現FLT3L。在一些實施方案中，表現在如本文所述的外源調節元件(例如，啟動子)的控制下。趨化介素和FLT3L可增加抗原呈遞細胞的活性。抗原呈遞細胞可將許多腫瘤抗原呈遞給宿主免疫細胞，使得宿主免疫細胞將識別這些腫瘤抗原並殺死這些腫瘤細胞，即使這些腫瘤細胞不表現由CAR或TCR識別的抗原。在一些實施方案中，趨化介素(例如，CXCL10)和/或FLT3L可增強記憶T細胞功能，並因此提供對腫瘤的長期保護。在一些實施方案中，宿主免疫細胞可在受試者被工程化細胞治療之後至少1、至少2、至少3、至少4、至少5、至少6、至少7、至少9、至少10、至少11或至少12個月後有效地殺死腫瘤細胞。In some embodiments, the engineered cells can express chemokines such as CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, XCL1 or XCL2. In some embodiments, the chemokine is CXCL10 or XCL1. In some embodiments, the engineered cells express FLT3L. In some embodiments, expression is under the control of an exogenous regulatory element (eg, a promoter) as described herein. Chemokines and FLT3L increase the activity of antigen-presenting cells. Antigen-presenting cells can present many tumor antigens to host immune cells, such that the host immune cells will recognize these tumor antigens and kill these tumor cells, even if these tumor cells do not express the antigen recognized by CAR or TCR. In some embodiments, chemokines (eg, CXCL10) and/or FLT3L can enhance memory T cell function and thus provide long-term protection against tumors. In some embodiments, the host immune cells can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, at least 11, or at least 11 after treatment of the subject with the engineered cells. Effectively kills tumor cells after at least 12 months.

因此，在一個方面，本公開提供了一種改善免疫細胞療法(例如，TCR-T、CAR-T)的方法。在一些實施方案中，除了TCR或CAR之外，免疫細胞還被工程化以表現IL-12(例如，膜拴系IL12)。在一些實施方案中，細胞被進一步工程化以表現趨化介素(例如，CXCL10)和/或FLT3L。重組載體 Accordingly, in one aspect, the present disclosure provides a method of improving immune cell therapy (eg, TCR-T, CAR-T). In some embodiments, immune cells are engineered to express IL-12 (eg, membrane-tethered IL12) in addition to the TCR or CAR. In some embodiments, the cells are further engineered to express chemokines (eg, CXCL10) and/or FLT3L. recombinant vector

本公開還提供了包括本文公開的分離的多核苷酸(例如編碼本文公開的多肽的多核苷酸)的重組載體(例如表現載體)、向其中引入重組載體(即，使得宿主細胞含有多核苷酸和/或包含多核苷酸的載體)的宿主細胞、以及通過重組技術生產重組多肽或其片段。The present disclosure also provides recombinant vectors (eg, expression vectors) comprising an isolated polynucleotide disclosed herein (eg, a polynucleotide encoding a polypeptide disclosed herein), into which the recombinant vector is introduced (ie, such that a host cell contains the polynucleotide) and/or a polynucleotide-containing vector), and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

載體是當將載體引入宿主細胞時能夠將一種或多種目的多核苷酸遞送至宿主細胞的構建體。“表現載體”能夠在已經引入表現載體的宿主細胞中遞送一種或多種目的多核苷酸和將其表現為編碼的多肽。因此，通過在目的多核苷酸的整合位點處或附近或側翼與載體內或宿主細胞基因組中的調控元件諸如啟動子、增強子和/或聚A尾巴可操作地連接使得目的多核苷酸將在與表現載體一起引入的宿主細胞中翻譯，將目的多核苷酸定位在載體中用於表現。A vector is a construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering one or more polynucleotides of interest and expressing it as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, by operably linking at or near or flanking the integration site of the polynucleotide of interest with regulatory elements such as promoters, enhancers and/or poly A tails within the vector or in the genome of the host cell such that the polynucleotide of interest will Translation in the host cell introduced with the expression vector locates the polynucleotide of interest in the vector for expression.

可以通過本領域已知的方法將載體引入宿主細胞中，例如電穿孔、化學轉染(例如DEAE-葡聚糖)、轉化、轉染、以及感染和/或轉導(例如用重組病毒)。因此，載體的非限制性實例包括病毒載體(其可以用於產生重組病毒)、裸DNA或RNA、質體、黏粒、噬菌體載體、以及與陽離子縮合劑相關的DNA或RNA表現載體。Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (eg, DEAE-dextran), transformation, transfection, and infection and/or transduction (eg, with recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plastids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

本公開提供了包含適合於對細胞進行遺傳修飾的核酸構建體的重組載體，其可以用於治療病理性疾病或病症。The present disclosure provides recombinant vectors comprising nucleic acid constructs suitable for genetic modification of cells, which can be used to treat pathological diseases or disorders.

任何載體或載體類型均可以用於將遺傳物質遞送至細胞。這些載體包括但不限於質體載體、病毒載體、細菌人工染色體(BAC)、酵母人工染色體(YAC)和人類人工染色體(HAC)。病毒載體可以包括但不限於重組反轉錄病毒載體、重組慢病毒載體、重組腺病毒載體、泡沫病毒載體、重組腺相關病毒(AAV)載體、雜交載體、和質體轉座子(例如，睡美人(sleeping beauty)轉座子系統和PiggyBac轉座子系統)或基於整合酶的載體系統。也可以結合本文所述的方法使用本領域已知的其他載體。Any vector or type of vector can be used to deliver genetic material to cells. These vectors include, but are not limited to, plastid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors can include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy viral vectors, recombinant adeno-associated virus (AAV) vectors, hybrid vectors, and plastid transposons (eg, Sleeping Beauty). (sleeping beauty) transposon system and PiggyBac transposon system) or integrase-based vector system. Other vectors known in the art can also be used in conjunction with the methods described herein.

在一些實施方案中，載體是病毒載體。可以將病毒載體在對病毒載體製造具有特異性的培養基中生長。根據本文所述的實施方案，可以使用任何合適的生長培養基和/或用於使病毒載體生長的補充劑。在一些實施方案中，使用MP71載體。In some embodiments, the vector is a viral vector. Viral vectors can be grown in media specific for viral vector production. Any suitable growth media and/or supplements for growing viral vectors may be used in accordance with the embodiments described herein. In some embodiments, the MP71 vector is used.

在一些實施方案中，所使用的載體是重組反轉錄病毒載體。反轉錄病毒載體能夠指導目的核酸分子的表現。反轉錄病毒以RNA形式存在於其病毒膜中，並且當在宿主細胞中複製時形成雙鏈DNA中間體。類似地，反轉錄病毒載體以RNA和雙鏈DNA兩種形式存在。反轉錄病毒載體還包括含有重組DNA片段的DNA形式和含有重組RNA片段的RNA形式。載體可以包括至少一種轉錄啟動子/增強子、或控制基因表現的其他元件。此類載體還可以包括包裝訊息、長末端重複序列(LTR)或其部分、以及適合於所使用的反轉錄病毒的正鏈和負鏈引物結合位點。長末端重複序列(LTR)是在反轉錄轉座子或通過反轉錄病毒RNA的反轉錄形成的前病毒DNA的末端發現的重複許多次(例如數百或數千次)的相同的DNA序列。病毒使用它們將其遺傳物質***宿主基因組中。任選地，載體還可以包括指導聚腺苷酸化的訊息、可選擇標記(諸如胺苄青黴素抗性、新黴素抗性、TK、潮黴素抗性、腐草黴素抗性、組胺醇抗性、或DHFR)、以及一個或多個限制位點和翻譯終止序列。例如，此類載體可以包括5' LTR、前導序列、tRNA結合位點、包裝訊息、第二鏈DNA合成的起點、和3' LTR或其一部分。另外，本文所用的反轉錄病毒載體還可以是指通過去除反轉錄病毒gag、pol和env基因並用目的基因替代而產生的重組載體。In some embodiments, the vector used is a recombinant retroviral vector. Retroviral vectors are capable of directing the expression of nucleic acid molecules of interest. Retroviruses exist in their viral membranes as RNA and form double-stranded DNA intermediates when replicated in host cells. Similarly, retroviral vectors exist in both RNA and double-stranded DNA forms. Retroviral vectors also include DNA forms containing recombinant DNA fragments and RNA forms containing recombinant RNA fragments. The vector may include at least one transcriptional promoter/enhancer, or other elements that control gene expression. Such vectors may also include packaging information, long terminal repeats (LTRs) or portions thereof, and binding sites for plus-strand and minus-strand primers appropriate to the retrovirus used. Long terminal repeats (LTRs) are identical DNA sequences found at the ends of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA that are repeated many times (eg, hundreds or thousands of times). Viruses use them to insert their genetic material into the host genome. Optionally, the vector may also include a message directing polyadenylation, a selectable marker (such as ampicillin resistance, neomycin resistance, TK, hygromycin resistance, phleomycin resistance, histamine alcohol resistance, or DHFR), and one or more restriction sites and translation termination sequences. For example, such a vector can include a 5' LTR, a leader sequence, a tRNA binding site, packaging information, an origin for second strand DNA synthesis, and a 3' LTR or a portion thereof. In addition, the retroviral vector used herein may also refer to a recombinant vector produced by removing the retroviral gag, pol and env genes and replacing them with a gene of interest.

在一些實施方案中，載體或構建體可以含有驅動一個或多個核酸分子的表現的單個啟動子。在一些實施方案中，此類啟動子可以是多順反子(雙順反子或三順反子)。例如，在一些實施方案中，轉錄單元可以是工程化為含有IRES(內部核糖體進入位點)的雙順反子單元，其允許基因產物(例如編碼TCR和經修飾的IL-12的α鏈和/或β鏈)通過來自單一啟動子的消息共表現。另選地，在一些情況下，單一啟動子可引導RNA的表現，所述RNA在單一開放閱讀框(ORF)中含有兩個或三個基因(例如，編碼TCR的α鏈和/或β鏈)，所述兩個或三個基因通過編碼自切割肽(例如，P2A或T2A)的序列或蛋白酶識別位點(例如弗林蛋白酶(furin))彼此分開。因此，ORF編碼單個多蛋白，所述多蛋白在翻譯期間(在2A(例如T2A)的情況下)或之後被切割成單個蛋白質。在一些情況下，肽(諸如T2A)可以引起核糖體跳過2A元件C末端處肽鍵的合成(核糖體跳躍)，從而導致在2A序列末端與下游相鄰肽之間分開。In some embodiments, a vector or construct may contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters may be polycistronic (bicistronic or tricistronic). For example, in some embodiments, the transcription unit can be a bicistronic unit engineered to contain an IRES (internal ribosome entry site) that allows for gene products (eg, the alpha chain encoding TCR and modified IL-12) and/or beta strands) are co-expressed by messages from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (eg, encoding the alpha and/or beta chains of the TCR) in a single open reading frame (ORF) ), the two or three genes are separated from each other by a sequence encoding a self-cleaving peptide (eg, P2A or T2A) or a protease recognition site (eg, furin). Thus, the ORF encodes a single polyprotein that is cleaved into individual proteins either during translation (in the case of 2A (eg T2A)) or after. In some cases, a peptide (such as T2A) can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in separation between the end of the 2A sequence and the downstream adjacent peptide.

可以結合如本文所述的載體使用各種細胞系。可以用於表現多肽的示例性真核細胞包括但不限於COS細胞，包括COS 7細胞；293細胞，包括293-6E細胞；CHO細胞，包括CHO-S、DG44。Lec13 CHO細胞和FUT8 CHO細胞；PER.C6?細胞；和NSO細胞。在一些實施方案中，特定的真核宿主細胞基於其對結合分子進行所需的翻譯後修飾的能力來選擇。例如，在一些實施方案中，CHO細胞產生的多肽的唾液酸化位準高於在293細胞中產生的相同多肽。在一個方面，本公開涉及包含如本文所述的載體或載體對的細胞。在一些實施方案中，細胞是T細胞。Various cell lines can be used in conjunction with the vectors as described herein. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells and FUT8 CHO cells; PER.C6™ cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modification of the binding molecule. For example, in some embodiments, a polypeptide produced by CHO cells has a higher level of sialylation than the same polypeptide produced in 293 cells. In one aspect, the present disclosure relates to cells comprising a vector or vector pair as described herein. In some embodiments, the cells are T cells.

在一些情況下，某些TCR可能表現出不良的表現或活性，部分原因是與內源性TCR鏈的錯誤配對和/或競爭和/或其他因素。解決這些挑戰的一種方法是設計具有小鼠恆定結構域的重組TCR，以防止與內源性人TCR a鏈或b鏈錯誤配對。然而，將重組TCR與小鼠序列一起使用可能帶來免疫反應的風險。在一些實施方案中，例如通過基因編輯在編碼一個或多個TCR鏈的內源基因上引入遺傳破壞。In some cases, certain TCRs may exhibit poor performance or activity, in part due to mispairing and/or competition with endogenous TCR chains and/or other factors. One approach to address these challenges is to design recombinant TCRs with mouse constant domains to prevent mispairing with endogenous human TCR a- or b-chains. However, the use of recombinant TCRs with mouse sequences may pose a risk of immune responses. In some embodiments, genetic disruption is introduced at an endogenous gene encoding one or more TCR chains, eg, by gene editing.

如圖1A至圖1B所示，本文提供了小鼠和人IL-12表現載體，其包含編碼IL-12B(p40)和IL-12A(p35)的核酸序列。在一些實施方案中，編碼IL-12B和IL-12A的核酸序列通過接頭序列連接。在一些實施方案中，載體還包含連接在IL-12A之後的編碼免疫球蛋白鉸鏈區、兩個或三個恆定結構域(例如，一個或兩個CH2以及CH3)和跨膜區(例如，CD4跨膜區)的序列。As shown in Figures 1A-1B, provided herein are mouse and human IL-12 expression vectors comprising nucleic acid sequences encoding IL-12B (p40) and IL-12A (p35). In some embodiments, the nucleic acid sequences encoding IL-12B and IL-12A are linked by a linker sequence. In some embodiments, the vector further comprises an immunoglobulin-encoding hinge region, two or three constant domains (eg, one or both CH2 and CH3), and a transmembrane region (eg, CD4) linked after IL-12A transmembrane region).

在一些實施方案中，編碼IL-12的載體還包含在IL-12B之前編碼靶向腫瘤的抗體(例如，NHS-76)的重鏈可變區(vH)和輕鏈可變區(vL)的序列。在一些實施方案中，編碼vH和vL的核酸序列通過接頭序列連接。在一些實施方案中，編碼vL和IL-12B的核酸序列通過接頭序列連接。In some embodiments, the vector encoding IL-12 further comprises variable heavy (vH) and variable light (vL) regions encoding a tumor-targeting antibody (eg, NHS-76) prior to IL-12B the sequence of. In some embodiments, the nucleic acid sequences encoding vH and vL are joined by a linker sequence. In some embodiments, the nucleic acid sequences encoding vL and IL-12B are joined by a linker sequence.

在一些實施方案中，本文所述的載體包含編碼訊息肽序列的序列。在一些實施方案中，編碼訊息肽序列的序列與編碼IL-12B的序列連接。在一些實施方案中，編碼訊息肽序列的序列與編碼靶向腫瘤的抗體的vH的序列連接。在一些實施方案中，載體還包含在訊息肽序列之前編碼接頭肽序列(例如，P2A)的序列。In some embodiments, the vectors described herein comprise a sequence encoding a message peptide sequence. In some embodiments, the sequence encoding the message peptide sequence is linked to the sequence encoding IL-12B. In some embodiments, the sequence encoding the message peptide sequence is linked to the sequence encoding the vH of the tumor-targeting antibody. In some embodiments, the vector further comprises a sequence encoding a linker peptide sequence (eg, P2A) preceding the message peptide sequence.

如圖1C至圖1D中，在一些實施方案中，本文所述的載體還包含編碼T細胞受體(TCR)或嵌合抗原受體(CAR)的序列。在一些實施方案中，編碼TCR或CAR的序列在編碼訊息肽序列的序列之前連接。1C-1D, in some embodiments, the vectors described herein further comprise a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the sequence encoding the TCR or CAR is ligated before the sequence encoding the message peptide sequence.

在一些實施方案中，載體還包含編碼趨化介素例如CXCL-8、CCL2、CCL3、CCL4、CCL11、CXCL10、XCL1或XCL2的序列。在一些實施方案中，趨化介素是CXCL10或XCL1。在一些實施方案中，載體還包含編碼FMS樣酪胺酸激酶3配體(FLT3L)的序列。在一些實施方案中，XCL1胺基酸序列與SEQ ID NO: 46或47至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同。在一些實施方案中，CXCL10胺基酸序列與SEQ ID NO: 48或49至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同。在一些實施方案中，Flt3L胺基酸序列與SEQ ID NO: 50或51至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同。在一些實施方案中，這些序列通過編碼2A自切割肽的序列與其他序列分開。In some embodiments, the vector further comprises a sequence encoding a chemokine such as CXCL-8, CCL2, CCL3, CCL4, CCL11, CXCL10, XCL1 or XCL2. In some embodiments, the chemokine is CXCL10 or XCL1. In some embodiments, the vector further comprises a sequence encoding an FMS-like tyrosine kinase 3 ligand (FLT3L). In some embodiments, the XCL1 amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 94%, 95%, 96%, 97% identical to SEQ ID NO: 46 or 47. 98% or 99% the same. In some embodiments, the CXCL10 amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% identical to SEQ ID NO: 48 or 49, 98% or 99% the same. In some embodiments, the Flt3L amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% identical to SEQ ID NO: 50 or 51, 98% or 99% the same. In some embodiments, these sequences are separated from other sequences by the sequence encoding the 2A self-cleaving peptide.

本公開還提供了編碼人IL-12融合蛋白的核酸。在一些實施方案中，編碼本文所述的IL-12融合蛋白的核酸列出於SEQ ID NO: 7中，或者為與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的核酸序列。在一些實施方案中，編碼本文所述的膜拴系IL-12融合蛋白的核酸列出於SEQ ID NO: 9中，或者為與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的核酸序列。在一些實施方案中，編碼本文所述的膜拴系IL-12融合蛋白的核酸列出於SEQ ID NO: 11中，或者為與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的核酸序列。在一些實施方案中，編碼本文所述的可溶性IL-12融合蛋白的核酸列出於SEQ ID NO: 13中，或者為與其具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的核酸序列。The present disclosure also provides nucleic acids encoding human IL-12 fusion proteins. In some embodiments, the nucleic acid encoding the IL-12 fusion protein described herein is set forth in SEQ ID NO: 7, or has at least 80%, 85%, 90%, 91%, 92%, 93% therewith , 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 9, or has at least 80%, 85%, 90%, 91%, 92% therewith , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 11, or has at least 80%, 85%, 90%, 91%, 92% therewith , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a soluble IL-12 fusion protein described herein is set forth in SEQ ID NO: 13, or is at least 80%, 85%, 90%, 91%, 92%, 93% therewith %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences.

如本文所用，術語“接頭”(L)或“接頭結構域”或“接頭區域”是指將任何結構域/區域連接在一起的長度為約1至100個胺基酸的寡聚或多肽區域。接頭可以由柔性殘基(如甘胺酸和絲胺酸)構成，使得相鄰的蛋白質結構域可以相對於彼此自由移動。當期望確保兩個相鄰結構域彼此之間不發生空間干擾時，可以使用較長的接頭。接頭可以是可切割的或不可切割的。可切割接頭的實例包括2A接頭(例如P2A、T2A)、2A樣接頭或其功能等同物、以及它們的組合。在一些實施方案中，接頭包括小核糖核酸病毒2A樣接頭；豬捷申病毒(P2A)、明脈扁刺蛾β四體病毒(Thosea asigna virus)(T2A)的CHYSEL序列；或它們的組合、變體和功能等同物。其他接頭對本領域技術人員將是顯而易見的，並且可以在本文所述的方法中使用。As used herein, the term "linker" (L) or "linker domain" or "linker region" refers to an oligomeric or polypeptide region of about 1 to 100 amino acids in length that joins any domain/region together . Linkers can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used when it is desired to ensure that two adjacent domains do not sterically interfere with each other. Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg, P2A, T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker; the CHYSEL sequence of porcine Teshin virus (P2A), Thosea asigna virus (T2A); or a combination thereof, Variants and functional equivalents. Other linkers will be apparent to those skilled in the art and can be used in the methods described herein.

在一些實施方案中，接頭肽序列包含至少或約5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、30、40或50個胺基酸殘基。在一些實施方案中，接頭序列包含至少或約4、5、6、7、8、9、10、11、12、13、14、15、16、20、25、30或40個甘胺酸殘基。在一些實施方案中，接頭序列包含甘胺酸和絲胺酸殘基兩者、或由其組成。在一些實施方案中，接頭序列包含以下序列、或由其組成：所述序列與SEQ ID NO: 17、GGGGSGGGGS(SEQ ID NO: 18)或GGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 19)至少或約70%、至少或約75%、至少或約80%、至少或約85%、至少或約90%、至少或約95%、至少或約99%、或100%相同。在一些實施方案中，接頭序列包含GGGGS(SEQ ID NO: 20)的至少1、2、3、4、5或6個重複。In some embodiments, the linker peptide sequence comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker sequence comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues base. In some embodiments, the linker sequence comprises or consists of both glycine and serine residues. In some embodiments, the linker sequence comprises or consists of a sequence that is at least or about 70% identical to SEQ ID NO: 17, GGGGSGGGGS (SEQ ID NO: 18), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19), At least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100% identical. In some embodiments, the linker sequence comprises at least 1, 2, 3, 4, 5, or 6 repeats of GGGGS (SEQ ID NO: 20).

本公開還提供了核酸序列，所述核酸序列包含編碼IL-12、經修飾的IL-12、CAR、TCR、它們的抗原結合片段和/或TCR衍生的結合分子(包括例如本文所述的它們的功能部分和功能變體、多肽或蛋白質)中的任一者的核苷酸序列。如本文所用，“核酸”可以包括“多核苷酸”、“寡核苷酸”和“核酸分子”，並且通常意指DNA或RNA的聚合物，其可以是單鏈或雙鏈的、合成的或獲得自天然來源，其可以含有天然、非天然或改變的核苷酸。此外，核酸包含互補DNA(cDNA)。通常優選的是，核酸不包含任何***、缺失、倒位和/或取代。然而，如本文所討論的，在一些情形下，核酸包含一個或多個***、缺失、倒位和/或取代可能是合適的。The disclosure also provides nucleic acid sequences comprising encoding IL-12, modified IL-12, CAR, TCR, antigen-binding fragments thereof, and/or TCR-derived binding molecules (including, for example, those described herein) The nucleotide sequence of any of the functional parts and functional variants, polypeptides or proteins). As used herein, "nucleic acid" may include "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which may be single- or double-stranded, synthetic Or obtained from natural sources, which may contain natural, non-natural or altered nucleotides. Furthermore, nucleic acids comprise complementary DNA (cDNA). It is generally preferred that the nucleic acid does not contain any insertions, deletions, inversions and/or substitutions. However, as discussed herein, in some cases it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions and/or substitutions.

可以使用本領域已知的程序基於化學合成和/或酶促連接反應來構建如本文所述的核酸。例如，可以使用天然存在的核苷酸或各種經修飾的核苷酸來化學地合成核酸。在一些任何此類實施方案中，核苷酸序列是密碼子優化的。Nucleic acids as described herein can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, naturally occurring nucleotides or various modified nucleotides can be used to chemically synthesize nucleic acids. In some of any such embodiments, the nucleotide sequence is codon-optimized.

本公開還提供了核酸，所述核酸包含與本文所述的任何核酸的核苷酸序列互補的核苷酸序列或者在嚴格條件下與本文所述的任何核酸的核苷酸序列雜交的核苷酸序列。The present disclosure also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleoside that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein acid sequence.

在一些實施方案中，編碼α鏈的核苷酸序列和編碼β鏈的核苷酸序列被引起核糖體跳躍的肽序列分開。在一些實施方案中，引起核糖體跳躍的肽是P2A或T2A肽。在一些實施方案中，核酸是合成的。在一些實施方案中，核酸是cDNA。In some embodiments, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome hopping. In some embodiments, the peptide that causes ribosome skipping is a P2A or T2A peptide. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.

在一些實施方案中，載體可以另外包括編碼檢查點抑制劑(CPI)(例如抑制蛋白)的核酸序列。在一些實施方案中，檢查點抑制劑是例如本文所述的任何抗體或其抗原結合片段。在一些實施方案中，抗體或其抗原結合片段可以特異性地結合PD-1、PD-L1、PD-L2、2B4(CD244)、4-1BB、A2aR、B7.1、B7.2、B7-H2、B7-H3、B7-H4、B7-H6、BTLA、嗜乳脂蛋白、CD160、CD48、CTLA4、GITR、gp49B、HHLA2、HVEM、ICOS、ILT-2、ILT-4、KIR家族受體、LAG-3、OX-40、PIR-B、SIRPα(CD47)、TFM-4、TIGIT、TIM-1、TIM-3、TIM-4、或VISTA。在一些實施方案中，抑制蛋白是scFv(例如抗PD-1 scFv)。在一些實施方案中，載體可以另外包括編碼雙功能陷阱融合蛋白的核酸序列。在一些實施方案中，雙功能陷阱蛋白靶向PD-1和TGF-β二者。在一些實施方案中，雙功能陷阱蛋白靶向PD-L1和TGF-β二者。在一些實施方案中，雙功能融合蛋白設計成阻斷PD-L1和螯合(sequester)TGF-β。基於人IgG1單株抗體(mAb)阿維魯單抗(avelumab)，M7824(MSB0011395C)包含與人抗PD-L1 scFv的C末端連接的人TGF-β受體II(TGFβRII)的細胞外結構域。在一些實施方案中，雙功能融合蛋白包含與人抗PD-1 scFv的C末端連接的人TGF-β受體II(TGFβRII)的細胞外結構域。In some embodiments, the vector may additionally include a nucleic acid sequence encoding a checkpoint inhibitor (CPI) (eg, an arrestin). In some embodiments, the checkpoint inhibitor is, for example, any antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7- H2, B7-H3, B7-H4, B7-H6, BTLA, Butyrophilin, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG -3, OX-40, PIR-B, SIRPα (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, or VISTA. In some embodiments, the inhibitory protein is an scFv (eg, an anti-PD-1 scFv). In some embodiments, the vector may additionally include a nucleic acid sequence encoding a bifunctional trap fusion protein. In some embodiments, the bifunctional trap protein targets both PD-1 and TGF-beta. In some embodiments, the bifunctional trap protein targets both PD-L1 and TGF-beta. In some embodiments, the bifunctional fusion protein is designed to block PD-L1 and sequester TGF-beta. Human IgG1 based monoclonal antibody (mAb) avelumab, M7824 (MSB0011395C) contains the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-L1 scFv . In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-1 scFv.

在一些任何此類實施方案中，IL-12(例如，膜拴系IL-12)、CAR、TCR或它們的抗原結合片段由已經進行密碼子優化的核苷酸序列編碼。在某些實施方案中，所述多肽包含訊息肽。在任何此類實施方案的一些實施方案中，多肽和/或融合蛋白是重組的。In some of any such embodiments, the IL-12 (eg, membrane-tethered IL-12), CAR, TCR, or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized. In certain embodiments, the polypeptide comprises a message peptide. In some embodiments of any such embodiments, the polypeptide and/or fusion protein is recombinant.

本公開還提供了與本文所述的任何核苷酸序列為至少1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同的核酸序列、以及與本文所述的任何胺基酸序列為至少1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同的胺基酸序列。在一些實施方案中，本公開涉及編碼本文所述的任何肽的核苷酸序列、或由本文所述的任何核苷酸序列編碼的任何胺基酸序列。The present disclosure also provides that any nucleotide sequence described herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleic acid sequences, and at least 1%, 2%, 3%, 4% with any amino acid sequence described herein , 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein, or any amino acid sequence encoded by any of the nucleotide sequences described herein.

在一些實施方案中，核酸序列為至少或約10、20、30、40、50、60、70、80、90、100、110、120、130、150、200、250、300、350、400、500、或600個核苷酸。在一些實施方案中，胺基酸序列為至少或約5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、300、400、500、600、700、800、或900個胺基酸殘基。在一些實施方案中，核酸序列為小於10、20、30、40、50、60、70、80、90、100、110、120、130、150、200、250、300、350、400、500、或600個核苷酸。在一些實施方案中，胺基酸序列為小於5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、300、400、500、600、700、800、或900個胺基酸殘基。In some embodiments, the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.

為了確定兩個胺基酸序列或兩個核酸序列的同一性百分比，出於最佳比較目的，對序列進行比對(例如，可以在第一和第二胺基酸或核酸序列中的一者或兩者中引入缺口以進行最佳比對，並且出於比較目的可以忽略非同源序列)。然後比較在相應的胺基酸位置或核苷酸位置的胺基酸殘基或核苷酸。當第一序列中的位置被與第二序列中的相應位置相同的胺基酸殘基或核苷酸佔據時，則分子在所述位置是相同的。考慮了缺口數量和每個缺口的長度(需要將其引入以實現兩個序列的最佳比對)，兩個序列之間的同一性百分比是由序列所共享的相同位置的數量的函數。用於製備工程化細胞的方法 To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (eg, one of the first and second amino acid or nucleic acid sequences may be or both to introduce gaps for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. A molecule is identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. Taking into account the number of gaps and the length of each gap (which needs to be introduced to achieve optimal alignment of the two sequences), the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Methods for making engineered cells

本公開提供了用於製備、製造工程化細胞和/或使用工程化細胞來治療病理性疾病或病症的方法或過程。The present disclosure provides methods or processes for preparing, manufacturing, and/or using the engineered cells to treat a pathological disease or disorder.

可以從樣品(諸如生物樣品，例如從受試者獲得或來源的生物樣品)中分離用於引入本文所述的蛋白質(例如，IL-12、TCR和/或CAR)的細胞。在一些實施方案中，從其分離細胞的受試者是患有疾病或病症或需要細胞療法、或將向其施用細胞療法的受試者。在一些實施方案中，受試者是需要特定治療干預(諸如針對其分離、處理細胞和/或對細胞進行工程化的過繼細胞療法)的人。Cells used to introduce the proteins described herein (eg, IL-12, TCR and/or CAR) can be isolated from a sample, such as a biological sample, eg, obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of, or to which cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

因此，在一些實施方案中，所述細胞是原代細胞，例如原代人細胞。樣品包括直接取自受試者的組織、流體和其他樣品，以及來自一個或多個處理步驟(諸如分離、離心、基因工程化(例如用病毒載體轉導)、洗滌和/或孵育)的樣品。生物樣品可以是直接從生物來源獲得的樣品或經過處理的樣品。生物樣品包括但不限於體液(諸如血液、血漿、血清、腦脊液、滑液、尿液和汗液)、組織和器官樣品，包括由其衍生的經處理樣品。Thus, in some embodiments, the cells are primary cells, eg, primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples from one or more processing steps such as isolation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation . A biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.

在一些方面，細胞從其中衍生或分離的樣品是血液或血液衍生的樣品，或者是或源自單采術或白細胞分離術產物。示例性樣品包括全血、外周血單核細胞(PBMC)、白細胞、骨髓、胸腺、組織活檢、腫瘤、白血病、淋巴瘤、淋巴結、腸相關淋巴組織、黏膜相關淋巴組織、脾、其他淋巴組織、肝、肺、胃、腸、結腸、腎、胰腺、***、骨、***、子宮頸、睾丸、卵巢、扁桃體或其他器官和/或由其衍生的細胞。在細胞療法(例如過繼細胞療法)的背景下，樣品包括來自自體和同種異體來源的樣品。In some aspects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, Liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs and/or cells derived therefrom. In the context of cell therapy (eg, adoptive cell therapy), samples include samples from autologous and allogeneic sources.

在一些實施方案中，所述細胞衍生自細胞系，例如T細胞系。在一些實施方案中，所述細胞獲得自異種來源，例如獲得自小鼠、大鼠、或非人靈長類動物。在一些實施方案中，所述細胞從小鼠淋巴結中分離。In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, eg, from a mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.

在一些實施方案中，洗滌從受試者收集的血細胞，例如以去除血漿級分並將細胞置於適當的緩衝液或介質中以用於隨後的加工步驟。在一些實施方案中，用磷酸鹽緩衝鹽水(PBS)洗滌所述細胞。在一些實施方案中，洗滌溶液缺乏鈣和/或鎂和/或許多或所有二價陽離子。在一些方面，通過半自動化“流通式”離心完成洗滌步驟。在一些方面，通過切向流過濾(TFF)完成洗滌步驟。在一些實施方案中，洗滌後將細胞重懸於多種生物相容性緩衝液(諸如，例如不含Ca2+ /Mg2+ 的PBS)中。在某些實施方案中，去除血細胞樣品的組分並將所述細胞直接重懸於培養基中。在一些實施方案中，所述方法包括基於密度的細胞分離方法，如通過裂解紅細胞並通過Percoll或Ficoll梯度進行離心來從外周血製備白細胞。In some embodiments, blood cells collected from the subject are washed, eg, to remove plasma fractions and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is devoid of calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished by semi-automated "flow-through" centrifugation. In some aspects, the washing step is accomplished by tangential flow filtration (TFF). In some embodiments, cells are resuspended in various biocompatible buffers (such as, for example, PBS without Ca 2+ /Mg 2+ ) after washing. In certain embodiments, components of the blood cell sample are removed and the cells are directly resuspended in culture medium. In some embodiments, the method includes a density-based cell separation method, such as the preparation of leukocytes from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

在一些實施方案中，所述方法包括以下中的一個或多個步驟：例如，從患者血液中分離T細胞；用病毒載體轉導群體T細胞，所述病毒載體包括編碼基因工程化抗原受體的核酸構建體；體外擴增經轉導的細胞；和/或將擴增的細胞輸注到患者體內，在那裡工程化T細胞將尋找並破壞抗原陽性的腫瘤細胞。在一些實施方案中，核酸構建體還包括編碼抑制蛋白的序列。在一些實施方案中，這些工程化T細胞可以阻斷PD-1/PD-L1免疫抑制並增強抗腫瘤免疫反應。所述方法還包括：用含有核酸構建體的病毒載體轉染T細胞。In some embodiments, the method comprises one or more of the following steps: eg, isolating T cells from a patient's blood; transducing a population of T cells with a viral vector comprising encoding a genetically engineered antigen receptor expand the transduced cells in vitro; and/or infuse the expanded cells into a patient, where the engineered T cells will seek out and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further includes a sequence encoding an arrestin. In some embodiments, these engineered T cells can block PD-1/PD-L1 immunosuppression and enhance anti-tumor immune responses. The method also includes transfecting the T cells with the viral vector containing the nucleic acid construct.

在一些實施方案中，所述方法涉及將本文所述的任何載體體外或離體引入細胞中。在一些實施方案中，載體是病毒載體，並且所述引入通過轉導來進行。在一些實施方案中，將細胞轉導持續至少或約1小時、2小時、3小時、4小時、5小時、6小時、12小時、18小時、1天、2天、3天、4天、5天、6天、1周、2周、3周或更長時間。在一些實施方案中，所述方法還涉及將一種或多種藥劑引入細胞中，其中所述一種或多種藥劑中的每一種獨立地能夠誘導T細胞受體α鏈恆定區(TRAC)基因和/或T細胞受體β鏈恆定區(TRBC)基因的遺傳破壞。在一些實施方案中，所述一種或多種藥劑是抑制核酸(例如siRNA)。在一些實施方案中，所述一種或多種藥劑是包含DNA靶向蛋白和核酸酶或RNA指導的核酸酶的融合蛋白(例如，成簇的規律間隔短回文核酸(CRISPR)相關的核酸酶)。In some embodiments, the methods involve introducing any of the vectors described herein into cells in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introduction is by transduction. In some embodiments, the cells are transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or more. In some embodiments, the method further involves introducing into the cell one or more agents, wherein each of the one or more agents is independently capable of inducing a T cell receptor alpha chain constant region (TRAC) gene and/or Genetic disruption of the T cell receptor beta chain constant region (TRBC) gene. In some embodiments, the one or more agents are inhibitory nucleic acids (eg, siRNA). In some embodiments, the one or more agents are fusion proteins comprising a DNA targeting protein and a nuclease or RNA-guided nuclease (eg, Clustered Regularly Interspaced Short Palindromic Nucleic Acids (CRISPR)-related nucleases) .

在一些實施方案中，細胞是腫瘤浸潤性淋巴細胞，並且細胞用編碼IL-12或經修飾的IL-12的載體轉染。在一些實施方案中，細胞是T細胞，並且細胞用編碼IL-12(例如，經修飾的IL-12)和TCR的載體或編碼IL-12(例如，經修飾的IL-12)和CAR的載體轉染。In some embodiments, the cells are tumor-infiltrating lymphocytes, and the cells are transfected with a vector encoding IL-12 or modified IL-12. In some embodiments, the cells are T cells and the cells are treated with a vector encoding IL-12 (eg, modified IL-12) and a TCR or a vector encoding IL-12 (eg, modified IL-12) and a CAR vector transfection.

熟練技術人員通過使用任何標準方法諸如磷酸鈣、電穿孔、脂質體介導的轉移、顯微注射、生物彈顆粒遞送系統、或任何其他已知方法可以實現T細胞的轉染。在一些實施方案中，使用磷酸鈣方法進行T細胞的轉染。Transfection of T cells can be accomplished by the skilled artisan using any standard method such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, bioelastic particle delivery systems, or any other known method. In some embodiments, transfection of T cells is performed using the calcium phosphate method.

本公開提供了創建個性化抗腫瘤免疫療法的方法。基因工程化T細胞可以從患者的血細胞產生。然後，將這些工程化T細胞作為細胞療法產品重新輸注到患者體內。The present disclosure provides methods for creating personalized anti-tumor immunotherapies. Genetically engineered T cells can be generated from a patient's blood cells. These engineered T cells are then re-infused into the patient as a cell therapy product.

製備工程化細胞並將這些工程化細胞施用至受試者的方法是本領域已知的，並且描述於例如美國專利 10,174,098和Draper等人，“Targeting of HPV-16+ Epithelial Cancer Cells By Tcr Gene Engineered t Cells Directed Against e6.” Clinical Cancer Research 21.19 (2015): 4431-4439中，這兩個文獻全文以引用方式併入。治療方法 Methods of preparing engineered cells and administering these engineered cells to a subject are known in the art and are described, for example, in US Pat. No. 10,174,098 and Draper et al., "Targeting of HPV-16+ Epithelial Cancer Cells By Tcr Gene Engineered t Cells Directed Against e6." Clinical Cancer Research 21.19 (2015): 4431-4439, both of which are incorporated by reference in their entirety. treatment method

本文公開的方法可以用於各種治療目的。在一個方面，本公開提供了用於治療受試者的癌症的方法，降低受試者中腫瘤體積隨時間增加的速率的方法，降低發生轉移的風險的方法、或降低受試者中發生額外轉移的風險的方法。在一些實施方案中，治療可以停止、減慢、延緩或抑制癌症的進展。在一些實施方案中，治療可以導致受試者中癌症的一種或多種症狀的數量、嚴重性和/或持續時間的減少。The methods disclosed herein can be used for various therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, methods of reducing the rate of increase in tumor volume over time in a subject, methods of reducing the risk of developing metastases, or reducing the occurrence of extraneous lesions in a subject method of transferring risk. In some embodiments, the treatment can stop, slow, delay or inhibit the progression of the cancer. In some embodiments, the treatment can result in a reduction in the number, severity and/or duration of one or more symptoms of cancer in the subject.

在一個方面，本公開的特徵在於方法，其包括將治療有效量的表現IL-12(例如經修飾的IL-12)、TCR、CAR、它們的抗原結合片段和TCR衍生的結合分子的工程化細胞施用至有需要的受試者(例如，患有或鑒定為或診斷為患有癌症的受試者)。In one aspect, the disclosure features methods comprising engineering a therapeutically effective amount of expressing IL-12 (eg, modified IL-12), TCR, CAR, antigen-binding fragments thereof, and TCR-derived binding molecules The cells are administered to a subject in need (eg, a subject suffering from or identified as or diagnosed with cancer).

在一些實施方案中，受試者患有實體瘤(例如，異質實體瘤或同質實體瘤)。在一些實施方案中，所述受試者患有乳腺癌(例如三陰性乳腺癌)、類癌、宮頸癌、子宮內膜癌、神經膠質瘤、頭頸癌、肝癌、肺癌、小細胞肺癌、淋巴瘤、黑色素瘤、卵巢癌、胰腺癌、***癌、腎癌、結直腸癌、胃癌、睾丸癌、甲狀腺癌、膀胱癌、尿道癌或血液系統惡性腫瘤。在一些實施方案中，所述癌症是不可切除的黑色素瘤或轉移性黑色素瘤、非小細胞肺癌(NSCLC)、小細胞肺癌(SCLC)、膀胱癌或轉移性激素難治性***癌。在一些實施方案中，癌症是宮頸癌、頭頸癌、口咽癌、肛門癌、陰莖癌、***癌或外陰癌。In some embodiments, the subject has a solid tumor (eg, a heterogeneous solid tumor or a homogeneous solid tumor). In some embodiments, the subject has breast cancer (eg, triple negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma tumor, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is cervical cancer, head and neck cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

在一些實施方案中，受試者患有異質癌症。在一些實施方案中，受試者患有同質癌症。在一些實施方案中，與由異質癌症中的大多數(例如，至少50%、60%、70%、80%或90%)癌細胞表現的一種或多種抗原相比，異質癌症具有至少1%、2%、3%、4%、5%、6%、7%、8%、9%、10%或更多的表現不同抗原的癌細胞。在一些實施方案中，TCR、CAR、它們的抗原結合片段和/或TCR衍生的結合分子可結合由異質癌症中的部分(例如，至少50%、60%、70%、80%、90%、95%或更多)或所有細胞表現的一種或多種抗原。在一些實施方案中，該一種或多種抗原不由異質癌症中的細胞的部分(例如，小於50%、40%、30%、20%、10%或5%)表現。在一些實施方案中，該一種或多種抗原被去活化(例如，切割或諸如免疫逃逸的機制)，使得TCR、CAR、它們的抗原結合片段和/或TCR衍生的結合分子不再能夠識別。在一些實施方案中，異質癌症中的細胞的部分(例如，小於50%、40%、30%、20%、10%或5%)可表現免疫檢查點分子(例如，PD-L1)以抑制免疫反應。腫瘤抗原異質性和抗原逃逸的詳細描述可見於O’Rourke等人，“A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma.” Science translational medicine 9.399 (2017): eaaa0984；Majzner和Crystal，"Tumor antigen escape from CAR T-cell therapy." Cancer discovery 8.10 (2018): 1219-1226；這些文獻中的每一者全文以引用方式併入本文。In some embodiments, the subject has a heterogeneous cancer. In some embodiments, the subject has a homogeneous cancer. In some embodiments, the heterogeneous cancer has at least 1% of the antigen or antigens expressed by the majority (eg, at least 50%, 60%, 70%, 80%, or 90%) of cancer cells in the heterogeneous cancer , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of cancer cells expressing different antigens. In some embodiments, TCRs, CARs, antigen-binding fragments thereof, and/or TCR-derived binding molecules can bind to a portion (eg, at least 50%, 60%, 70%, 80%, 90%, 95% or more) or one or more antigens expressed by all cells. In some embodiments, the one or more antigens are not expressed by a fraction (eg, less than 50%, 40%, 30%, 20%, 10%, or 5%) of cells in a heterogeneous cancer. In some embodiments, the one or more antigens are deactivated (eg, cleavage or a mechanism such as immune escape) such that the TCR, CAR, antigen-binding fragments thereof and/or TCR-derived binding molecules are no longer recognized. In some embodiments, a fraction (eg, less than 50%, 40%, 30%, 20%, 10%, or 5%) of cells in a heterogeneous cancer can express an immune checkpoint molecule (eg, PD-L1) to inhibit immune response. A detailed description of tumor antigen heterogeneity and antigen escape can be found in O'Rourke et al., "A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma." Science translational medicine 9.399 ( 2017): eaaa0984; Majzner and Crystal, "Tumor antigen escape from CAR T-cell therapy." Cancer discovery 8.10 (2018): 1219-1226; each of these documents are incorporated herein by reference in their entirety.

在一些實施方案中，與不表現IL-12的類似工程化TCR或CAR細胞相比，由本文所述的工程化細胞表現的IL-12(例如，經修飾的IL-12)可以將治療異源性癌症改善(例如殺傷癌細胞或減少腫瘤體積)至少或約10%、20%、30%、40%、50%、60%、70%、80%、90%、100%、2倍、3倍、5倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍或更多。In some embodiments, the IL-12 (eg, modified IL-12) expressed by the engineered cells described herein can be therapeutically specific compared to similarly engineered TCR or CAR cells that do not express IL-12. At least or about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more.

在一些實施方案中，本文公開的組合物和方法可以用於治療處於患癌症風險的患者。癌症患者可以通過本領域已知的各種方法來鑒定。In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Cancer patients can be identified by various methods known in the art.

此外，本公開提供了用於治療受試者的感染或感染相關病症的方法。在一些實施方案中，治療可以停止、減慢、延緩或抑制疾病的進展。這些方法通常涉及將治療有效量的本文公開的基因工程化細胞施用至有需要的受試者。在一些實施方案中，待治療的疾病或病症是感染性疾病或病症，諸如但不限於病毒、反轉錄病毒、細菌和原生動物感染、免疫缺陷、人乳頭狀瘤病毒(HPV)、巨細胞病毒(CMV)、愛潑斯坦巴爾病毒(EBV)、腺病毒、BK多瘤病毒。Furthermore, the present disclosure provides methods for treating an infection or an infection-related disorder in a subject. In some embodiments, the treatment can stop, slow, delay or inhibit the progression of the disease. These methods generally involve administering to a subject in need thereof a therapeutically effective amount of a genetically engineered cell disclosed herein. In some embodiments, the disease or disorder to be treated is an infectious disease or disorder, such as, but not limited to, viral, retrovirus, bacterial and protozoal infections, immunodeficiency, human papilloma virus (HPV), cytomegalovirus (CMV), Epstein Barr virus (EBV), adenovirus, BK polyoma virus.

如本文所用，“有效量”意指足以實現有益或期望結果(包括停止、減慢、延緩或抑制疾病(例如癌症)的進展)的量或劑量。有效量將根據例如將要向其施用治療劑和/或治療組合物的受試者的年齡和體重、症狀的嚴重性和施用途徑而變化，並且因此可以在個體基礎上確定施用。As used herein, an "effective amount" means an amount or dose sufficient to achieve beneficial or desired results, including halting, slowing, delaying or inhibiting the progression of a disease (eg, cancer). The effective amount will vary depending, for example, on the age and weight of the subject to which the therapeutic agent and/or therapeutic composition will be administered, the severity of symptoms and the route of administration, and thus administration can be determined on an individual basis.

如本文所用，術語“延遲疾病的發展”是指推遲、阻礙、減慢、延緩、穩定、抑制和/或延期疾病(例如癌症)的發展。該延遲可以具有不同的時間長度，這取決於病史和/或所治療的個體。對於本領域技術人員顯而易見的是，足夠或顯著的延遲實際上可以涵蓋預防，因為個體不會患上疾病。例如，可能延遲晚期癌症，諸如轉移的發展。As used herein, the term "delaying the development of a disease" refers to delaying, retarding, slowing, delaying, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer). This delay can be of varying lengths, depending on the medical history and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, since the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

有效量可以在一次或多次給藥中進行施用。舉例來說，組合物的有效量是足以改善、停止、穩定、逆轉、抑制、減慢和/或延遲患者癌症進展的量，或者是足以在體外改善、停止、穩定、逆轉、減慢和/或延遲細胞(例如，活檢的細胞、本文所述的任何癌細胞、或細胞系(例如癌細胞系))的增殖的量。如本領域中所理解的，有效量可以根據尤其是患者病史以及其他因素諸如所使用的組合物的類型(和/或劑量)而變化。An effective amount can be administered in one or more administrations. For example, an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay the progression of cancer in a patient, or to ameliorate, stop, stabilize, reverse, slow and/or in vitro or an amount that delays the proliferation of cells (eg, biopsied cells, any of the cancer cells described herein, or cell lines (eg, cancer cell lines)). As understood in the art, the effective amount may vary depending on, inter alia, the patient's medical history, as well as other factors such as the type (and/or dosage) of the composition used.

可以根據經驗確定施用的有效量和時間表，並且進行這種確定在本領域技術範圍內。本領域技術人員將理解，必須施用的劑量將根據例如將接受治療的哺乳動物、施用途徑、向哺乳動物施用的治療劑和其他藥物的特定類型而變化。選擇適當劑量的指南可以在文獻中找到。此外，治療不一定導致對疾病或病症的100%或完全治療或預防。有多種具有不同程度治療效果的治療/預防方法可用，本領域普通技術人員將其視為潛在有利的治療手段。Effective amounts and schedules of administration can be determined empirically, and it is within the skill in the art to make such determinations. Those skilled in the art will appreciate that the dosage that must be administered will vary depending on, for example, the mammal to be treated, the route of administration, the particular type of therapeutic and other drugs administered to the mammal. Guidelines for choosing an appropriate dose can be found in the literature. Furthermore, treatment does not necessarily result in 100% or complete treatment or prevention of the disease or disorder. There are a variety of therapeutic/preventive methods available with varying degrees of therapeutic effect, which are considered potentially beneficial treatments by those of ordinary skill in the art.

在一些方面，本公開還提供了診斷哺乳動物中的疾病/病症的方法，其中所述TCR、CAR、抗原結合片段、TCR衍生的結合分子與從受試者獲得的一個或多個樣品發生相互作用以形成複合物，其中所述樣品可以包含一種或多種細胞、多肽、蛋白質、核酸、抗體、或抗原結合部分；血液、全細胞、其裂解物、或全細胞裂解物的級分，例如細胞核或細胞質級分、全蛋白級分、或其核酸級分，其中對複合物的檢測指示哺乳動物中病症的存在，其中所述病症是癌症或感染。此外，對複合物的檢測可以通過任何數量的本領域已知的方式，但不限於ELISA、流式細胞術、螢光原位雜交(FISH)、聚合酶鏈反應(PCR)、微陣列、南方點墨法、電泳、噬菌體分析、層析等。因此，治療方法可以還包括例如通過確定受試者是否患有感染或癌症來確定受試者是否可以從本文公開的治療中受益。In some aspects, the present disclosure also provides methods of diagnosing a disease/disorder in a mammal, wherein the TCR, CAR, antigen-binding fragment, TCR-derived binding molecule interacts with one or more samples obtained from a subject Act to form a complex, wherein the sample may comprise one or more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen-binding moieties; blood, whole cells, lysates thereof, or fractions of whole cell lysates, such as nuclei or a cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction thereof, wherein detection of the complex is indicative of the presence of a disorder in the mammal, wherein the disorder is cancer or infection. Furthermore, detection of complexes can be by any number of means known in the art, but not limited to ELISA, flow cytometry, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), microarray, Southern Spotting, electrophoresis, phage analysis, chromatography, etc. Thus, methods of treatment can further include determining whether a subject can benefit from the treatments disclosed herein, eg, by determining whether the subject has an infection or cancer.

在本文所述的任何方法中，工程化細胞和、和/或至少一種另外的治療劑可以每週至少一次(例如，每週一次、每週兩次、每週三次、每週四次、每天一次、每天兩次或每天三次)施用至受試者。在一些實施方案中，將至少兩種不同的工程化細胞(例如表現不同結合分子的細胞)以同一組合物(例如液體組合物)施用。在一些實施方案中，將工程化細胞和至少一種另外的治療劑以同一組合物(例如液體組合物)施用。在一些實施方案中，將工程化細胞和至少一種另外的治療劑以兩種不同的組合物施用。在一些實施方案中，將所述至少一種另外的治療劑以丸劑、片劑或膠囊劑施用。在一些實施方案中，將所述至少一種另外的治療劑以持續釋放的口服配製品施用。In any of the methods described herein, the engineered cells and/or at least one additional therapeutic agent can be at least once a week (eg, once a week, twice a week, three times a week, four times a week, daily once, twice daily, or three times daily) to the subject. In some embodiments, at least two different engineered cells (eg, cells expressing different binding molecules) are administered in the same composition (eg, a liquid composition). In some embodiments, the engineered cells and the at least one additional therapeutic agent are administered in the same composition (eg, a liquid composition). In some embodiments, the engineered cells and at least one additional therapeutic agent are administered in two different compositions. In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

在一些實施方案中，可以在將工程化細胞施用至受試者之前、同時或之後，將一種或多種另外的治療劑施用至受試者。In some embodiments, one or more additional therapeutic agents can be administered to a subject before, concurrently with, or after administration of the engineered cells to the subject.

在一些實施方案中，可以將一種或多種另外的治療劑施用至受試者。所述另外的治療劑可以是檢查點抑制劑(CPI)。在一些實施方案中，檢查點抑制劑是抑制蛋白，例如抗體或其抗原結合片段。檢查點抑制劑可以抑制或阻斷一個或多個免疫檢查點，包括例如PD-1、PD-L1、PD-L2、2B4(CD244)、4-1BB、A2aR、B7.1、B7.2、B7-H2、B7-H3、B7-H4、B7-H6、BTLA、嗜乳脂蛋白、CD160、CD48、CTLA4、GITR、gp49B、HHLA2、HVEM、ICOS、ILT-2、ILT-4、KIR家族受體、LAG-3、OX-40、PIR-B、SIRPα(CD47)、TFM-4、TIGIT、TIM-1、TIM-3、TIM-4、VISTA及其組合。在一些實施方案中，抑制蛋白阻斷PD-1或PD-Ll。在各種實施方案中，抑制蛋白包含抗PD-1 scFv。抑制蛋白能夠導致群體的T細胞中PD-1或PD-L1的表現降低和/或抑制PD-1或PD-L1的上調、和/或在物理上阻礙PD-1/PD-L1複合物的形成和隨後的訊息轉導。在一些實施方案中，抑制蛋白阻斷PD-1。在一些實施方案中，另外的治療劑是抗OX40抗體、抗PD-L1抗體、抗PD-L2抗體、抗LAG-3抗體、抗TIGIT抗體、抗BTLA抗體、抗CTLA-4抗體、或抗GITR抗體。在一些實施方案中，另外的治療劑是抗CTLA4抗體(例如易普利姆瑪)、抗CD20抗體(例如利妥昔單抗)、抗EGFR抗體(例如西妥昔單抗)、抗CD319抗體(例如埃羅妥珠單抗(elotuzumab))或抗PD1抗體(例如納武單抗)。In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent may be a checkpoint inhibitor (CPI). In some embodiments, the checkpoint inhibitor is an inhibitory protein, such as an antibody or antigen-binding fragment thereof. Checkpoint inhibitors can inhibit or block one or more immune checkpoints including, for example, PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, Butteroprotein, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors , LAG-3, OX-40, PIR-B, SIRPα (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, and combinations thereof. In some embodiments, the arrestin blocks PD-1 or PD-L1. In various embodiments, the arrestin comprises an anti-PD-1 scFv. Arrestins can result in decreased expression of PD-1 or PD-L1 in a population of T cells and/or inhibit the upregulation of PD-1 or PD-L1, and/or physically block the PD-1/PD-L1 complex formation and subsequent message transduction. In some embodiments, arrestin blocks PD-1. In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody Antibody. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (eg, ipilimumab), an anti-CD20 antibody (eg, rituximab), an anti-EGFR antibody (eg, cetuximab), an anti-CD319 antibody (eg elotuzumab) or anti-PD1 antibody (eg nivolumab).

在一些實施方案中，另外的治療劑是雙功能陷阱融合蛋白。雙功能陷阱蛋白可以靶向免疫檢查點和TGF-β負調控途徑二者。除了表現免疫檢查點外，腫瘤微環境還含有其他免疫抑制分子。特別令人感興趣的是在癌症中具有多種功能的細胞介素TGF-β(TGFB)。TGF-β在腫瘤發展的早期阻止腫瘤細胞的增殖並促進分化和凋亡。然而，在腫瘤進展期間，由於TGF-β受體表現的喪失或下游訊息傳導元件的突變而引起腫瘤TGF-β不敏感性。然後，TGF-β通過其對血管生成、上皮-間充質轉化(EMT)的誘導和免疫抑制的作用來促進腫瘤進展。高的TGF-β血清位準和在腫瘤上TGF-β受體(TGFβR)表現的喪失與不良預後相關。TGFβ靶向療法已展示出有限的臨床活性。在一些實施方案中，雙功能陷阱蛋白靶向PD-1和TGF-β二者。在一些實施方案中，雙功能陷阱蛋白靶向PD-L1和TGF-β二者。在一些實施方案中，雙功能融合蛋白設計成阻斷PD-L1和螯合(sequester)TGF-β。基於人IgG1單株抗體(mAb)阿維魯單抗(avelumab)，M7824(MSB0011395C)包含與人抗PD-L1 scFv的C末端連接的人TGF-β受體II(TGFβRII)的細胞外結構域。在一些實施方案中，雙功能融合蛋白包含與人抗PD-1 scFv的C末端連接的人TGF-β受體II(TGFβRII)的細胞外結構域。這些雙功能陷進融合蛋白例如在以下文獻中進行了描述：Knudson等人，“M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine.” Oncoimmunology 7.5 (2018): e1426519；該文獻全文以引用方式併入本文。在一些實施方案中，用表現本文所述的TCR或抗原結合分子的細胞和一種或多種雙功能陷阱融合蛋白治療受試者。In some embodiments, the additional therapeutic agent is a bifunctional trap fusion protein. Bifunctional trap proteins can target both immune checkpoints and TGF-beta negative regulatory pathways. In addition to expressing immune checkpoints, the tumor microenvironment also contains other immunosuppressive molecules. Of particular interest is the interleukin TGF-beta (TGFB), which has multiple functions in cancer. TGF-β prevents tumor cell proliferation and promotes differentiation and apoptosis in the early stages of tumor development. However, during tumor progression, tumor TGF-β insensitivity occurs due to loss of TGF-β receptor expression or mutation of downstream signaling elements. TGF-β then promotes tumor progression through its effects on angiogenesis, induction of epithelial-mesenchymal transition (EMT), and immunosuppression. High TGF-beta serum levels and loss of TGF-beta receptor (TGFbetaR) expression on tumors are associated with poor prognosis. TGFβ-targeted therapy has demonstrated limited clinical activity. In some embodiments, the bifunctional trap protein targets both PD-1 and TGF-beta. In some embodiments, the bifunctional trap protein targets both PD-L1 and TGF-beta. In some embodiments, the bifunctional fusion protein is designed to block PD-L1 and sequester TGF-beta. Human IgG1 based monoclonal antibody (mAb) avelumab, M7824 (MSB0011395C) contains the extracellular domain of human TGF-β receptor II (TGFβRII) linked to the C-terminus of a human anti-PD-L1 scFv . In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-1 scFv. These bifunctional trap fusion proteins are described, for example, in Knudson et al., "M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine. "Oncoimmunology 7.5 (2018): e1426519; the entirety of which is incorporated herein by reference." In some embodiments, a subject is treated with a cell expressing a TCR or antigen binding molecule described herein and one or more bifunctional trap fusion proteins.

在一些實施方案中，所述另外的治療劑可以包含一種或多種抑制劑，所述一種或多種抑制劑選自由以下組成的組：B-Raf的抑制劑、EGFR抑制劑、MEK抑制劑、ERK抑制劑、K-Ras抑制劑、c-Met抑制劑、間變性淋巴瘤激酶(ALK)抑制劑、磷脂醯肌醇3激酶(PI3K)抑制劑、Akt抑制劑、mTOR抑制劑、PI3K/mTOR雙重抑制劑、布魯頓氏酪胺酸激酶(BTK)抑制劑、以及異檸檬酸脫氫酶1(IDH1)和/或異檸檬酸脫氫酶2(IDH2)的抑制劑。在一些實施方案中，所述另外的治療劑是吲哚胺2,3-二加氧酶-1)(IDO1)的抑制劑(例如艾卡哚司他(epacadostat))。在一些實施方案中，所述另外的治療劑可以包含一種或多種抑制劑，所述一種或多種抑制劑選自由以下組成的組：HER3抑制劑、LSD1抑制劑、MDM2抑制劑、BCL2抑制劑、CHK1抑制劑、活化的hedgehog訊息傳導途徑的抑制劑，以及選擇性地降解***受體的藥劑。In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, MEK inhibitors, ERK inhibitors Inhibitor, K-Ras inhibitor, c-Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol 3 kinase (PI3K) inhibitor, Akt inhibitor, mTOR inhibitor, PI3K/mTOR dual Inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and inhibitors of isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (eg, epacadostat). In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

在一些實施方案中，所述另外的治療劑可以包含一種或多種治療劑，所述一種或多種治療劑選自由以下組成的組：曲貝替定、白蛋白結合型紫杉醇(nab-paclitaxel)、Trebananib、帕唑帕尼、西地尼布、帕博昔布、依維莫司、氟嘧啶、IFL、瑞戈非尼、Reolysin、力比泰(Alimta)、Zykadia、索坦(Sutent)、坦西莫司、阿昔替尼、依維莫司、索拉非尼、Votrient、帕唑帕尼、IMA-901、AGS-003、卡博替尼、長春氟寧、Hsp90抑制劑、Ad-GM-CSF、替莫唑胺(Temazolomide)、IL-2、IFNa、長春鹼、沙立度胺(Thalomid)、達卡巴嗪、環磷醯胺、來那度胺、氮雜胞苷、來那度胺、硼替佐米、氨柔比星(amrubicine)、卡非佐米、普拉曲沙和恩紮妥林。In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, Trebananib, pazopanib, cediranib, pembocoxib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsimo Division, Axitinib, Everolimus, Sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, Cabozantinib, Vinflunine, Hsp90 Inhibitor, Ad-GM-CSF , temozolomide (Temazolomide), IL-2, IFNa, vinblastine, thalidomide (Thalomid), dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib , amrubicine, carfilzomib, pralatrexate, and enzastaurin.

在一些實施方案中，所述另外的治療劑可以包含一種或多種治療劑，所述一種或多種治療劑選自由以下組成的組：佐劑、TLR激動劑、腫瘤壞死因子(TNF)α、IL-1、HMGB1、IL-10拮抗劑、IL-4拮抗劑、IL-13拮抗劑、IL-17拮抗劑、HVEM拮抗劑、ICOS激動劑、靶向CX3CL1的治療、靶向CXCL9的治療、靶向CXCL10的治療、靶向CCL5的治療、LFA-1激動劑、ICAM1激動劑和選擇素(Selectin)激動劑。在一些實施方案中，所述另外的治療劑可以是CXCL10、Flt3L或XCL1。In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of adjuvant, TLR agonist, tumor necrosis factor (TNF) alpha, IL -1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, IL-17 antagonist, HVEM antagonist, ICOS agonist, therapy targeting CX3CL1, therapy targeting CXCL9, target CXCL10-targeted therapy, CCL5-targeted therapy, LFA-1 agonists, ICAM1 agonists, and Selectin agonists. In some embodiments, the additional therapeutic agent may be CXCL10, Flt3L, or XCL1.

在一些實施方案中，將卡鉑、白蛋白結合型紫杉醇、紫杉醇、順鉑、培美曲塞、吉西他濱、FOLFOX或FOLFIRI施用至受試者。在一些實施方案中，所述另外的治療劑選自天冬醯胺酶、白消安、卡鉑、順鉑、柔紅黴素、多柔比星、氟尿嘧啶、吉西他濱、羥基脲、胺甲喋呤、紫杉醇、利妥昔單抗、長春鹼、長春新鹼和/或其組合。組合物和配製品 In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, carbamazepine urea, paclitaxel, rituximab, vinblastine, vincristine, and/or combinations thereof. Compositions and Formulations

本公開提供了含有通過本文公開的方法生產的工程化細胞及其群體的組合物(包括藥物組合物和治療組合物)。還提供了用於向受試者(例如患者)施用工程化細胞及其組合物的方法(例如治療方法)。The present disclosure provides compositions (including pharmaceutical compositions and therapeutic compositions) containing engineered cells and populations thereof produced by the methods disclosed herein. Also provided are methods (eg, therapeutic methods) for administering the engineered cells and compositions thereof to a subject (eg, a patient).

提供了包括用於施用的工程化細胞的組合物，包括藥物組合物和配製品，諸如包括用於以給定劑量或其部分施用的細胞數量的單位劑型組合物。藥物組合物和配製品可以包括一種或多種任選的藥學上可接受的載體或賦形劑。在一些實施方案中，所述組合物包括至少一種另外的治療劑。Compositions comprising engineered cells for administration are provided, including pharmaceutical compositions and formulations, such as unit dosage compositions comprising the number of cells for administration in a given dose or fraction thereof. Pharmaceutical compositions and formulations may include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

藥學上可接受的載體是指藥物組合物中除活性成分以外的成分。藥學上可接受的載體不干擾活性成分並且對受試者無毒。藥學上可接受的載體可以包括但不限於緩衝液、賦形劑、穩定劑或防腐劑。藥物配製品是指將不同的物質和/或藥劑組合以產生最終藥物產品的過程。配製品研究涉及開發對於患者可接受的藥物製劑。另外，是這樣的製劑，其處於使得其中所含活性成分的生物活性有效的形式，並且不含對給予配製品的受試者具有不可接受的毒性的另外的組分。A pharmaceutically acceptable carrier refers to ingredients other than the active ingredient in a pharmaceutical composition. A pharmaceutically acceptable carrier does not interfere with the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers or preservatives. Pharmaceutical formulation refers to the process of combining different substances and/or pharmaceutical agents to produce a final drug product. Formulation research involves the development of pharmaceutical formulations that are acceptable to patients. Additionally, are formulations that are in a form that renders the biological activity of the active ingredient contained therein effective and that are free of additional components that would have unacceptable toxicity to the subject to whom the formulation is administered.

在一些實施方案中，載體的選擇部分地由特定細胞(例如T細胞或NK細胞)和/或由施用方法來確定。多種合適的配製品是可用的。例如，藥物組合物可以含有防腐劑。合適的防腐劑可以包括例如對羥基苯甲酸甲酯、對羥基苯甲酸丙酯、苯甲酸鈉和苯紮氯銨。在一些實施方案中，使用兩種或更多種防腐劑的混合物。所述防腐劑或其混合物通常以按總組合物的重量計約0.0001%至約2%的量存在。載體描述於例如Remington’s Pharmaceutical Sciences 第16版, Osol, A.編輯 (1980)中。藥學上可接受的載體在所用的劑量和濃度下通常對接受者無毒，並且包括但不限於：緩衝劑，如磷酸鹽、檸檬酸鹽和其他有機酸；抗氧化劑，包括抗壞血酸和甲硫胺酸；防腐劑(如十八烷基二甲基苄基氯化銨；六甲氯銨；苯紮氯銨；苄索氯銨；苯酚、丁醇或苄醇；烷基對羥基苯甲酸酯，如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯；兒茶酚；間苯二酚；環己醇；3-戊醇；和間甲酚)；低分子量(少於約10個殘基)多肽；蛋白質，如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，如聚乙烯吡咯烷酮；胺基酸，如甘胺酸、麩醯胺酸、天冬醯胺、組胺酸、精胺酸或離胺酸；單醣、雙醣和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，如EDTA；糖類，如蔗糖、甘露醇、海藻糖或山梨醇；成鹽抗衡離子，如鈉；金屬絡合物(例如鋅-蛋白質絡合物)；和/或非離子表面活性劑，如聚乙二醇(PEG)。In some embodiments, the choice of vector is determined in part by the particular cell (eg, T cells or NK cells) and/or by the method of administration. A variety of suitable formulations are available. For example, pharmaceutical compositions may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservatives or mixtures thereof are generally present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine ; preservatives (eg octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides ; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartamine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as Sodium; metal complexes (eg, zinc-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG).

合適的緩衝劑包括例如檸檬酸、檸檬酸鈉、磷酸、磷酸鉀和各種其他酸和鹽。在一些實施方案中，使用兩種或更多種緩衝劑的混合物。所述緩衝劑或其混合物通常以按總組合物的重量計約0.001%至約4%的量存在。用於製備可給予的藥物組合物的方法是已知的。示例性方法在例如以下文獻中有更詳細的描述：Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 第21版 (2005年5月1日)。Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. The buffer or mixtures thereof are generally present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Ed. (May 1, 2005).

配製品可以包括水性溶液。所述配製品或組合物還可以含有可用於正在用工程化細胞治療的特定適應症、疾病或病症的超過一種活性成分，優選地具有與所述細胞互補的活性的那些成分，其中各自的活性不會彼此產生不利影響。此類活性成分以有效用於既定目的的量以合適的方式組合存在。因此，在一些實施方案中，藥物組合物還可以包括其他藥物活性藥劑或藥物，諸如檢查點抑制劑、融合蛋白、化學治療劑，例如天冬醯胺酶、白消安、卡鉑、順鉑、柔紅黴素、多柔比星、氟尿嘧啶、吉西他濱、羥基脲、胺甲喋呤、紫杉醇、利妥昔單抗、長春鹼和/或長春新鹼。Formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated with the engineered cells, preferably those ingredients having activities complementary to the cells, wherein the respective activities will not adversely affect each other. Such active ingredients are present in suitable combinations in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition may also include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, eg, asparaginase, busulfan, carboplatin, cisplatin , Daunorubicin, Doxorubicin, Fluorouracil, Gemcitabine, Hydroxyurea, Ammethopterin, Paclitaxel, Rituximab, Vinblastine and/or Vincristine.

在一些實施方案中，藥物組合物含有有效治療或預防疾病或病症的量(諸如治療有效量或預防有效量)的細胞。在一些實施方案中，通過定期評估所治療的受試者來監測治療或預防功效。所需劑量可以通過細胞的單次推注施用、通過細胞的多次推注施用或通過細胞的連續輸注施用來遞送。In some embodiments, the pharmaceutical composition contains the cells in an amount effective to treat or prevent a disease or disorder, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodically evaluating the treated subject. The desired dose can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

細胞和組合物可以使用標準給藥技術、配製品和/或裝置來施用。所述細胞的施用可以是自體的或異源的。例如，免疫反應T細胞或祖細胞可以獲得自一名受試者，並且在根據本文所述的各種實施方案將其進行遺傳修飾之後，將其施用至同一受試者或不同的相容受試者。外周血衍生的免疫反應T細胞或其後代(例如，體內、離體或體外衍生的)可以通過局部注射施用，包括導管施用、全身注射、局部注射、靜脈內注射或腸胃外施用。通常，當施用治療性組合物(例如，含有遺傳修飾的免疫反應細胞的藥物組合物)時，通常將其配製成單位劑量可注射形式(溶液、懸浮液、乳液)。Cells and compositions can be administered using standard administration techniques, formulations and/or devices. Administration of the cells can be autologous or allogeneic. For example, immune reactive T cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject after genetic modification thereof according to various embodiments described herein By. Peripheral blood-derived immunoreactive T cells or progeny thereof (eg, in vivo, ex vivo or in vitro derived) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. Typically, when a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immune-responsive cells) is administered, it is usually formulated in a unit dose injectable form (solution, suspension, emulsion).

本文公開的配製品包括用於口服、靜脈內、腹膜內、皮下、經肺、透皮、肌內、鼻內、經頰、舌下或栓劑施用的那些。在一些實施方案中，腸胃外地施用所述細胞群。如本文所用，術語“腸胃外”包括靜脈內、肌內、皮下、直腸、***和腹膜內施用。在一些實施方案中，通過靜脈內、腹膜內或皮下注射使用外周全身遞送將細胞施用至受試者。The formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the population of cells is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

在一些實施方案中，組合物作為無菌液體製劑提供，所述無菌液體製劑例如為等滲水性溶液、懸浮液、乳液、分散體或黏性組合物，其在一些方面可以緩衝至選擇的pH。液體製劑一般比凝膠、其他黏性組合物和固體組合物製備起來更容易。另外地，液體組合物稍微更方便施用，特別是通過注射。在另一方面，黏性組合物可以配製在適當的黏度範圍內，以提供與特定組織的更長的接觸時間。液體或黏性組合物可以包含載體，所述載體可以是溶劑或分散介質，所述溶劑或分散介質含有例如水、鹽水、磷酸鹽緩衝鹽水、多元醇(例如，甘油、丙二醇、液體聚乙二醇)及其合適的混合物。In some embodiments, the compositions are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. In another aspect, the viscous composition can be formulated in an appropriate viscosity range to provide longer contact times with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) alcohol) and suitable mixtures thereof.

無菌可注射溶液可以通過將細胞摻入溶劑中來製備，例如與合適的載體、稀釋劑或賦形劑(如無菌水、生理鹽水、葡萄糖、右旋糖等)混合。所述組合物可以含有輔助物質，諸如潤濕劑、分散劑或乳化劑(例如甲基纖維素)、pH緩衝劑、膠凝或黏度增強添加劑、防腐劑、調味劑和/或顏料，這取決於施用途徑和所需的製劑。在一些方面，可以參考標準文本來製備合適的製劑。Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, with an appropriate carrier, diluent, or excipient (eg, sterile water, physiological saline, dextrose, dextrose, and the like). The compositions may contain auxiliary substances such as wetting agents, dispersing agents or emulsifying agents (eg methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents and/or pigments, depending on the on the route of administration and the desired formulation. In some aspects, reference can be made to standard texts for the preparation of suitable formulations.

可以添加增強所述組合物的穩定性和無菌性的各種添加劑，包括抗微生物防腐劑、抗氧化劑、螯合劑和緩沖劑。可以通過各種抗細菌劑和抗真菌劑(例如對羥基苯甲酸酯、氯丁醇、苯酚和山梨酸)來確保防止微生物作用。可以通過使用延遲吸收的藥劑(例如單硬脂酸鋁和明膠)實現可注射藥物形式的延長吸收。Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.

用於進行體內 施用的配製品通常是無菌的。可以例如通過經無菌濾膜過濾容易地實現無菌性。 Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile membranes.

本文所述的組合物或藥物組合物可以與施用說明書一起包含在容器、包裝或分配器中。施用方法 The compositions or pharmaceutical compositions described herein can be included in a container, pack or dispenser with instructions for administration. Application method

還提供了施用細胞、群體和組合物的方法，以及此類細胞、群體和組合物治療或預防疾病、病症和障礙(包括癌症)的用途。在一些實施方案中，本文所述的方法可以降低發生本文所述的疾病、病症和障礙的風險。Also provided are methods of administering cells, populations, and compositions, as well as the use of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, the methods described herein can reduce the risk of developing the diseases, conditions, and disorders described herein.

在一些實施方案中，將本文所述的細胞、群體和組合物施用至受試者或患者，所述受試者或患者患有將要例如通過過繼細胞療法(諸如過繼T細胞療法)治療的特定疾病或病症。在一些實施方案中，將通過所提供的方法製備的細胞和組合物(諸如孵育和/或其他處理步驟後的工程化組合物和最終生產組合物)施用至受試者，諸如患有疾病或病症或具有患上疾病或病症的風險的受試者。在一些方面，所述方法由此治療疾病或病症(例如，改善疾病或病症的一種或多種症狀)，諸如通過減輕表現由工程化T細胞識別的抗原的癌症中的腫瘤負荷來治療。In some embodiments, the cells, populations, and compositions described herein are administered to a subject or patient suffering from a specific disease to be treated, eg, by adoptive cell therapy, such as adoptive T cell therapy disease or condition. In some embodiments, cells and compositions prepared by the provided methods (such as engineered compositions and final production compositions after incubation and/or other processing steps) are administered to a subject, such as suffering from a disease or A disorder or a subject at risk of developing a disease or disorder. In some aspects, the methods thereby treat a disease or disorder (eg, ameliorate one or more symptoms of the disease or disorder), such as by reducing tumor burden in cancers that express antigens recognized by engineered T cells.

用於施用過繼細胞療法的細胞的方法是已知的，並且可以與所提供的方法和組合物結合使用。例如，過繼T細胞療法的方法描述於例如U.S. 2003/0170238；美國專利 號4,690,915；Rosenberg, “Cell transfer immunotherapy for metastatic solid cancer—what clinicians need to know.” Nature reviews Clinical oncology 8.10 (2011): 577；Themeli等人, “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Nature biotechnology 31.10 (2013): 928；Tsukahara等人, “CD19 target-engineered T cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.” Biochemical and biophysical research communications 438.1 (2013): 84-89；Davila等人, “CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.” PloS one 8.4 (2013)；這些文獻中的每一者全文以引用方式併入本文。Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in U.S. 2003/0170238; U.S. Patent No. 4,690,915; Rosenberg, "Cell transfer immunotherapy for metastatic solid cancer—what clinicians need to know." Nature reviews Clinical oncology 8.10 (2011): 577; Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Nature biotechnology 31.10 (2013): 928; Tsukahara et al., “CD19 target-engineered T cells accumulate at tumor lesions in human B -cell lymphoma xenograft mouse models.” Biochemical and biophysical research communications 438.1 (2013): 84-89; Davila et al., “CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia." PloS one 8.4 (2013); each of these documents is incorporated herein by reference in its entirety.

在一些實施方案中，通過自體轉移進行細胞療法(例如過繼T細胞療法)，其中從將要接受細胞療法的受試者中或從衍生自這樣的受試者的樣品中分離和/或以其他方式製備T細胞。因此，在一些方面，所述細胞源自於需要治療的受試者(例如患者)，並且在分離和處理後將所述細胞施用至同一受試者。In some embodiments, cell therapy (eg, adoptive T cell therapy) is performed by autologous transfer, wherein isolation and/or other way to prepare T cells. Thus, in some aspects, the cells are derived from a subject (eg, a patient) in need of treatment, and the cells are administered to the same subject after isolation and treatment.

在一些實施方案中，通過同種異體轉移進行細胞療法(例如過繼T細胞療法)，其中從除將要接受或最終接受細胞療法的受試者以外的受試者(例如第一受試者)分離和/或以其他方式製備T細胞。在此類實施方案中，細胞隨後被施用至同一物種的不同受試者(例如第二受試者)。在一些實施方案中，所述第一受試者和第二受試者在遺傳上是相同的。在一些實施方案中，所述第一受試者和第二受試者在遺傳上是相似的。在一些實施方案中，所述第二受試者與所述第一受試者表現相同的HLA類別或超類型。In some embodiments, cell therapy (eg, adoptive T cell therapy) is performed by allogeneic transfer, wherein the isolation and /or otherwise prepare T cells. In such embodiments, the cells are subsequently administered to a different subject (eg, a second subject) of the same species. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject exhibits the same HLA class or supertype as the first subject.

在一些實施方案中，在施用細胞或含有細胞的組合物之前，已經用靶向疾病或病症(例如腫瘤)的治療劑對受試者進行過治療。在一些方面，受試者對其他治療劑是難治的或無反應。在一些實施方案中，受試者例如在用另一種治療性干預進行治療後具有持續性或復發性疾病，所述治療性干預包括化學療法、放射線和/或造血幹細胞移植(HSCT)(例如同種異體HSCT)。在一些實施方案中，儘管受試者已經對另一種療法產生抗性，但施用仍有效地治療受試者。In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or disorder (eg, tumor) prior to administration of the cells or cell-containing composition. In some aspects, the subject is refractory or unresponsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease, eg, following treatment with another therapeutic intervention including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) (eg, allogeneic allogeneic HSCT). In some embodiments, the administration is effective to treat the subject despite the subject having developed resistance to another therapy.

在一些實施方案中，受試者對其他治療劑有反應，並且用所述治療劑進行治療降低了疾病負荷。在一些方面，受試者最初對所述治療劑有反應，但是隨著時間的推移展現出疾病或病症的復發。在一些實施方案中，受試者尚未復發。在一些此類實施方案中，確定受試者處於復發的風險中，諸如處於高復發風險中，因此預防性地施用細胞，例如以降低復發的可能性或防止復發。在一些實施方案中，受試者尚未接受用另一種治療劑的先前治療。In some embodiments, the subject is responsive to other therapeutic agents, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject initially responds to the therapeutic agent, but exhibits recurrence of the disease or disorder over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, eg, to reduce the likelihood of relapse or to prevent relapse. In some embodiments, the subject has not received prior treatment with another therapeutic agent.

在一些實施方案中，細胞以所需劑量施用，所述所需劑量在一些方面包括所需劑量或數量的細胞或一種或多種細胞類型和/或所需比率的細胞類型。因此，在一些實施方案中，細胞劑量基於細胞總數(或每kg體重的細胞數量)和所需的單個群體或亞型的比率，諸如CD4+與CD8+的比率。在一些實施方案中，細胞劑量基於所需的單個群體中的細胞或單個細胞類型的總數(或每kg體重的細胞數量)。在一些實施方案中，劑量基於此類特徵的組合，諸如所需的總細胞數、所需比率、和單個群體中所需的細胞總數。In some embodiments, the cells are administered in a desired dose, which in some aspects includes a desired dose or number of cells or one or more cell types and/or a desired ratio of cell types. Thus, in some embodiments, the cell dosage is based on the total number of cells (or the number of cells per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dose of cells is based on the desired total number of cells or individual cell types in a single population (or number of cells per kg body weight). In some embodiments, the dosage is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in a single population.

在一些實施方案中，以所需劑量的總細胞(諸如所需劑量的T細胞)的耐受差異或在所述耐受差異之內施用細胞的群體或亞型(諸如CD8+和CD4+ T細胞)。在一些實施方案中，所需劑量是所需細胞數或被施用所述細胞的受試者的每單位體重的所需細胞數(例如細胞/kg)。在一些實施方案中，所需劑量等於或高於最小細胞數或每單位體重的最小細胞數。在一些實施方案中，在以所需劑量施用的總細胞中，單個群體或亞型以等於或接近所需輸出比率(諸如CD4+與CD8+的比率)(例如在這種比率的一定耐受差異或誤差內)存在。In some embodiments, populations or subtypes of cells (such as CD8+ and CD4+ T cells) are administered with a desired dose of total cells (such as a desired dose of T cells) for a tolerance differential or within the tolerance differential . In some embodiments, the desired dose is the desired number of cells or the desired number of cells per unit body weight of the subject to which the cells are administered (eg, cells/kg). In some embodiments, the desired dose is equal to or higher than the minimum number of cells or the minimum number of cells per unit body weight. In some embodiments, the individual populations or subtypes are at or near the desired output ratio (such as the ratio of CD4+ to CD8+) in total cells administered at the desired dose (eg, some tolerated difference in this ratio or error) exists.

在一些實施方案中，以所需劑量的一種或多種單個細胞群體或亞型(諸如所需劑量的CD4+細胞和/或所需劑量的CD8+細胞)的耐受差異或在所述耐受差異之內施用細胞。在一些實施方案中，所需劑量是所需的所述亞型或群體的細胞數或所需的被施用所述細胞的受試者的每單位體重的此類細胞數(例如細胞/kg)。在一些實施方案中，所需劑量等於或高於最小的所述群體或亞型的細胞數或每單位體重的最小的所述群體或亞型的細胞數。In some embodiments, the difference in tolerance to or between a desired dose of one or more individual cell populations or subtypes (such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells) intracellular administration of cells. In some embodiments, the desired dose is the desired number of cells of the subtype or population or the desired number of such cells per unit body weight of the subject to which the cells are administered (eg, cells/kg) . In some embodiments, the desired dose is equal to or higher than the minimum number of cells of said population or subtype or the minimum number of cells of said population or subtype per unit body weight.

因此，在一些實施方案中，劑量基於所需的總細胞固定劑量和所需比率，和/或基於所需固定劑量的一種或多種(例如每一種)單個亞型或亞群。因此，在一些實施方案中，劑量基於所需固定劑量或最小劑量的T細胞和所需的CD4+與CD8+細胞的比率，和/或基於所需固定劑量或最小劑量的CD4+和/或CD8+細胞。Thus, in some embodiments, the dose is based on the desired fixed dose of total cells and the desired ratio, and/or based on one or more (eg, each) individual subtypes or subpopulations of the desired fixed dose. Thus, in some embodiments, the dosage is based on the desired fixed or minimum dose of T cells and the desired ratio of CD4+ to CD8+ cells, and/or based on the desired fixed or minimum dose of CD4+ and/or CD8+ cells.

在某些實施方案中，將細胞、或細胞的單個群體或亞型以約100萬至約1000億個細胞(諸如，例如100萬至約500億個細胞(例如，約500萬個細胞、約2500萬個細胞、約5億個細胞、約10億個細胞、約50億個細胞、約200億個細胞、約300億個細胞、約400億個細胞或由前述值中的任兩個值所限定的範圍)，諸如約1000萬至約1000億個細胞(例如，約2000萬個細胞、約3000萬個細胞、約4000萬個細胞、約6000萬個細胞、約7000萬個細胞、約8000萬個細胞、約9000萬個細胞、約100億個細胞、約250億個細胞、約500億個細胞、約750億個細胞、約900億個細胞或由前述值中的任兩個值所限定的範圍)，並且在一些情況下約1億個細胞至約500億個細胞(例如，約1.2億個細胞、約2.5億個細胞、約3.5億個細胞、約4.5億個細胞、約6.5億個細胞、約8億個細胞、約9億個細胞、約30億個細胞、約300億個細胞、約450億個細胞或在這些範圍之間的任何值)施用至受試者。In certain embodiments, cells, or a single population or subtype of cells, are divided into about 1 million to about 100 billion cells (such as, for example, 1 million to about 50 billion cells (eg, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or any two of the foregoing values defined range), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or any two of the foregoing values defined range), and in some cases from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or any value in between these ranges) are administered to the subject.

在一些實施方案中，總細胞的劑量和/或單個細胞亞群的劑量在以下範圍內：在為或約104 與為或約109 個細胞/千克(kg)體重之間，諸如在105 與106 個細胞/kg體重之間，例如，至少或至少約或為或約1×105 個細胞/kg體重、1.5×105 個細胞/kg體重、2×105 個細胞/kg體重、或1×106 個細胞/kg體重。例如，在一些實施方案中，以在為或約104 與為或約109 個T細胞/千克(kg)體重之間，諸如在105 與106 個T細胞/kg體重之間，例如，至少或至少約或者為或約1×105 個T細胞/kg體重、1.5×105 個T細胞/kg體重、2×105 個T細胞/kg體重、或1×106 個T細胞/kg體重，或在其一定誤差範圍內施用細胞。In some embodiments, the dose of total cells and/or the dose of individual cell subsets is in the range between at or about 10 and at or about 10 cells per kilogram (kg) body weight, such as at 10 Between 5 and 10 6 cells/kg body weight, eg, at least or at least about or at or about 1 x 10 5 cells/kg body weight, 1.5 x 10 5 cells/kg body weight, 2 x 10 5 cells/kg Body weight, or 1×10 6 cells/kg body weight. For example, in some embodiments, at between at or about 10 and at or about 10 T cells/kilogram (kg) body weight, such as between 10 and 10 T cells/kg body weight, eg , at least or at least about or at or about 1 x 10 5 T cells/kg body weight, 1.5 x 10 5 T cells/kg body weight, 2 x 10 5 T cells/kg body weight, or 1 x 10 6 T cells/kg body weight /kg body weight, or administer cells within a certain margin of error.

在一些實施方案中，以在為或約104 與為或約109 個CD4+和/或CD8+細胞/千克(kg)體重之間，諸如在105 與106 個CD4+和/或CD8+細胞/kg體重之間，例如，至少或至少約或者為或約1×105 個CD4+和/或CD8+細胞/kg體重、1.5×105 個CD4+和/或CD8+細胞/kg體重、2×105 個CD4+和/或CD8+細胞/kg體重、或1×106 個CD4+和/或CD8+細胞/kg體重，或在其一定誤差範圍內施用細胞。In some embodiments, at between at or about 10 and at or about 10 CD4+ and/or CD8+ cells/kilogram (kg) body weight, such as between 10 and 10 CD4+ and/or CD8+ cells/ Between kg body weight, eg, at least or at least about or at or about 1 x 10 5 CD4+ and/or CD8+ cells/kg body weight, 1.5 x 10 5 CD4+ and/or CD8+ cells/kg body weight, 2 x 10 5 cells CD4+ and/or CD8+ cells/kg body weight, or 1 x 106 CD4+ and/or CD8+ cells/kg body weight, or cells were administered within a certain margin of error.

在一些實施方案中，以大於、和/或至少約1×106 、約2.5×106 、約5×106 、約7.5×106 、或約9×106 個CD4+細胞、和/或至少約1×106 、約2.5×106 、約5×106 、約7.5×106 、或約9×106 個CD8+細胞、和/或至少約1×106 、約2.5×106 、約5×106 、約7.5×106 、或約9×106 個T細胞，或在其一定誤差範圍內施用細胞。在一些實施方案中，以在約108 與1012 個之間或在約1010 與1011 個之間的T細胞、在約108 與1012 個之間或在約1010 與1011 個之間的CD4+細胞，和/或在約108 與1012 個之間或在約1010 與1011 個之間的CD8+細胞，或在其一定誤差範圍內施用細胞。In some embodiments, with greater than, and/or at least about 1×10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9×10 6 CD4+ cells, and/or At least about 1×10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9×10 6 CD8+ cells, and/or at least about 1×10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9×10 6 T cells, or within a certain margin of the administered cells. In some embodiments, with between about 10 8 and 10 12 , or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 , or between about 10 10 and 10 11 between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4+ cells, and/or between about 10 8 and 10 12 , or between about 10 10 and 10 11 , or administer the cells within a margin of error.

在一些實施方案中，在多種細胞群或亞型(諸如CD4+和CD8+細胞或亞型)的所需輸出比率的耐受範圍下或在其耐受範圍內施用細胞。在一些方面，所需比率可以是特定比率或可以是比率範圍。例如，在一些實施方案中，所需比率(例如，CD4+與CD8+細胞的比率)在為或約1:5與為或約5:1之間(或大於約1:5且小於約5:1)，或在為或約1:3與為或約3:1之間(或大於約1:3且小於約3:1)，諸如在為或約2:1與為或約1:5之間(或大於約1:5且小於約2:1)，諸如為或約5:1、4.5:1、4:1、3.5:1、3:1、2.5:1、2:1、1.9:1、1.8:1、1.7:1、1.6:1、1.5:1、1.4:1、1.3:1、1.2:1、1.1:1、1:1、1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2、1:2.5、1:3、1:3.5、1:4、1:4.5或1:5。在一些方面，耐受差異在所需比率的約1%、約2%、約3%、約4%、約5%、約10%、約15%、約20%、約25%、約30%、約35%、約40%、約45%、約50%內，包括這些範圍之間的任何值。在一些方面，本文所述的TCR提供了改善的表現和活性，從而即使在低的效應子與靶標(E:T)比率下也提供治療效果。In some embodiments, the cells are administered at or within the tolerable range of the desired output ratio of the various cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. For example, in some embodiments, the desired ratio (eg, ratio of CD4+ to CD8+ cells) is between at or about 1:5 and at or about 5:1 (or greater than about 1:5 and less than about 5:1 ), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1), such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9: 1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1: 5. In some aspects, the tolerance difference is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30% of the desired ratio %, about 35%, about 40%, about 45%, about 50%, including any value between these ranges. In some aspects, the TCRs described herein provide improved performance and activity, thereby providing therapeutic effects even at low effector-to-target (E:T) ratios.

對療法的最佳反應可能取決於工程化重組受體(諸如TCR)在細胞表面上一致且可靠地表現和/或結合靶抗原的能力。例如，在一些情況下，當在一些情況下在細胞(諸如人T細胞)中表現，用於細胞療法時，某些重組受體(例如TCR)的特性可能影響重組受體的表現和/或活性。在一些情境下，特定重組受體(例如TCR)的表現位準可能很低，並且表現此類重組受體的工程化細胞(諸如人T細胞)的活性可能由於表現不良或訊息傳導活性不佳而受到限制。在一些情況下，重組受體的表現的一致性和/或效率以及受體的活性在可用的治療方法的某些細胞或某些細胞群中受到限制。在一些情況下，需要大量的工程化T細胞(高的效應子與靶標(E:T)比率)來展現出功能活性。在一些實施方案中，所需比率(E:T比率)在為或約1:10與為或約10:1之間(或大於約1:10且小於約10:1)、或在為或約1:1與為或約10:1之間(或大於約1:1且小於約5:1)，諸如在為或約2:1與為或約10:1之間。在一些實施方案中，E:T比率為大於或約1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、或10:1。The optimal response to therapy may depend on the ability of engineered recombinant receptors, such as TCRs, to consistently and reliably express and/or bind target antigens on the cell surface. For example, in some cases, properties of certain recombinant receptors (eg, TCRs) may affect the expression and/or properties of certain recombinant receptors (eg, TCRs) when expressed in cells (such as human T cells) in some cases for use in cell therapy active. In some contexts, the level of expression of particular recombinant receptors (eg, TCRs) may be low, and the activity of engineered cells (such as human T cells) expressing such recombinant receptors may be due to poor expression or poor signaling activity and restricted. In some cases, the consistency and/or efficiency of expression of recombinant receptors and the activity of the receptors are limited in certain cells or certain populations of cells for available therapeutic methods. In some cases, large numbers of engineered T cells (high effector-to-target (E:T) ratios) are required to exhibit functional activity. In some embodiments, the desired ratio (E:T ratio) is between at or about 1:10 and at or about 10:1 (or greater than about 1:10 and less than about 10:1), or at or Between about 1:1 and at or about 10:1 (or greater than about 1:1 and less than about 5:1), such as between at or about 2:1 and at or about 10:1. In some embodiments, the E:T ratio is greater than or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

為了預防或治療疾病，適當的劑量可能取決於待治療的疾病類型、細胞或重組受體的類型、疾病的嚴重性和病程、細胞是否針對預防或治療目的而被施用、先前療法、受試者的臨床病史和對細胞的反應以及主治醫師的決斷。在一些實施方案中，適合將組合物和細胞一次或在一系列治療中施用至受試者。For prophylactic or therapeutic purposes, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are being administered for prophylactic or therapeutic purposes, previous therapy, the subject clinical history and response to cells and the discretion of the attending physician. In some embodiments, the compositions and cells are suitably administered to a subject at one time or over a series of treatments.

本文所述的細胞可以通過任何合適的手段施用，例如通過推注輸注，通過注射例如靜脈內或皮下注射、眼內注射、眼周注射、視網膜下注射、玻璃體內注射、經中隔注射、鞏膜下注射、脈絡膜內注射、前房注射、結膜下(subconjectval)注射、結膜下(subconjuntival)注射、眼球筋膜囊下(sub-Tenon)注射、球後注射、球周注射或後近鞏膜(posterior juxtascleral)遞送。在一些實施方案中，將它們通過腸胃外、肺內和鼻內施用以及(如果需要用於局部治療的話)病灶內施用。腸胃外輸注包括肌肉內、靜脈內、動脈內、腹膜內或皮下施用。在一些實施方案中，將給定劑量通過細胞的單次推注給藥來施用。在一些實施方案中，給定劑量通過細胞的多次推注給藥例如在不超過3天的時間段內來施用，或通過細胞的連續輸注給藥。The cells described herein can be administered by any suitable means, eg, by bolus infusion, by injection, eg, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, scleral injection Sub-, intra-choroidal, anterior chamber, subconjectval, subconjuntival, sub-Tenon, retrobulbar, peribulbar, or posterior juxtascleral) delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus injection of cells. In some embodiments, a given dose is administered by multiple bolus administration of cells, eg, over a period of not more than 3 days, or by continuous infusion of cells.

在一些實施方案中，所述細胞作為組合治療的一部分施用，如與另一種治療性干預如抗體或工程化細胞或受體或藥劑(如細胞毒性劑或治療劑)同時施用或以任何順序依次施用。在一些實施方案中，所述細胞與一種或多種另外的治療劑共同施用或與另一種治療性干預結合施用(同時或以任何順序依次施用)。在一些情境下，將所述細胞與另一種療法在時間上足夠接近地共同施用，使得所述細胞群增強一種或多種另外的治療劑的效果，或反之亦然。在一些實施方案中，在一種或多種另外的治療劑之前施用細胞。在一些實施方案中，在一種或多種另外的治療劑之後施用細胞。在一些實施方案中，一種或多種另外的藥劑包括細胞介素(如IL-2)，例如以增強持久性。在一些實施方案中，所述方法包括施用化學治療劑。In some embodiments, the cells are administered as part of a combination therapy, such as concurrently or sequentially in any order with another therapeutic intervention such as an antibody or engineered cell or receptor or agent (eg, a cytotoxic or therapeutic agent). apply. In some embodiments, the cells are co-administered with one or more additional therapeutic agents or in combination with another therapeutic intervention (simultaneously or sequentially in any order). In some cases, the cells are co-administered with another therapy sufficiently close in time that the population of cells enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include an interferon (eg, IL-2), eg, to enhance persistence. In some embodiments, the method includes administering a chemotherapeutic agent.

在一些實施方案中，在施用細胞後，例如通過許多已知方法中的任一種測量工程化細胞群的生物學活性。待評估的參數包括工程化T細胞與抗原的特異性結合，其在體內例如通過成像進行評估，或者離體例如通過ELISA或流式細胞術進行評估。在某些實施方案中，可以使用本領域已知的任何合適的方法測量工程化細胞破壞靶細胞的能力，所述方法諸如描述於例如Kochenderfer等人，“Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor.” Journal of immunotherapy (Hagerstown, Md.: 1997) 32.7 (2009): 689以及Hermans等人, “The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targetsin vitro andin vivo .” Journal of immunological methods 285.1 (2004): 25-40中。在某些實施方案中，通過測定一種或多種細胞介素(諸如CD107a、IFNγ、IL-2和TNF)的表現和/或分泌來測量細胞的生物活性。在一些方面，通過評估臨床結果(諸如腫瘤負荷或負擔的減少)來測量生物活性。In some embodiments, the biological activity of the engineered cell population is measured following administration of the cells, eg, by any of a number of known methods. Parameters to be assessed include the specific binding of engineered T cells to antigen, assessed in vivo, eg, by imaging, or ex vivo, eg, by ELISA or flow cytometry. In certain embodiments, the ability of an engineered cell to destroy a target cell can be measured using any suitable method known in the art, such as described in, eg, Kochenderfer et al., "Construction and pre-clinical evaluation of an anti- CD19 chimeric antigen receptor.” Journal of immunotherapy (Hagerstown, Md.: 1997) 32.7 (2009): 689 and Hermans et al., “The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets in in vitro and in vivo .” Journal of immunological methods 285.1 (2004): 25-40. In certain embodiments, the biological activity of the cells is measured by assaying the expression and/or secretion of one or more cytokines, such as CD107a, IFNy, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or burden.

提供了重複給藥方法，其中給予第一劑量的細胞，隨後給予一個或多個第二連續劑量。當以過繼療法方法施用至受試者時，通常設計細胞的多個劑量的時間安排和大小以增加如本文所述的工程化細胞的功效和/或活性和/或功能。所述方法涉及施用第一劑量，通常隨後是一個或多個連續劑量，在不同劑量之間具有特定的時間範圍。Repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, the timing and size of multiple doses of cells are typically designed to increase the efficacy and/or activity and/or function of engineered cells as described herein. The method involves administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.

在過繼細胞療法的情境下，給定“劑量”的施用包括以單一組合物和/或單次不間斷給藥的方式(例如以單次注射或連續輸注的方式)施用給定量或數量的細胞，並且還涵蓋在指定時間段(例如不超過3天)內以在多種單獨組合物或多次單獨輸注中提供的分割劑量的方式施用給定量或數量的細胞。因此，在一些情境下，第一或連續劑量是在單個時間點給予或開始的對指定數量的細胞的單次或連續施用。然而，在一些情境下，將第一或連續劑量在受限的時間段(例如不超過三天)內以多次注射或輸注的方式施用，諸如每天一次施用持續三天或兩天或者通過在一天的時間段內多次輸注的方式施用。In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells in a single composition and/or in a single uninterrupted administration (eg, in a single injection or continuous infusion). , and also encompass the administration of a given amount or number of cells in divided doses provided in multiple separate compositions or multiple separate infusions over a specified period of time (eg, no more than 3 days). Thus, in some contexts, a first or continuous dose is a single or continuous administration of a specified number of cells administered or initiated at a single time point. However, in some situations, the first or consecutive doses are administered as multiple injections or infusions over a limited period of time (eg, no more than three days), such as once daily for three or two days or by Administered by multiple infusions over a period of one day.

第一劑量的細胞以單一藥物組合物施用。在一些實施方案中，連續劑量的細胞以單一藥物組合物施用。The first dose of cells is administered in a single pharmaceutical composition. In some embodiments, consecutive doses of cells are administered in a single pharmaceutical composition.

在一些實施方案中，第一劑量的細胞以共同含有第一劑量的細胞的多種組合物施用。在一些實施方案中，連續劑量的細胞以共同含有連續劑量的細胞的多種組合物施用。在一些方面，可以在不超過3天的時間段內以多種組合物施用另外的連續劑量。In some embodiments, the first dose of cells is administered in multiple compositions that together contain the first dose of cells. In some embodiments, consecutive doses of cells are administered in multiple compositions that together contain consecutive doses of cells. In some aspects, additional consecutive doses of the various compositions may be administered over a period of not more than 3 days.

關於先前劑量，諸如第一劑量，術語“連續劑量”是指在先前(例如第一)劑量之後暫時沒有向受試者施用任何介入劑量的情況下向同一受試者施用的劑量。但是，所述術語不涵蓋在單個分割劑量內包含的一系列輸注或注射中的第二次、第三次和/或等等注射或輸注。因此，除非另有說明，否則在一天、兩天或三天時間段內的第二次輸注不被視為如本文所用的“連續”劑量。同樣，在“連續”劑量的意義的情境下，在分割劑量內的一系列多次劑量中的第二次、第三次等等也不被視為“介入”劑量。因此，除非另有說明，否則只要在第一或先前劑量開始後的三天時間段內發生了第二次或隨後的注射或輸注，即使受試者在第一劑量開始後接受了細胞的第二次或隨後的注射或輸注，在開始第一或先前劑量之後大於三天的一定時間段內施用的劑量都被視為“連續”劑量。With respect to a previous dose, such as a first dose, the term "successive dose" refers to a dose administered to the same subject without any intervening dose being administered to the subject temporarily following the previous (eg, first) dose. However, the term does not cover the second, third and/or etc. injections or infusions in a series of infusions or injections contained within a single divided dose. Thus, unless otherwise stated, a second infusion over a one, two or three day period is not considered a "continuous" dose as used herein. Likewise, the second, third, etc. of a series of multiple doses within a divided dose are not considered "interventional" doses in the context of a "continuous" dose. Therefore, unless otherwise stated, as long as the second or subsequent injection or infusion occurs within a three-day period after the start of the first or previous dose, even if the subject received the first dose of cells after the start of the first dose Second or subsequent injections or infusions, doses administered over a period of time greater than three days after initiation of the first or previous dose, are considered "continuous" doses.

因此，除非另有說明，否則在長達3天的時間段內多次施用相同細胞被視為單一劑量，並且在初次施用的3天內施用細胞不被視為連續劑量，並且也不被視為出於確定第二劑量是否與第一劑量“連續”的目的使用的介入劑量。Therefore, unless otherwise stated, multiple administrations of the same cells over a period of up to 3 days are considered a single dose, and administration of cells within 3 days of the initial administration is not considered a consecutive dose and is not considered The intervention dose used for the purpose of determining whether the second dose is "consecutive" with the first dose.

在一些實施方案中，在一些方面，使用與關於在第一劑量與第一連續劑量之間的時間安排指南相同的時間安排指南，例如通過施用第一和多個連續劑量，給予多個連續劑量。In some embodiments, in some aspects, the same timing guidelines as for timing between the first dose and the first consecutive dose are used, eg, by administering the first and multiple consecutive doses, multiple consecutive doses are administered .

在一 些實施 方案中，在第一劑量與第一連續劑量、或者第一與多個連續劑量之間的時間安排為使得在大於約5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天或更長時間的時間段內給予每個連續劑量。在一些實施方案中，在施用第一劑量或緊接先前劑量之後少於約28天的時間段內給予連續劑量。另外的多個另外一個或多個連續劑量也被稱為後續劑量或後續連續劑量。 In some embodiments , the timing between the first dose and the first consecutive dose, or the first and multiple consecutive doses, is such that the dose is less than about 5 days, 6 days, 7 days, 8 days, 9 days , 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days Each successive dose is administered over a period of 27 days, 28 days, or longer. In some embodiments, consecutive doses are administered for a period of less than about 28 days following administration of the first dose or immediately preceding dose. Additional multiples of one or more consecutive doses are also referred to as subsequent doses or subsequent consecutive doses.

將細胞的第一劑量和/或一個或多個連續劑量的大小通常設計為提供改善的功效和/或降低的毒性風險。在一些方面，第一劑量或任何連續劑量的劑量量或大小是如上所述的任何劑量或量。在一些實施方案中，在第一劑量或任何連續劑量中的細胞數在約0.5×106 個細胞/kg受試者體重與5×106 個細胞/kg之間、在約0.75×106 個細胞/kg與3×106 個細胞/kg之間、或在約1×106 個細胞/kg與2×106 個細胞/kg之間。The first dose of cells and/or one or more consecutive doses are typically sized to provide improved efficacy and/or reduced risk of toxicity. In some aspects, the dose amount or size of the first dose or any successive doses is any dose or amount as described above. In some embodiments, the number of cells in the first dose or any successive doses is between about 0.5 x 10 6 cells/kg subject body weight and 5 x 10 6 cells/kg, between about 0.75 x 10 6 cells/kg Between cells/kg and 3×10 6 cells/kg, or between about 1×10 6 cells/kg and 2×10 6 cells/kg.

如本文所用，“第一劑量”用於描述在施用連續或後續劑量之前的給定劑量的時間安排。所述術語不一定暗示受試者以前從未接受過一定劑量的細胞療法，或者甚至受試者以前沒有接受過一定劑量的相同細胞或表現相同重組受體或靶向相同抗原的細胞。As used herein, "first dose" is used to describe the timing of a given dose prior to administration of successive or subsequent doses. The term does not necessarily imply that the subject has never received a dose of cell therapy before, or even that the subject has not previously received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.

在一些實施方案中，可以在延長的時間段內(例如，在至少1周、2周、3周、1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、1年、2年、3年、4年或5年的時間段內)向受試者施用多個劑量。熟練的醫療專業人員可以使用本文所述的用於診斷或跟蹤治療有效性(例如觀察至少一種癌症症狀)的任何方法來確定治療期的長度。實施例 In some embodiments, over an extended period of time (eg, at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) to the subject dose. A skilled medical professional can determine the length of a treatment period using any of the methods described herein for diagnosing or tracking treatment effectiveness (eg, observing at least one cancer symptom). Example

在以下實施例中進一步描述本發明，所述實施例並不限制申請專利範圍中所描述的本發明的範圍。實施例 1. 構建體設計 The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Construct Design

小鼠和人IL-12表現載體(圖1A至圖1B)被設計成含有被肽接頭分開的IL-12B(p40)和IL-12A(p35)。對於膜拴系IL-12，在IL-12A之後添加具有兩個(膜拴系IL-12；mt-IL-12；或smt)或三個(長膜拴系IL-12；長mt-IL-12；或lmt)恆定結構域(CH2和CH3)的免疫球蛋白鉸鏈區以及CD4跨膜區。表現可溶性IL-12的載體也被設計成含有靶向腫瘤壞死的人IgG1即NHS-76的重鏈可變區(VH)和輕鏈可變區(VL)兩者(NHS76-IL-12)。Mouse and human IL-12 expression vectors (FIGS. 1A-1B) were designed to contain IL-12B (p40) and IL-12A (p35) separated by a peptide linker. For membrane-tethered IL-12, add two (membrane-tethered IL-12; mt-IL-12; or smt) or three (long membrane-tethered IL-12; long mt-IL) after IL-12A -12; or lmt) the immunoglobulin hinge region of the constant domains (CH2 and CH3) and the CD4 transmembrane region. A vector expressing soluble IL-12 was also designed to contain both the heavy chain variable region (VH) and light chain variable region (VL) of a human IgG1 targeting tumor necrosis, NHS-76 (NHS76-IL-12) .

為了產生與TCR或CAR組合表現IL-12的淋巴細胞，將編碼TCR(圖1C)或CAR(圖1D)的另外的核酸序列添加到表現載體中。該另外的序列和編碼IL-12B或NHS-76 vH的序列被P2A肽序列分開。To generate lymphocytes expressing IL-12 in combination with TCR or CAR, additional nucleic acid sequences encoding TCR (FIG. 1C) or CAR (FIG. 1D) were added to the expression vector. This additional sequence and the sequence encoding IL-12B or NHS-76 vH are separated by the P2A peptide sequence.

小鼠IL-12構建體示於圖1A中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 2和SEQ ID NO: 1。小鼠膜拴系IL-12構建體示於圖1A中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 3和SEQ ID NO: 4。小鼠長膜拴系IL-12構建體示於圖1A中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 5和SEQ ID NO: 6。人IL-12構建體示於圖1B中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 7和SEQ ID NO: 8。人膜拴系IL-12構建體示於圖1B中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 9和SEQ ID NO: 10。人長膜拴系IL-12構建體示於圖1B中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 11和SEQ ID NO: 12。人NHS76-IL-12構建體示於圖1B中，並且其核酸序列和對應的胺基酸序列分別為SEQ ID NO: 13和SEQ ID NO: 14。實施例 2. 工程化以表現 IL-12 的淋巴細胞分泌 IFNγ The mouse IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The mouse membrane-tethered IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The mouse long membrane tethered IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The human IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The human membrane-tethered IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The human long membrane tethered IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The human NHS76-IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Example 2. Lymphocytes engineered to express IL-12 secrete IFNγ

將純化的原代小鼠淋巴細胞工程化以表現單獨的L202 TCR(L202)(抗LMP2 TCR)、L202加小鼠膜拴系IL-12(樣品名稱：mt12)或L202加小鼠NHS-76-IL-12(樣品名稱：NHS76)。然後，轉導後兩天，將指示的淋巴細胞群與LLC-HLA-A2-Peplinker(LLW)靶細胞共培養過夜。使用針對mIL12 p40(目錄#430707)和mIFNγ(目錄#430801)的BioLegend MAX ELISA試劑盒定量小鼠IL-12(mIL12)和小鼠IFNγ(mIFNγ)向培養基中的分泌。圖2A至圖2B中的結果表明小鼠IL-12和IFNγ在mt12和NHS76細胞中均以較高位準表現，並且在與靶細胞共培養後細胞介素濃度進一步增加。小鼠膜拴系IL-12和NHS76-IL-12的胺基酸序列分別示於SEQ ID NO: 4和SEQ ID NO: 14中。實施例 3. IL-12 表現增加幼稚淋巴細胞中的 IFNγ 產生 Purified primary mouse lymphocytes were engineered to express L202 TCR alone (L202) (anti-LMP2 TCR), L202 plus mouse membrane-tethered IL-12 (sample name: mt12), or L202 plus mouse NHS-76 - IL-12 (sample name: NHS76). Then, two days after transduction, the indicated lymphocyte populations were co-cultured overnight with LLC-HLA-A2-Peplinker (LLW) target cells. The secretion of mouse IL-12 (mIL12) and mouse IFNγ (mIFNγ) into the culture medium was quantified using BioLegend MAX ELISA kits for mIL12 p40 (catalog #430707) and mIFNγ (catalog #430801). The results in Figures 2A-2B demonstrate that mouse IL-12 and IFNγ are expressed at higher levels in both mt12 and NHS76 cells, and the interleukin concentration is further increased after co-culture with target cells. The amino acid sequences of mouse membrane-tethered IL-12 and NHS76-IL-12 are shown in SEQ ID NO: 4 and SEQ ID NO: 14, respectively. Example 3. IL-12 expression increases IFNγ production in naive lymphocytes

從淋巴結中分離的幼稚小鼠淋巴細胞未被轉導(樣品名稱：UT)，或被轉導以表現單獨的L202 TCR(樣品名稱：L202)、L202連同膜拴系IL-12(樣品名稱：mt12)或L202連同小鼠NHS76-IL-12(樣品名稱：NHS76)。將指示的淋巴細胞群與Jurkat靶細胞共培養過夜。然後使用小鼠IFNγ ELISA MAX試劑盒(BioLegend)在培養基中測量小鼠IFNγ。如圖3所示，結果表明，IL-12表現刺激淋巴細胞在與靶細胞一起溫育時的IFNγ產生，並且IL-12表現反式活化幼稚淋巴細胞。實施例 4 ：小鼠淋巴細胞中的裝甲 TCR-T 的表面上的 IL-12 表現 Naive mouse lymphocytes isolated from lymph nodes were either not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 together with membrane-tethered IL-12 (sample name: L202) mt12) or L202 together with mouse NHS76-IL-12 (sample name: NHS76). The indicated lymphocyte populations were co-cultured with Jurkat target cells overnight. Mouse IFNy was then measured in the culture medium using the Mouse IFNy ELISA MAX kit (BioLegend). As shown in Figure 3, the results show that IL-12 appears to stimulate IFNy production by lymphocytes when incubated with target cells, and that IL-12 appears to transactivate naive lymphocytes. Example 4 : IL-12 expression on the surface of armored TCR-T in mouse lymphocytes

從淋巴結中分離原代小鼠淋巴細胞，並且用單獨的L202 TCR或L202連同mt-IL-12或小鼠NHS76-IL-12轉導5天。然後用3uL的抗mIL12抗體PE-Cy7對指示的細胞類型進行染色，以評估IL-12的表面表現。如圖4A至圖4D所示，約52%的mt-IL-12 PBMC表現出人IL-12的細胞表面表現，而IL-12在未轉導、L202或L202-NHS76-IL-12細胞的表面上不表現。人膜拴系IL-12和NHS76-IL-12的胺基酸序列分別示於SEQ ID NO: 10和SEQ ID NO: 14中。實施例 5 ： NHS-76-IL-12 小鼠淋巴細胞的細胞內 IL-12 表現 Primary mouse lymphocytes were isolated from lymph nodes and transduced with L202 TCR alone or L202 together with mt-IL-12 or mouse NHS76-IL-12 for 5 days. The indicated cell types were then stained with 3 uL of the anti-mIL12 antibody PE-Cy7 to assess the surface expression of IL-12. As shown in Figures 4A to 4D, approximately 52% of mt-IL-12 PBMCs exhibited cell surface expression of human IL-12, while IL-12 was expressed in untransduced, L202 or L202-NHS76-IL-12 cells It doesn't appear on the surface. The amino acid sequences of human membrane-tethered IL-12 and NHS76-IL-12 are shown in SEQ ID NO: 10 and SEQ ID NO: 14, respectively. Example 5 : Intracellular IL-12 expression in NHS-76-IL-12 mouse lymphocytes

從淋巴結中分離小鼠淋巴細胞，然後用NHS76-IL-12轉導2天。如用人Fab(圖5A)或抗IL12抗體PE-Cy7(圖5B)染色所測量的，用NHS76-IL-12轉導的淋巴細胞表現出細胞內IL-12表現。實施例 6 ：人 PBMC 的 TCRb 和人 IL-12 表面染色 Mouse lymphocytes were isolated from lymph nodes and then transduced with NHS76-IL-12 for 2 days. Lymphocytes transduced with NHS76-IL-12 exhibited intracellular IL-12 expression as measured by staining with human Fab (FIG. 5A) or anti-IL12 antibody PE-Cy7 (FIG. 5B). Example 6 : TCRb and human IL-12 surface staining of human PBMC

如所指示的，用單獨的L202 TCR或與人mt-IL-12或人長mt-IL-12組合轉導人PBMC。感染後12天，分別通過用抗TCRb抗體(TCRb-PE)和抗IL-12抗體(IL-12-PE-Cy7)染色來測量TCR和IL-12表面表現。圖6A至圖6H中的結果表明，具有L202轉導的細胞對於每種細胞類型都具有TCR表面表現，而在用膜拴系形式的IL-12轉導的PBMC上檢測到表面IL-12。人膜拴系IL-12(mt-IL-12)和人長膜拴系IL-12(人長mt-IL-12)的胺基酸序列分別示於SEQ ID NO: 10和SEQ ID NO: 12中。實施例 7 ：與靶細胞共培養的表現 mt-IL-12 的 PBMC 中的 CD69 表現 Human PBMCs were transduced with L202 TCR alone or in combination with human mt-IL-12 or human long mt-IL-12 as indicated. 12 days after infection, TCR and IL-12 surface expression was measured by staining with anti-TCRb antibody (TCRb-PE) and anti-IL-12 antibody (IL-12-PE-Cy7), respectively. The results in Figures 6A-6H demonstrate that cells transduced with L202 have TCR surface expression for each cell type, whereas surface IL-12 was detected on PBMCs transduced with a membrane-tethered form of IL-12. The amino acid sequences of human membrane tethered IL-12 (mt-IL-12) and human long membrane tethered IL-12 (human long mt-IL-12) are shown in SEQ ID NO: 10 and SEQ ID NO: , respectively 12. Example 7 : CD69 expression in mt - IL-12 expressing PBMC co-cultured with target cells

如所指示的，感染後第12天，將未轉導的人PBMC或工程化以表現L202 TCR、L202加人膜拴系IL-12(smt)、或L202加人長膜拴系IL-12(lmt)的PBMC與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養過夜。然後，通過用APC抗CD69抗體染色來評估T細胞活化。On day 12 post-infection, as indicated, untransduced human PBMCs were either engineered to express L202 TCR, L202 plus human membrane-tethered IL-12 (smt), or L202 plus human long membrane-tethered IL-12 (lmt) PBMCs were co-cultured overnight with A375-HLA-A2-peplinker (LLW) melanoma target cells. Then, T cell activation was assessed by staining with APC anti-CD69 antibody.

圖7A是示出在與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養的人PBMC中的CD69表現的圖。圖7B是示出在沒有共培養情況下的人PBMC中的CD69表現的圖。如圖7A至圖7B所示，表現IL-12的PBMC與L202或未轉導的T細胞相比，當它們與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養時，具有更強的CD69表現。實施例 8 ： IL-12 裝甲的 PBMC 中的 IFNγ 表現 Figure 7A is a graph showing CD69 expression in human PBMC co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells. Figure 7B is a graph showing CD69 expression in human PBMC without co-culture. As shown in Figures 7A-7B, IL-12-expressing PBMCs had more IL-12 than L202 or untransduced T cells when they were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells Strong CD69 expression. Example 8 : IFNγ expression in IL-12 armored PBMCs

將未轉導的人PBMC或由L202 TCR-T、L202 TCR-T加人膜拴系IL-12(smt)或L202 TCR-T加人長膜拴系IL-12(lmt)轉導的PBMC與A375-HLA-A2-peplinker(LLW)靶細胞共培養過夜，然後用hIFNγ-FITC染色以測量T細胞活化。如圖8A至圖8H所示，結果顯示L202 TCR-T細胞中約20%的IFNγ陽性率相對於表現膜拴系IL-12(smt或lmt)的L202 TCR-T細胞中大於40%的IFNγ表現，表明IL-12的細胞表面表現增強TCR-T細胞活化。實施例 9 ： IL-12 裝甲的 mTCRb+ 和 mTCRb- 細胞中的 CD69 表現 Untransduced human PBMCs or PBMCs transduced with L202 TCR-T, L202 TCR-T plus human membrane tethered IL-12 (smt) or L202 TCR-T plus human long membrane tethered IL-12 (lmt) Co-culture with A375-HLA-A2-peplinker (LLW) target cells overnight followed by staining with hIFNγ-FITC to measure T cell activation. As shown in Figures 8A to 8H, the results show that about 20% IFNγ positivity in L202 TCR-T cells relative to greater than 40% IFNγ in L202 TCR-T cells expressing membrane-tethered IL-12 (smt or lmt) expression, indicating that the cell surface expression of IL-12 enhances TCR-T cell activation. Example 9 : CD69 expression in IL-12 armored mTCRb+ and mTCRb- cells

進行實驗以確定IL12裝甲的細胞是否會增加兩種類型細胞的活化：具有IL12裝甲的細胞和附近的未裝甲免疫細胞。為了觀察IL-12裝甲的細胞對附近未裝甲細胞的影響，將50% L202 TCR轉導和50%未轉導的人PBMC的混合群體與A375-LLW黑色素瘤靶細胞共培養。L202 TCR轉導的人PBMC由單獨的L202、L202加人膜拴系IL-12(smt)或L202加人長膜拴系IL-12(lmt)轉導。L202 TCR具有小鼠TCRb。Experiments were conducted to determine whether IL12-armoured cells increased the activation of two types of cells: cells with IL12 armour and nearby unarmoured immune cells. To observe the effect of IL-12 armored cells on nearby unarmored cells, a mixed population of 50% L202 TCR-transduced and 50% untransduced human PBMCs was co-cultured with A375-LLW melanoma target cells. L202 TCR-transduced human PBMCs were transduced with L202 alone, L202 plus human membrane tethered IL-12 (smt) or L202 plus human long membrane tethered IL-12 (lmt). The L202 TCR has mouse TCRb.

通過針對CD69染色來測量TCRb+和TCRb-群體中的CD3、CD8雙陽性T細胞的活化。在圖9A中，mTCRb+群體指示具有IL12裝甲的細胞。CD69是活化的標記物。TCRb+細胞的活化對於L202 TCR-T和裝甲L202 TCR-T細胞相當(圖9A)。在圖9B中，CD69在mTCRb-群體上的表現指示周圍細胞(未裝甲細胞)的活化。未裝甲細胞也被活化。膜拴系IL-12的添加進一步增加了不表現mTCRb的PBMC的活化(圖9B)。實施例 10 ： TCRb+ 細胞中的 IFNγ 活化通過添加膜拴系 IL-12 而增加 Activation of CD3, CD8 double positive T cells in TCRb+ and TCRb- populations was measured by staining for CD69. In Figure 9A, the mTCRb+ population indicates cells with IL12 armor. CD69 is a marker of activation. Activation of TCRb+ cells was comparable for L202 TCR-T and armored L202 TCR-T cells (Figure 9A). In Figure 9B, the expression of CD69 on the mTCRb-population indicates activation of surrounding cells (unarmored cells). Unarmored cells were also activated. Addition of membrane-tethered IL-12 further increased activation of PBMCs that do not express mTCRb (Figure 9B). Example 10 : IFNγ activation in TCRb+ cells is increased by addition of membrane-tethered IL-12

將未轉導的人PBMC細胞、工程化以表現L202 TCR-T、L202 TCR-T加人膜拴系IL-12(mtIL12)或L202 TCR-T加人長膜拴系IL-12(長mtIL12)的人PBMC細胞與未轉導的人PBMC細胞以1:1的比率混合。Untransduced human PBMC cells, engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12), or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) ) were mixed with untransduced human PBMC cells at a ratio of 1:1.

然後將這些細胞與A375-LLW靶細胞共培養並且隨後使用BD Perm/Wash緩衝液進行細胞內IFNγ FITC染色。細胞在淋巴細胞上門控，然後是單細胞、CD3+細胞、CD8+細胞和TCRb+細胞。如圖10A至圖10H所示，資料表明膜拴系IL-12的表現增強了TCR+ PBMC的T細胞活化。實施例 11 ： TCRb- 細胞中的 IFNγ 活化通過添加膜拴系 IL-12 而增加 These cells were then co-cultured with A375-LLW target cells and subsequently subjected to intracellular IFNy FITC staining using BD Perm/Wash buffer. Cells were gated on lymphocytes, followed by single cells, CD3+ cells, CD8+ cells and TCRb+ cells. As shown in Figures 10A-10H, the data demonstrate that expression of membrane-tethered IL-12 enhances T cell activation by TCR+ PBMCs. Example 11 : IFNγ activation in TCRb- cells is increased by addition of membrane-tethered IL-12

將未轉導的人PBMC細胞、工程化以表現L202 TCR-T、L202 TCR-T加人膜拴系IL-12(mtIL12)或L202 TCR-T加人長膜拴系IL-12(長mtIL12)的人PBMC細胞與未轉導的人PBMC細胞以1:1的比率混合。Untransduced human PBMC cells, engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12), or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) ) were mixed with untransduced human PBMC cells at a ratio of 1:1.

然後將這些細胞與A375-LLW靶細胞共培養，並且隨後使用BD Perm/Wash緩衝液進行細胞內IFNγ FITC染色。細胞在淋巴細胞上門控，然後是單細胞、CD3+細胞、CD8+細胞和TCRb-細胞。如圖11A至圖11H所示，資料表明膜拴系IL-12的表現增強了TCR- PBMC的T細胞活化。實施例 12 ： IL-12 裝甲的 TCR-T 細胞的表型分析 These cells were then co-cultured with A375-LLW target cells and subsequently subjected to intracellular IFNy FITC staining using BD Perm/Wash buffer. Cells were gated on lymphocytes, followed by single cells, CD3+ cells, CD8+ cells and TCRb- cells. As shown in Figures 11A-11H, the data demonstrate that expression of membrane-tethered IL-12 enhances T-cell activation of TCR-PBMCs. Example 12 : Phenotypic Analysis of IL-12 Armored TCR-T Cells

在感染後第12天，對未轉導(UT)、L202 TCR-T(L202)、表現人膜拴系IL-12(mt-IL12)的L202 TCR-T或表現人長膜拴系IL-12(lmt-IL12)的L202 TCR-T的PBMC進行CD4(hCD4-太平洋藍)和CD8(hCD8-PerCP-Cy5.5)染色。結果示於圖12A至圖12D中。與未轉導的和L202 TCR-T細胞相比，IL-12裝甲的L202 TCR-T細胞表現出顯著更高的CD4表現，表明IL-12富集CD4+ T細胞群體。實施例 13 ：用於定量中樞記憶和效應記憶 T 細胞的流式細胞術染色 On day 12 post-infection, treatment with untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane-tethered IL-12 (mt-IL12), or human long membrane-tethered IL- 12 (lmt-IL12) PBMCs of L202 TCR-T were stained for CD4 (hCD4-Pacific Blue) and CD8 (hCD8-PerCP-Cy5.5). The results are shown in Figures 12A to 12D. IL-12-armoured L202 TCR-T cells exhibited significantly higher CD4 expression compared to untransduced and L202 TCR-T cells, indicating an IL-12-enriched CD4+ T cell population. Example 13 : Flow cytometry staining for quantification of central and effector memory T cells

對上述實驗中未轉導的CD4+ PBMC進行效應記憶標記物CD45RO(hCD45RO-FITC抗體)和中樞記憶標記物CCR7(CCR7-R-PE抗體)染色。如圖13所示，流式細胞術分析顯示，約41%的PBMC是效應記憶細胞，而58%是中樞記憶細胞。實施例 14 ： IL-12 裝甲的 CD4+ TCR-T 細胞的記憶表型 Untransduced CD4+ PBMCs in the above experiments were stained for the effector memory marker CD45RO (hCD45RO-FITC antibody) and the central memory marker CCR7 (CCR7-R-PE antibody). As shown in Figure 13, flow cytometry analysis revealed that approximately 41% of PBMCs were effector memory cells, while 58% were central memory cells. Example 14 : Memory Phenotype of IL-12 Armored CD4+ TCR-T Cells

對CD4+未轉導(UT)、L202 TCR-T(L202)、表現人膜拴系IL-12(mt-IL12)的L202 TCR-T或表現人長膜拴系IL-12(lmt-IL12)的L202 TCR-T的PBMC進行CD45RO和CCR7染色。如圖14A至圖14D所示，結果表明，表面結合的IL-12的表現使CD4+ PBMC朝效應記憶表型偏移。實施例 15 ： IL-12 裝甲的 CD8+ TCR-T 細胞的記憶表型 For CD4+ untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane tethered IL-12 (mt-IL12) or expressing human long membrane tethered IL-12 (lmt-IL12) L202 TCR-T PBMCs were stained for CD45RO and CCR7. As shown in Figures 14A-14D, the results demonstrate that the expression of surface-bound IL-12 biases CD4+ PBMCs towards the effector memory phenotype. Example 15 : Memory Phenotype of IL-12 Armored CD8+ TCR-T Cells

對CD8+未轉導(UT)、L202 TCR-T(L202)、表現人膜拴系IL-12(mt-IL12)的L202 TCR-T或表現人長膜拴系IL-12(lmt-IL12)的L202 TCR-T的PBMC進行CD45RO和CCR7染色。如圖15A至圖15D所示，結果表明，表面結合的IL-12的表現使CD8+ PBMC朝效應記憶表型偏移。實施例 16 ： IL-12 不從表現膜拴系 IL-12 的細胞中釋放 For CD8+ untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane tethered IL-12 (mt-IL12) or expressing human long membrane tethered IL-12 (lmt-IL12) L202 TCR-T PBMCs were stained for CD45RO and CCR7. As shown in Figures 15A-15D, the results demonstrate that the expression of surface-bound IL-12 biases CD8+ PBMCs towards an effector memory phenotype. Example 16 : IL-12 is not released from cells expressing membrane-tethered IL-12

Jurkat細胞未轉導(UT)，或被轉導以表現IL-12、NHS76-IL-12或膜拴系IL-12。在轉導後第5天，將1×106 個細胞鋪板於12孔板中的1ml細胞培養基中。在24小時後，將細胞培養物離心並收集上清液。通過酶聯免疫吸附測定(ELISA)測量上清液中IL-12的濃度。如圖16所示，表現膜拴系IL-12的Jurkat細胞在細胞培養基中具有最低位準的IL-12。相比之下，表現IL-12或NHS76-IL-12的Jurkat細胞在培養基中具有高的細胞外IL-12位準(例如，3000pg/mL-5000pg/mL)。結果表明，具有CH2和CH3結構域以及CD4跨膜區的膜拴系IL-12可安全地拴系到細胞膜上，並且不會釋放到細胞培養基中。實施例 17. 構建體設計 Jurkat cells were either untransduced (UT), or transduced to express IL-12, NHS76-IL-12 or membrane-tethered IL-12. On day 5 post-transduction, 1 x 106 cells were plated in 1 ml of cell culture medium in 12-well plates. After 24 hours, the cell culture was centrifuged and the supernatant was collected. The concentration of IL-12 in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). As shown in Figure 16, Jurkat cells expressing membrane-tethered IL-12 had the lowest levels of IL-12 in the cell culture medium. In contrast, Jurkat cells expressing IL-12 or NHS76-IL-12 had high levels of extracellular IL-12 in culture medium (eg, 3000 pg/mL-5000 pg/mL). The results show that membrane-tethered IL-12 with CH2 and CH3 domains and CD4 transmembrane region can be safely tethered to the cell membrane and is not released into the cell culture medium. Example 17. Construct Design

進一步修飾小鼠和人IL-12表現載體(圖17A至圖17B)。將編碼小鼠Flt3L(mFlt3L)、小鼠CXCL10(mCXCL10)或小鼠XCL1(mXCL1)的序列添加到構建體中(圖17A)。mFlt3L、mCXCL10或mXCL1通過2A自切割肽與膜拴系IL-12分開。The mouse and human IL-12 expression vectors were further modified (Figures 17A-17B). Sequences encoding mouse Flt3L (mFlt3L), mouse CXCL10 (mCXCL10) or mouse XCL1 (mXCL1) were added to the construct (Figure 17A). mFlt3L, mCXCL10 or mXCL1 are separated from membrane-tethered IL-12 by a 2A self-cleaving peptide.

類似地，將編碼人Flt3L(hFlt3L)、人CXCL10(mCXCL10)或人XCL1(hXCL1)的序列添加到構建體中(圖17B)。hFlt3L、hCXCL10或hXCL1通過2A自切割肽與膜拴系IL-12分開。Similarly, sequences encoding human Flt3L (hFlt3L), human CXCL10 (mCXCL10) or human XCL1 (hXCL1) were added to the construct (Figure 17B). hFlt3L, hCXCL10 or hXCL1 are separated from membrane-tethered IL-12 by a 2A self-cleaving peptide.

編碼TCR或CAR的序列可添加在編碼Flt3L、CXCL10或XCL1的序列之前或在編碼膜拴系CD4的序列之後。這些序列可被2A自切割肽分開。The sequence encoding the TCR or CAR can be added before the sequence encoding Flt3L, CXCL10 or XCL1 or after the sequence encoding membrane-tethered CD4. These sequences can be separated by the 2A self-cleaving peptide.

在該實施例中，將編碼抗EGFRvIII CAR的序列添加到這些構建體中。抗EGFRvIII CAR是本領域已知的，並且描述於例如US10570214B2中，該專利全文以引用方式併入本文。從淋巴結中分離原代小鼠淋巴細胞，並用這些構建體進行轉導。這些細胞首先被帶His標籤的EGFRvIII肽(SEQ ID NO: 45)標記，然後被用於His標籤的標記二抗標記。如圖18所示，這些構建體的轉導效率在26%-53%的範圍內。然後使用效率來確定CAR+細胞的數量，使得在實驗中使用等量的CAR+細胞用於比較目的。實施例 18. 具有單一裝甲的 EGFRvIII CAR 有效地殺死靶細胞 In this example, sequences encoding anti-EGFRvIII CARs were added to these constructs. Anti-EGFRvIII CARs are known in the art and are described, for example, in US10570214B2, which is incorporated herein by reference in its entirety. Primary mouse lymphocytes were isolated from lymph nodes and transduced with these constructs. These cells were first labeled with the His-tagged EGFRvIII peptide (SEQ ID NO: 45) and then with a labeled secondary antibody for His-tagging. As shown in Figure 18, the transduction efficiencies of these constructs ranged from 26%-53%. Efficiency was then used to determine the number of CAR+ cells so that an equal amount of CAR+ cells was used in the experiments for comparison purposes. Example 18. EGFRvIII CAR with a single armor efficiently kills target cells

小鼠淋巴細胞用編碼(1)抗EGFRvIII CAR(“EGFRvIII”)、(2) IL12和抗EGFRvIII CAR(“EGFRvIII-IL12”)、(3) CXCL10和抗EGFRvIII CAR(“EGFRvIII-CxCL10”)、(4) Flt3L和抗EGFRvIII CAR(“EGFRvIII-FLt3L”)、(5) CXCL10、IL12和抗EGFRvIII CAR(“EGFRvIII-CxCL10-IL12”)或(5)Flt3L、IL12和抗EGFRvIII CAR(“EGFRvIII-Flt3L-IL12”)的構建體轉導。將轉導的淋巴細胞與KLUC和KLuc-EGFRvIII靶細胞以不同的效應子與靶細胞比率共培養24小時。如圖19所示，所有轉導的淋巴細胞有效地殺死靶細胞。實施例 19. 共培養中的細胞 介素分泌 Mouse lymphocytes were encoded with (1) anti-EGFRvIII CAR ("EGFRvIII"), (2) IL12 and anti-EGFRvIII CAR ("EGFRvIII-IL12"), (3) CXCL10 and anti-EGFRvIII CAR ("EGFRvIII-CxCL10"), (4) Flt3L and anti-EGFRvIII CAR ("EGFRvIII-FLt3L"), (5) CXCL10, IL12 and anti-EGFRvIII CAR ("EGFRvIII-CxCL10-IL12") or (5) Flt3L, IL12 and anti-EGFRvIII CAR ("EGFRvIII- Flt3L-IL12") construct for transduction. Transduced lymphocytes were co-cultured with KLUC and KLuc-EGFRvIII target cells at different effector to target cell ratios for 24 hours. As shown in Figure 19, all transduced lymphocytes efficiently killed target cells. Example 19. Cytokinin secretion in co-culture

將轉導的小鼠淋巴細胞(CAR+)與相等數量的Kluc或Kluc-EGFRvIII(“Kluc vIII”)腫瘤細胞一起培養。測量細胞介素分泌。當CAR+細胞與KLUC vIII靶細胞共培養時，IFNg分泌更高(圖20A)。類似地，當CAR+細胞與KLUC vIII靶細胞共培養時,IL12分泌更高(圖20B)。還測量了CXCL10分泌(圖20C)。實施例 20. EGFRvIII 裝甲的 CAR 的體內功效研究 Transduced mouse lymphocytes (CAR+) were cultured with equal numbers of Kluc or Kluc-EGFRvIII ("Kluc vIII") tumor cells. Interferon secretion was measured. IFNg secretion was higher when CAR+ cells were co-cultured with KLUC vIII target cells (Figure 20A). Similarly, IL12 secretion was higher when CAR+ cells were co-cultured with KLUC vIII target cells (FIG. 20B). CXCL10 secretion was also measured (Figure 20C). Example 20. In vivo efficacy studies of EGFRvIII armored CARs

在第-39天時，用6×106 個KLUC細胞和KLUC vIII細胞接種C57BL/6小鼠。在第-4天時，隨後以10mL/kg的給藥體積向小鼠腹膜內注射150mg/kg環磷醯胺。在第0天，將由各種構建體轉導的細胞轉移到小鼠中(圖21)。在小鼠被這些CAR-T細胞處理之前，平均腫瘤大小為約106mm3 。On day -39, C57BL/6 mice were inoculated with 6 x 106 KLUC cells and KLUC vIII cells. On day -4, mice were then injected intraperitoneally with 150 mg/kg cyclophosphamide in a dosing volume of 10 mL/kg. On day 0, cells transduced with various constructs were transferred into mice (Figure 21). Before mice were treated with these CAR-T cells, the average tumor size was about 106 mm 3 .

圖22A至圖22F示出了實驗的結果。如圖22A至圖22F所示，表現抗EGFRvIII CAR、CXCL10和IL12的T細胞(圖22B)、表現抗EGFRvIII CAR、Flt3和IL12的T細胞(圖22C)和表現抗EGFRvIII CAR和IL12的T細胞(圖22F)有效地殺死腫瘤細胞。相比之下，不表現IL12的T細胞不是很有效。圖23示出了不同組小鼠的存活曲線。在圖23中，EGFRvIII+CxCl10+IL12組和EGFRvIII+flt3+IL12組具有相同的曲線。22A to 22F show the results of the experiments. As shown in Figures 22A-22F, T cells expressing anti-EGFRvIII CAR, CXCL10 and IL12 (Figure 22B), T cells expressing anti-EGFRvIII CAR, Flt3 and IL12 (Figure 22C) and T cells expressing anti-EGFRvIII CAR and IL12 (FIG. 22F) Effectively kills tumor cells. In contrast, T cells that did not express IL12 were not very effective. Figure 23 shows survival curves of different groups of mice. In Figure 23, the EGFRvIII+CxCl10+IL12 group and the EGFRvIII+flt3+IL12 group have the same curve.

在如圖22B、圖22C和圖22F所示的那些小鼠中，它們中的大多數變得無腫瘤。在第94天(即，T細胞轉移後94天)，所有無腫瘤的小鼠在右側用2.5×106 個KLUC細胞並且在左側用2.5×106 個KLUCvIII細胞再次攻擊。這些小鼠在攻擊之前約66天無腫瘤。 表1 組 大小 腫瘤細胞類型 腫瘤細胞數量 未處理 4 KLUC/KLUCvIII 5 x106 EGFRvIII Car+CxCl10+IL12 6 KLUC/KLUCvIII 5 x106 EGFRvIII Car+flt3+IL12 6 KLUC/KLUCvIII 5 x106 EGFRvIII Car+IL12 5 KLUC/KLUCvIII 5 x106 Of those mice shown in Figures 22B, 22C and 22F, most of them became tumor free. On day 94 (ie, 94 days after T cell transfer), all tumor-free mice were rechallenged with 2.5 x 106 KLUC cells on the right and 2.5 x 106 KLUCvIII cells on the left. These mice were tumor free for approximately 66 days prior to challenge. Table 1 Group size tumor cell type number of tumor cells not processed 4 KLUC/KLUCvIII 5x10 6 EGFRvIII Car+CxCl10+IL12 6 KLUC/KLUCvIII 5x10 6 EGFRvIII Car+flt3+IL12 6 KLUC/KLUCvIII 5x10 6 EGFRvIII Car+IL12 5 KLUC/KLUCvIII 5x10 6

圖24A示出了在小鼠植入腫瘤細胞後第52天抗原陰性(EGFRvIII陰性)無腫瘤動物的百分比。在這些小鼠中，組C001是對照組。小鼠先前未通過細胞療法進行治療。C001-IL12組的小鼠先前用表現IL12和抗EGFRvIII CAR(“C001-IL12”)的細胞處理。C001-CXCL10-IL12組的小鼠先前用表現CXCL10、IL12和抗EGFRvIII CAR的細胞處理。C001-Flt3L-IL12組的小鼠先前用表現Flt3L、IL12和抗EGFRvIII CAR的細胞處理。結果顯示，CXCL10和Flt3L可進一步改善免疫反應。不希望受理論束縛，假設CXCL10和Flt3L可增加抗原呈遞細胞的活性。抗原呈遞細胞可將一些其他腫瘤抗原呈遞給宿主免疫細胞，使得宿主免疫細胞將識別這些腫瘤抗原並殺死這些腫瘤細胞，即使這些腫瘤細胞不表現由CAR或TCR識別的抗原。圖24B示出了在小鼠被腫瘤細胞植入後的再次攻擊研究的存活曲線。結果再次顯示，CXCL10和Flt3L可進一步改善免疫反應。其他實施方案 Figure 24A shows the percentage of antigen-negative (EGFRvIII negative) tumor-free animals at day 52 after tumor cell implantation in mice. In these mice, group C001 was the control group. The mice had not been previously treated with cell therapy. Mice in the C001-IL12 group were previously treated with cells expressing IL12 and an anti-EGFRvIII CAR ("C001-IL12"). Mice in the C001-CXCL10-IL12 group were previously treated with cells expressing CXCL10, IL12 and anti-EGFRvIII CAR. Mice in the C001-Flt3L-IL12 group were previously treated with cells expressing Flt3L, IL12 and anti-EGFRvIII CAR. The results showed that CXCL10 and Flt3L could further improve the immune response. Without wishing to be bound by theory, it is hypothesized that CXCL10 and Flt3L may increase the activity of antigen presenting cells. Antigen-presenting cells can present some other tumor antigens to host immune cells, such that the host immune cells will recognize these tumor antigens and kill these tumor cells, even if these tumor cells do not express the antigen recognized by CAR or TCR. Figure 24B shows survival curves for a rechallenge study after mice were engrafted with tumor cells. The results again showed that CXCL10 and Flt3L further improved the immune response. Other implementations

應當理解，儘管已經結合本發明的具體實施方式描述了本發明，但是前述描述旨在說明而不限制本發明的範圍，本發明的範圍由所附申請專利範圍的範圍限定。其他方面、優點和修改在以下申請專利範圍的範圍內。It should be understood that while the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

無without

在附圖中展示了示例性實施方案。旨在將本文公開的實施方案和附圖視為說明性的而非限制性的。Exemplary embodiments are shown in the accompanying drawings. The embodiments and figures disclosed herein are intended to be regarded in an illustrative rather than a restrictive sense.

圖1A是示出小鼠IL-12表現載體的示意圖。P2A編碼2A自切割肽；mSP編碼小鼠訊息肽；mP40編碼小鼠IL-12B(β鏈)；接頭編碼肽接頭；mP35編碼小鼠IL-12A(α鏈)；鉸鏈編碼免疫球蛋白鉸鏈區；mCH2編碼小鼠免疫球蛋白IgG4重鏈恆定結構域CH2；mCH3編碼小鼠免疫球蛋白IgG4重鏈恆定結構域CH3；mCD4 TM編碼小鼠CD4跨膜區；NHS-76 vH編碼靶向腫瘤壞死的人IgG1(NHS-76)的重鏈可變區(VH)；並且NHS-76 vL編碼NHS-76的輕鏈可變區(VL)。Figure 1A is a schematic diagram showing a mouse IL-12 expression vector. P2A encodes 2A self-cleaving peptide; mSP encodes mouse message peptide; mP40 encodes mouse IL-12B (beta chain); linker encodes peptide linker; mP35 encodes mouse IL-12A (alpha chain); hinge encodes immunoglobulin hinge region ; mCH2 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH2; mCH3 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH3; mCD4 TM encodes mouse CD4 transmembrane region; NHS-76 vH encodes target tumor necrosis The heavy chain variable region (VH) of human IgG1 (NHS-76); and NHS-76 vL encodes the light chain variable region (VL) of NHS-76.

圖1B是示出人IL-12表現載體的示意圖。P2A編碼2A自切割肽；hSP編碼人訊息肽；hP40編碼人IL-12B；接頭編碼肽接頭；hP35編碼人IL-12A；鉸鏈編碼免疫球蛋白鉸鏈區；hmCH2(人突變的CH2)編碼突變的人免疫球蛋白IgG4重鏈恆定結構域CH2；hCH3(人CH3)編碼人免疫球蛋白IgG4重鏈恆定結構域CH3；hCD4 TM編碼人CD4跨膜區；NHS-76 vH編碼NHS-76的VH；並且NHS-76 vL編碼NHS-76的VL。Figure IB is a schematic diagram showing a human IL-12 expression vector. P2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 (human mutated CH2) encodes mutated Human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 (human CH3) encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the VH of NHS-76; And NHS-76 vL encodes the VL of NHS-76.

圖1C是示出表現人IL-12與TCR組合的載體的示意圖。TCR編碼T細胞受體(例如，L202)；P2A編碼2A自切割肽；hSP編碼人訊息肽；hP40編碼人IL-12B；接頭編碼肽接頭；hP35編碼人IL-12A；鉸鏈編碼免疫球蛋白鉸鏈區；hmCH2編碼突變的人免疫球蛋白IgG4重鏈恆定結構域CH2；hCH3編碼人免疫球蛋白IgG4重鏈恆定結構域CH3；hCD4 TM編碼人CD4跨膜區；NHS-76 vH編碼NHS-76的VH；並且NHS-76 vL編碼NHS-76的VL。Figure 1C is a schematic diagram showing a vector expressing human IL-12 in combination with TCR. TCR encodes a T cell receptor (eg, L202); P2A encodes a 2A self-cleaving peptide; hSP encodes a human message peptide; hP40 encodes human IL-12B; linker encodes a peptide linker; hP35 encodes human IL-12A; hinge encodes an immunoglobulin hinge Region; hmCH2 encodes the mutated human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 encodes the human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the NHS-76 VH; and NHS-76 vL encodes the VL of NHS-76.

圖1D是示出表現人IL-12與CAR組合的載體的示意圖。CAR編碼嵌合抗原受體；P2A編碼2A自切割肽；hSP編碼人訊息肽；hP40編碼人IL-12B；接頭編碼肽接頭；hP35編碼人IL-12A；鉸鏈編碼免疫球蛋白鉸鏈區；hmCH2編碼突變的人免疫球蛋白IgG4重鏈恆定結構域CH2；hCH3編碼人免疫球蛋白IgG4重鏈恆定結構域CH3；hCD4 TM編碼人CD4跨膜區；NHS-76 vH編碼NHS-76的VH；並且NHS-76 vL編碼NHS-76的VL。Figure ID is a schematic diagram showing a vector expressing human IL-12 in combination with a CAR. CAR encodes chimeric antigen receptor; P2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 encodes Mutated human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the VH of NHS-76; and NHS -76 vL encodes the VL of NHS-76.

圖2A是示出如通過ELISA(酶聯免疫吸附測定)確定的培養基中的小鼠IL-12濃度的長條圖。純化的小鼠淋巴細胞未被轉導(樣品名稱：UT)，或被轉導以表現單獨的L202 TCR(樣品名稱：L202)、L202加小鼠膜栓系IL-12(樣品名稱：mt12)，或L202加NHS76-IL-12(樣品名稱：NHS76)。將淋巴細胞與LLC-HLA-A2-Peplinker(LLW)(靶細胞)共培養，或單獨培養(對照)。Figure 2A is a bar graph showing mouse IL-12 concentration in culture medium as determined by ELISA (enzyme-linked immunosorbent assay). Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus mouse membrane-tethered IL-12 (sample name: mt12) , or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells), or alone (control).

圖2B是示出如通過ELISA確定的培養基中的小鼠IFNγ濃度的長條圖。純化的小鼠淋巴細胞未被轉導(樣品名稱：UT)，或被轉導以表現單獨的L202 TCR(樣品名稱：L202)、L202加小鼠膜栓系IL-12(樣品名稱：mt12)，或L202加NHS76-IL-12(樣品名稱：NHS76)。將淋巴細胞與LLC-HLA-A2-Peplinker(LLW)(靶細胞)共培養，或單獨培養(對照)。Figure 2B is a bar graph showing mouse IFNy concentrations in culture medium as determined by ELISA. Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus mouse membrane-tethered IL-12 (sample name: mt12) , or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells), or alone (control).

圖3是示出如通過ELISA確定的培養基中的小鼠IFNγ濃度的長條圖。純化的小鼠淋巴細胞未被轉導(樣品名稱：UT)，或被轉導以表現單獨的L202 TCR(樣品名稱：L202)、L202加膜栓系IL-12(樣品名稱：mt12)，或L202加NHS76-IL-12(樣品名稱：NHS76)。將淋巴細胞與Jurkat細胞(靶細胞+小鼠淋巴細胞)共培養，或單獨培養(對照)。Figure 3 is a bar graph showing mouse IFNy concentrations in culture medium as determined by ELISA. Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus membrane-tethered IL-12 (sample name: mt12), or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with Jurkat cells (target cells + mouse lymphocytes), or alone (control).

圖4A是示出在未轉導的原代小鼠淋巴細胞(樣品名稱：未轉導)中的細胞表面IL-12表現的圖。Figure 4A is a graph showing cell surface IL-12 expression in untransduced primary mouse lymphocytes (sample name: untransduced).

圖4B是示出在被轉導以表現L202 TCR的原代小鼠淋巴細胞(樣品名稱：L202)中的細胞表面IL-12表現的圖。Figure 4B is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202) transduced to express L202 TCR.

圖4C是示出在被轉導以表現L202 TCR和人膜栓系IL-12的原代小鼠淋巴細胞(樣品名稱：L202+mtIL12)中的細胞表面IL-12表現的圖。Figure 4C is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202+mtIL12) transduced to express L202 TCR and human membrane-tethered IL-12.

圖4D是示出在被轉導以表現L202 TCR和NHS76-IL12的原代小鼠淋巴細胞(樣品名稱：L202+NHS-IL12)中的細胞表面IL-12表現的圖。Figure 4D is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202+NHS-IL12) transduced to express L202 TCR and NHS76-IL12.

圖5A是示出在未轉導的原代小鼠淋巴細胞(樣品名稱：NHS76-12 UT)或被轉導以表現NHS76-IL-12的原代小鼠淋巴細胞(樣品名稱：NHS76-12)中的人Fab(hFab)表現的圖。NHS76-IL12通過對人Fab染色來檢測。Figure 5A is a graph showing primary mouse lymphocytes either untransduced (sample name: NHS76-12 UT) or transduced to express NHS76-IL-12 (sample name: NHS76-12 ) is a graph of human Fab (hFab) performance. NHS76-IL12 was detected by staining for human Fab.

圖5B是示出在未轉導的原代小鼠淋巴細胞(樣品名稱：NHS76-12 UT)或被轉導以表現NHS76-IL-12的原代小鼠淋巴細胞(樣品名稱：NHS76-12)中的IL-12表現的圖。當對IL-12染色時，NHS76-IL12表現出峰值偏移。Figure 5B is a graph showing primary mouse lymphocytes either untransduced (sample name: NHS76-12 UT) or transduced to express NHS76-IL-12 (sample name: NHS76-12 ) in the graph of IL-12 expression. NHS76-IL12 exhibited a peak shift when stained for IL-12.

圖6A是示出未轉導的人PBMC(樣品名稱：未轉導)的TCRb表面染色的圖。Figure 6A is a graph showing TCRb surface staining of untransduced human PBMC (sample name: untransduced).

圖6B是示出被轉導以表現L202 TCR與人長膜栓系IL-12的組合的人PBMC(樣品名稱：L202+長mtIL12)的TCRb表面染色的圖。Figure 6B is a graph showing TCRb surface staining of human PBMC (sample name: L202+long mtIL12) transduced to express the combination of L202 TCR and human long-membrane-tethered IL-12.

圖6C是示出被轉導以表現L202 TCR與人膜栓系IL-12組合的人PBMC(樣品名稱：L202+mtIL12)的TCRb表面染色的圖。Figure 6C is a graph showing TCRb surface staining of human PBMCs (sample name: L202+mtIL12) transduced to express L202 TCR in combination with human membrane-tethered IL-12.

圖6D是示出被轉導以表現單獨的L202 TCR的人PBMC(樣品名稱：L202)的TCRb表面染色的圖。Figure 6D is a graph showing TCRb surface staining of human PBMC (sample name: L202) transduced to express the L202 TCR alone.

圖6E是示出未轉導的人PBMC(樣品名稱：未轉導)的人IL-12表面染色的圖。Figure 6E is a graph showing human IL-12 surface staining of untransduced human PBMC (sample name: untransduced).

圖6F是示出被轉導以表現L202 TCR與長mt-IL-12的組合的人PBMC(樣品名稱：L202+長mtIL12)的人IL-12表面染色的圖。Figure 6F is a graph showing human IL-12 surface staining of human PBMC (sample name: L202+long mtIL12) transduced to express the combination of L202 TCR and long mt-IL-12.

圖6G是示出被轉導以表現L202 TCR與mt-IL-12的組合的人PBMC(樣品名稱：L202+mtIL12)的人IL-12表面染色的圖。Figure 6G is a graph showing human IL-12 surface staining of human PBMC (sample name: L202+mtIL12) transduced to express the combination of L202 TCR and mt-IL-12.

圖6H是示出被轉導以表現單獨的L202 TCR的人PBMC(樣品名稱：L202)的人IL-12表面染色的圖。Figure 6H is a graph showing human IL-12 surface staining of human PBMC (sample name: L202) transduced to express the L202 TCR alone.

圖7A是示出在與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養的人PBMC中的CD69表現的圖。樣品包括未轉導的人PBMC(樣品名稱：UT A375LLW)；轉導以表現L202 TCR的人PBMC(樣品名稱：L202 A375LLW)；轉導以表現L202加人膜栓系IL-12的人PBMC(樣品名稱：smt A375LLW)；以及轉導以表現L202加人長膜栓系IL-12(lmt)的人PBMC(樣品名稱：lmt A375LLW)。Figure 7A is a graph showing CD69 expression in human PBMC co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells. Samples included untransduced human PBMC (sample name: UT A375LLW); human PBMC transduced to express L202 TCR (sample name: L202 A375LLW); human PBMC transduced to express L202 plus human membrane-tethered IL-12 ( Sample name: smt A375LLW); and human PBMC (sample name: lmt A375LLW) transduced to express L202 plus human long membrane tethered IL-12 (lmt).

圖7B是示出在沒有共培養情況下的人PBMC中的CD69表現的圖。樣品包括未轉導的人PBMC(樣品名稱：UT)；轉導以表現L202 TCR的人PBMC(樣品名稱：L202)；轉導以表現L202加人膜栓系IL-12的人PBMC(樣品名稱：smt)；以及轉導以表現L202加人長膜栓系IL-12的人PBMC(樣品名稱：lmt)。Figure 7B is a graph showing CD69 expression in human PBMC without co-culture. Samples included untransduced human PBMC (sample name: UT); human PBMC transduced to express L202 TCR (sample name: L202); human PBMC transduced to express L202 plus human membrane-tethered IL-12 (sample name) : smt); and human PBMCs (sample name: lmt) transduced to express L202 plus human long-membrane-tethered IL-12.

圖8A示出了流式細胞術結果，其示出了在未轉導的人PBMC中的人IFNγ(hIFNγ)表現。Figure 8A shows flow cytometry results showing human IFNy (hlFNy) expression in untransduced human PBMCs.

圖8B示出了流式細胞術結果，其示出了在被轉導表現L202 TCR的人PBMC中的人IFNγ(hIFNγ)表現。Figure 8B shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR.

圖8C示出了流式細胞術結果，其示出了在被轉導以表現L202 TCR加膜栓系IL-12的人PBMC中的人IFNγ(hIFNγ)表現。Figure 8C shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR plus membrane-tethered IL-12.

圖8D示出了流式細胞術結果，其示出了在被轉導以表現L202 TCR加長膜栓系IL-12的人PBMC中的人IFNγ(hIFNγ)表現。Figure 8D shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR lengthening membrane tethered IL-12.

圖8E示出了流式細胞術結果，其示出了在未轉導的人PBMC中的人IFNγ(hIFNγ)表現。將PBMC與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養。Figure 8E shows flow cytometry results showing human IFNy (hlFNy) expression in untransduced human PBMCs. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

圖8F示出了流式細胞術結果，其示出了在被轉導表現L202 TCR的人PBMC中的人IFNγ(hIFNγ)表現。將PBMC與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養。Figure 8F shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

圖8G示出了流式細胞術結果，其示出了在被轉導以表現L202 TCR加膜栓系IL-12的人PBMC中的人IFNγ(hIFNγ)表現。將PBMC與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養。Figure 8G shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR plus membrane-tethered IL-12. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

圖8H示出了流式細胞術結果，其示出了在被轉導以表現L202 TCR加長膜栓系IL-12的人PBMC中的人IFNγ(hIFNγ)表現。將PBMC與A375-HLA-A2-peplinker(LLW)黑色素瘤靶細胞共培養。Figure 8H shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR lengthening membrane-tethered IL-12. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

圖9A是示出在產生50% L202 TCR陽性(mTCRb+)PBMC(包括作為對照的未轉導的PBMC)和50%未轉導的人PBMC(mTCRb-)的混合群體後mTCRb+群體中CD69表現的圖。樣品包括未轉導的PBMC(樣品名稱：UT+A375)；轉導以表現L202 TCR的PBMC(樣品名稱：L202+A375)；轉導以表現L202加短膜栓系IL-12(smt)的PBMC(樣品名稱：smt+A375)；以及轉導以表現L202加長膜栓系IL-12的PBMC(樣品名稱：lmt+A375)。Figure 9A is a graph showing the expression of CD69 in the mTCRb+ population after generating a mixed population of 50% L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as controls) and 50% untransduced human PBMCs (mTCRb-) picture. Samples included untransduced PBMCs (sample name: UT+A375); PBMCs transduced to express L202 TCR (sample name: L202+A375); transduced to express L202 plus short membrane tethered IL-12 (smt) PBMCs (sample name: smt+A375); and PBMCs transduced to express L202 lengthening membrane-tethered IL-12 (sample name: lmt+A375).

圖9B是示出在產生50% L202 TCR陽性(mTCRb+)PBMC(包括作為對照的未轉導的PBMC)和50%未轉導的PBMC(mTCRb-)的混合群體後mTCRb群體中CD69表現的圖。樣品包括未轉導的PBMC(樣品名稱：UT+A375)；轉導以表現L202 TCR的PBMC(樣品名稱：L202+A375)；轉導以表現L202加短膜栓系IL-12(smt)的PBMC(樣品名稱：smt+A375)；以及轉導以表現L202加長膜栓系IL-12的PBMC(樣品名稱：lmt+A375)。Figure 9B is a graph showing CD69 expression in the mTCRb population after generating a mixed population of 50% L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as controls) and 50% untransduced PBMCs (mTCRb-) . Samples included untransduced PBMCs (sample name: UT+A375); PBMCs transduced to express L202 TCR (sample name: L202+A375); transduced to express L202 plus short membrane tethered IL-12 (smt) PBMCs (sample name: smt+A375); and PBMCs transduced to express L202 lengthening membrane-tethered IL-12 (sample name: lmt+A375).

圖10A示出了流式細胞術結果，其示出了在來自未轉導的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現Figure 10A shows flow cytometry results showing human IFNγ (hIFNγ) expression in mTCRb+ cells from untransduced PBMCs

圖10B示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。Figure 10B shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR.

圖10C示出了流式細胞儀結果，其示出了在來自被轉導以表現L202 TCR加人膜栓系IL-12的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。Figure 1OC shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12.

圖10D示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR加人長膜栓系IL-12的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。Figure 10D shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12.

圖10E示出了流式細胞術結果，其示出了在來自未轉導的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 10E shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from untransduced PBMCs. PBMCs were co-cultured with A375-LLW target cells.

圖10F示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 10F shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-LLW target cells.

圖10G示出了流式細胞儀結果，其示出了在來自被轉導以表現L202 TCR加人膜栓系IL-12的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 10G shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

圖10H示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR加人長膜栓系IL-12的PBMC的mTCRb+細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 10H shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

圖11A示出了流式細胞術結果，其示出了在來自未轉導的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現Figure 11A shows flow cytometry results showing human IFNγ (hIFNγ) expression in mTCRb-cells from untransduced PBMCs

圖11B示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。Figure 11B shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR.

圖11C示出了流式細胞儀結果，其示出了在來自被轉導以表現L202 TCR加人膜栓系IL-12的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。Figure 11C shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12.

圖11D示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR加人長膜栓系IL-12的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。Figure 1 ID shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12.

圖11E示出了流式細胞術結果，其示出了在來自未轉導的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 11E shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from untransduced PBMCs. PBMCs were co-cultured with A375-LLW target cells.

圖11F示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 11F shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-LLW target cells.

圖11G示出了流式細胞儀結果，其示出了在來自被轉導以表現L202 TCR加人膜栓系IL-12的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 11G shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

圖11H示出了流式細胞術結果，其示出了在來自被轉導以表現L202 TCR加人長膜栓系IL-12的PBMC的mTCRb-細胞中的人IFNγ(hIFNγ)表現。將PBMC與A375-LLW靶細胞共培養。Figure 11H shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

圖12A示出了流式細胞術結果，其示出了未轉導的PBMC(UT)中的CD4和CD8表現。Figure 12A shows flow cytometry results showing CD4 and CD8 expression in untransduced PBMC (UT).

圖12B示出了流式細胞術結果，其示出了L202 TCR-T PBMC(L202)中的CD4和CD8表現。Figure 12B shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMCs (L202).

圖12C示出了流式細胞術結果，其示出了在表現人膜拴系IL-12的L202 TCR-T PBMC(mt-IL12)中的CD4和CD8表現。Figure 12C shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

圖12D示出了流式細胞術結果，其示出了在表現人長膜拴系IL-12的L202 TCR-T PBMC(lmt-IL12)中的CD4和CD8表現。Figure 12D shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

圖13示出了流式細胞術結果，其示出了未轉導的PBMC中的效應記憶CD4+細胞和中樞記憶CD4+細胞的百分比。Figure 13 shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in untransduced PBMCs.

圖14A示出了流式細胞術結果，其示出了未轉導的PBMC(UT)中的效應記憶CD4+細胞和中樞記憶CD4+細胞的百分比。Figure 14A shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in untransduced PBMC (UT).

圖14B示出了流式細胞術結果，其示出了L202 TCR-T PBMC(L202)中效應記憶CD4+細胞和中樞記憶CD4+細胞的百分比。Figure 14B shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (L202).

圖14C示出了流式細胞術結果，其示出了表現人膜栓系IL-12的L202 TCR-T PBMC(mt-IL12)中的效應記憶CD4+細胞和中樞記憶CD4+細胞的百分比。Figure 14C shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

圖14D示出了流式細胞術結果，其示出了表現人長膜栓系IL-12的L202 TCR-T PBMC(lmt-IL12)中的效應記憶CD4+細胞和中樞記憶CD4+細胞的百分比。Figure 14D shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

圖15A是流式細胞術結果的圖像，其示出了未轉導的PBMC(UT)中的效應記憶CD8+細胞和中樞記憶CD4+細胞的百分比。Figure 15A is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in untransduced PBMC (UT).

圖15B是流式細胞術結果的圖像，其示出了L202 TCR-T PBMC(L202)中效應記憶CD8+細胞和中樞記憶CD4+細胞的百分比。Figure 15B is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (L202).

圖15C是流式細胞術結果的圖像，其示出了表現人膜拴系IL-12的L202 TCR-T PBMC(mt-IL12)中的效應記憶CD8+細胞和中樞記憶CD4+細胞的百分比。Figure 15C is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

圖15D是流式細胞術結果的圖像，其示出了表現人長膜拴系IL-12的L202 TCR-T PBMC(lmt-IL12)中的效應記憶CD8+細胞和中樞記憶CD4+細胞的百分比。Figure 15D is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

圖16示出了在未轉導的人PBMC(樣品名稱：UT)、轉導以表現人IL-12的人PBMC(樣品名稱：IL-12)、轉導以表現人NHS76-IL-12的人PBMC(樣品名稱：NHS76-IL-12)或轉導以表現人膜栓系IL-12的人PBMC(樣品名稱：Mt-IL-12)的細胞培養基中的IL-12濃度。Figure 16 shows the expression of human PBMC (sample name: UT), transduced to express human IL-12 (sample name: IL-12), transduced to express human NHS76-IL-12 IL-12 concentration in cell culture medium of human PBMC (sample name: NHS76-IL-12) or human PBMC (sample name: Mt-IL-12) transduced to express human membrane-tethered IL-12.

圖17A是示出具有小鼠Flt3L(mFlt3L)、小鼠CXCL10(mCXCL10)或小鼠XCL1(mXCL1)的小鼠IL-12表現載體的示意圖。2A編碼2A自切割肽；mSP編碼小鼠訊息肽；mP40編碼小鼠IL-12B(β鏈)；接頭編碼肽接頭；mP35編碼小鼠IL-12A(α鏈)；鉸鏈編碼免疫球蛋白鉸鏈區；mCH2編碼小鼠免疫球蛋白IgG4重鏈恆定結構域CH2；mCH3編碼小鼠免疫球蛋白IgG4重鏈恆定結構域CH3；並且mCD4 TM編碼小鼠CD4跨膜區。Figure 17A is a schematic diagram showing a mouse IL-12 expression vector with mouse Flt3L (mFlt3L), mouse CXCL10 (mCXCL10) or mouse XCL1 (mXCL1). 2A encodes 2A self-cleaving peptide; mSP encodes mouse message peptide; mP40 encodes mouse IL-12B (beta chain); linker encodes peptide linker; mP35 encodes mouse IL-12A (alpha chain); hinge encodes immunoglobulin hinge region mCH2 encodes the mouse immunoglobulin IgG4 heavy chain constant domain CH2; mCH3 encodes the mouse immunoglobulin IgG4 heavy chain constant domain CH3; and mCD4 TM encodes the mouse CD4 transmembrane region.

圖17B是示出具有人Flt3L(hFlt3L)、人CXCL10(mCXCL10)或人XCL1(hXCL1)的人IL-12表現載體的示意圖。2A編碼2A自切割肽；hSP編碼人訊息肽；hP40編碼人IL-12B；接頭編碼肽接頭；hP35編碼人IL-12A；鉸鏈編碼免疫球蛋白鉸鏈區；hmCH2(人突變的CH2)編碼突變的人免疫球蛋白IgG4重鏈恆定結構域CH2；hCH3(人CH3)編碼人免疫球蛋白IgG4重鏈恆定結構域CH3；hCD4 TM編碼人CD4跨膜區。Figure 17B is a schematic diagram showing human IL-12 expression vectors with human Flt3L (hFlt3L), human CXCL10 (mCXCL10) or human XCL1 (hXCL1). 2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 (human mutated CH2) encodes mutated Human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 (human CH3) encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region.

圖18是示出在未轉導的原代小鼠淋巴細胞和用各種構建體轉導的原代小鼠淋巴細胞中的CAR表現的圖。其中，Cxcl10-IL12是指編碼CXCL10、IL12和抗-EGFRvIII CAR的構建體；Flt3L-IL12是指編碼Flt3L、IL12和抗-EGFRvIII CAR的構建體；IL12是指編碼IL12和抗-EGFRvIII CAR的構建體，EGFRvIII是指編碼抗EGFRvIII CAR的構建體；CxCl10是指編碼CXCL10和抗EGFRvIII CAR的構建體；Flt3L是指編碼Flt3L和抗EGFRvIII CAR的構建體。Figure 18 is a graph showing CAR performance in untransduced primary mouse lymphocytes and primary mouse lymphocytes transduced with various constructs. Among them, Cxcl10-IL12 refers to the construct encoding CXCL10, IL12 and anti-EGFRvIII CAR; Flt3L-IL12 refers to the construct encoding Flt3L, IL12 and anti-EGFRvIII CAR; IL12 refers to the construct encoding IL12 and anti-EGFRvIII CAR EGFRvIII refers to the construct encoding anti-EGFRvIII CAR; CxCl10 refers to the construct encoding CXCL10 and anti-EGFRvIII CAR; Flt3L refers to the construct encoding Flt3L and anti-EGFRvIII CAR.

圖19示出了當未轉導的淋巴細胞(“UT”)或轉導的淋巴細胞與靶腫瘤細胞以不同的效應子與靶細胞比率共培養時競爭性殺傷的百分比。Figure 19 shows the percentage of competitive killing when untransduced lymphocytes ("UT") or transduced lymphocytes were co-cultured with target tumor cells at different effector to target cell ratios.

圖20A示出了當未轉導的淋巴細胞或轉導的淋巴細胞與Kluc細胞或Kluc-EGFRvIII(“Kluc vIII”)細胞共培養時的mIFNg分泌。淋巴細胞是未轉導(“UT”)的，或用編碼(1)抗EGFRvIII CAR(“EGFRvIII CAR”)、(2) IL12和抗EGFRvIII CAR(“EGFRvIII-IL12”)、(3) CXCL10和抗EGFRvIII CAR(“EGFRvIII-CxCL10”)、(4) Flt3L和抗EGFRvIII CAR(“EGFRvIII-FLt3L”)、(5) CXCL10、IL12和抗EGFRvIII CAR(“EGFRvIII-Cx12”)或(5)Flt3L、IL12和抗EGFRvIII CAR(“EGFRvIII-Ft12”)的構建體轉導。Figure 20A shows mIFNg secretion when untransduced lymphocytes or transduced lymphocytes are co-cultured with Kluc cells or Kluc-EGFRvIII ("Kluc vIII") cells. Lymphocytes were untransduced ("UT") or treated with encoding (1) anti-EGFRvIII CAR ("EGFRvIII CAR"), (2) IL12 and anti-EGFRvIII CAR ("EGFRvIII-IL12"), (3) CXCL10 and Anti-EGFRvIII CAR ("EGFRvIII-CxCL10"), (4) Flt3L and anti-EGFRvIII CAR ("EGFRvIII-FLt3L"), (5) CXCL10, IL12 and anti-EGFRvIII CAR ("EGFRvIII-Cx12") or (5) Flt3L, Constructs transduction of IL12 and anti-EGFRvIII CAR ("EGFRvIII-Ft12").

圖20B示出了未轉導的淋巴細胞或轉導的淋巴細胞與Kluc細胞或Kluc-EGFRvIII細胞共培養時的mIL12分泌。Figure 20B shows mIL12 secretion when untransduced lymphocytes or transduced lymphocytes are co-cultured with Kluc cells or Kluc-EGFRvIII cells.

圖20C示出了未轉導的淋巴細胞或轉導的淋巴細胞與Kluc細胞或Kluc-EGFRvIII細胞共培養時的CXCL10分泌。Figure 20C shows CXCL10 secretion when untransduced lymphocytes or transduced lymphocytes were co-cultured with Kluc cells or Kluc-EGFRvIII cells.

圖21是示出體內測試模型的研究設計的圖。Figure 21 is a diagram illustrating the study design of the in vivo testing model.

圖22A至圖22F示出了在用不同構建體轉導的不同淋巴細胞處理後不同小鼠中的腫瘤體積。Figures 22A-22F show tumor volumes in different mice following treatment with different lymphocytes transduced with different constructs.

圖23示出了在用不同的轉導淋巴細胞處理後不同小鼠的存活曲線。Figure 23 shows the survival curves of different mice after treatment with different transduced lymphocytes.

圖24A示出了再次攻擊研究中無腫瘤動物的百分比。Figure 24A shows the percentage of tumor-free animals in the rechallenge study.

圖24B示出了再次攻擊研究中不同小鼠的存活曲線。Figure 24B shows survival curves for different mice in the rechallenge study.

圖25列出了在本公開中所描述的序列。Figure 25 lists the sequences described in this disclosure.

Claims (81)

一種細胞，所述細胞表現 (a)  外源T細胞受體(TCR)或嵌合抗原受體(CAR)；以及 (b) IL-12。a cell that expresses (a) exogenous T cell receptor (TCR) or chimeric antigen receptor (CAR); and (b) IL-12. 如請求項1所述的細胞，其中所述IL-12是膜拴系IL-12。The cell of claim 1, wherein the IL-12 is membrane-tethered IL-12. 如請求項2所述的細胞，其中所述膜拴系IL-12包含CD4跨膜區。The cell of claim 2, wherein the membrane-tethered IL-12 comprises a CD4 transmembrane region. 如請求項3所述的細胞，其中所述膜拴系IL-12包含免疫球蛋白CH2結構域、免疫球蛋白CH3結構域和CD4跨膜區。The cell of claim 3, wherein the membrane-tethered IL-12 comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain and a CD4 transmembrane region. 如請求項3所述的細胞，其中所述膜拴系IL-12包含兩個或更多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和CD4跨膜區。The cell of claim 3, wherein the membrane-tethered IL-12 comprises two or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a CD4 transmembrane region. 如請求項4或5所述的細胞，其中所述免疫球蛋白CH2結構域是野生型免疫球蛋白恆定結構域。The cell of claim 4 or 5, wherein the immunoglobulin CH2 domain is a wild-type immunoglobulin constant domain. 如請求項4或5所述的細胞，其中所述免疫球蛋白CH2結構域的位置235處(EU編號)的胺基酸是Glu，並且所述免疫球蛋白CH2結構域的位置297處(EU編號)的胺基酸殘基是Gln。The cell of claim 4 or 5, wherein the amino acid at position 235 (EU numbering) of the immunoglobulin CH2 domain is Glu, and the immunoglobulin CH2 domain at position 297 (EU numbering) numbering) of the amino acid residue is GIn. 如請求項2-7中任一項所述的細胞，其中所述膜拴系IL-12還包含免疫球蛋白鉸鏈區。The cell of any one of claims 2-7, wherein the membrane-tethered IL-12 further comprises an immunoglobulin hinge region. 如請求項4-8中任一項所述的細胞，其中所述免疫球蛋白CH2結構域是人免疫球蛋白CH2結構域，所述免疫球蛋白CH3結構域是人免疫球蛋白CH3結構域，並且所述CD4跨膜區是人CD4跨膜區。The cell of any one of claims 4-8, wherein the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, and the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain, And the CD4 transmembrane region is a human CD4 transmembrane region. 如請求項1所述的細胞，其中所述IL-12是可溶性IL-12。The cell of claim 1, wherein the IL-12 is soluble IL-12. 如請求項1-10中任一項所述的細胞，其中所述IL-12與靶向腫瘤的抗體或其抗原結合片段連接。The cell of any one of claims 1-10, wherein the IL-12 is linked to a tumor-targeting antibody or antigen-binding fragment thereof. 如請求項11所述的細胞，其中所述靶向腫瘤的抗體或其抗原結合片段是單鏈可變片段(scFv)。The cell of claim 11, wherein the tumor-targeting antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). 如請求項11或12所述的細胞，其中所述靶向腫瘤的抗體是NHS76。The cell of claim 11 or 12, wherein the tumor-targeting antibody is NHS76. 如請求項1-13中任一項所述的細胞，其中所述細胞還表現CXCL10、XCL1或Flt3L。The cell of any one of claims 1-13, wherein the cell further expresses CXCL10, XCL1 or Flt3L. 如請求項1至14中任一項所述的細胞，其中所述TCR或CAR靶向BCMA、CD19、CD22、CD30、CD33、CD56、CD123(IL-3R)、CEA、IL13Ra2、ALPP、EBV相關抗原(例如，LMP2)、EGFR、EGFRvIII、GD2、GPC3、HER2、HPV相關抗原(例如，E6、E7)、MAGE(例如，MAGE-A3)、間皮素、MUC-1、NY-ESO-1、PSCA、PSMA、ROR1、WT1或Claudin 18.2。The cell of any one of claims 1 to 14, wherein the TCR or CAR targets BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (IL-3R), CEA, IL13Ra2, ALPP, EBV associated Antigens (eg, LMP2), EGFR, EGFRvIII, GD2, GPC3, HER2, HPV-associated antigens (eg, E6, E7), MAGE (eg, MAGE-A3), mesothelin, MUC-1, NY-ESO-1 , PSCA, PSMA, ROR1, WT1 or Claudin 18.2. 如請求項1-15中任一項所述的細胞，其中所述CAR包含細胞外結構域，其中所述細胞外結構域是單鏈可變片段(scFv)、配體(例如受體結合配體)或抗體模擬物。The cell of any one of claims 1-15, wherein the CAR comprises an extracellular domain, wherein the extracellular domain is a single-chain variable fragment (scFv), a ligand (eg, a receptor binding ligand) body) or antibody mimetics. 一種載體，所述載體包含： a)   第一核酸序列，所述第一核酸序列編碼IL-12α亞基和IL-12β亞基； b)   第二核酸序列，所述第二核酸序列編碼一個或多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和跨膜區， 其中所述第一核酸序列和所述第二核酸序列通過第一接頭序列連接。A carrier comprising: a) a first nucleic acid sequence encoding an IL-12α subunit and an IL-12β subunit; b) a second nucleic acid sequence encoding one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence. 如請求項17所述的載體，其中所述第一接頭序列編碼免疫球蛋白鉸鏈多肽序列。The vector of claim 17, wherein the first linker sequence encodes an immunoglobulin hinge polypeptide sequence. 如請求項17或18所述的載體，其中所述跨膜區是CD4跨膜區。The vector of claim 17 or 18, wherein the transmembrane region is a CD4 transmembrane region. 如請求項17或18所述的載體，其中所述跨膜區是免疫球蛋白跨膜區。The vector of claim 17 or 18, wherein the transmembrane region is an immunoglobulin transmembrane region. 如請求項17-20中任一項所述的載體，其中所述IL-12α亞基和所述IL-12β亞基通過接頭序列連接。The vector of any one of claims 17-20, wherein the IL-12α subunit and the IL-12β subunit are linked by a linker sequence. 如請求項17-21中任一項所述的載體，其中所述載體還包含編碼訊息肽的序列。The vector of any of claims 17-21, wherein the vector further comprises a sequence encoding a message peptide. 如請求項17-22中任一項所述的載體，其中所述IL-12α亞基是人IL-12α亞基，所述IL-12β亞基是人IL-12β亞基，所述免疫球蛋白CH2結構域是人免疫球蛋白CH2結構域，所述免疫球蛋白CH3結構域是人免疫球蛋白CH3結構域。The vector of any one of claims 17-22, wherein the IL-12α subunit is a human IL-12α subunit, the IL-12β subunit is a human IL-12β subunit, and the immunoglobulin The protein CH2 domain is a human immunoglobulin CH2 domain, and the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain. 如請求項17-23中任一項所述的載體，其中所述載體還包含編碼T細胞受體(TCR)或嵌合抗原受體(CAR)的第三核酸序列。The vector of any of claims 17-23, wherein the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 如請求項24所述的載體，其中所述第三核酸序列通過第二接頭序列與所述第一核酸連接。The vector of claim 24, wherein the third nucleic acid sequence is linked to the first nucleic acid through a second linker sequence. 如請求項25所述的載體，其中所述第二接頭序列編碼2A序列(例如P2A序列)。The vector of claim 25, wherein the second linker sequence encodes a 2A sequence (eg, a P2A sequence). 如請求項17-26中任一項所述的載體，其中所述第一核酸和所述第二核酸在調節元件(例如啟動子)的控制下；或其中所述第一核酸、所述第二核酸和所述第三核酸在調節元件(例如啟動子)的控制下。The vector of any one of claims 17-26, wherein the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter); or wherein the first nucleic acid, the second nucleic acid The second nucleic acid and the third nucleic acid are under the control of regulatory elements such as promoters. 如請求項17-27中任一項所述的載體，其中所述載體還包含編碼CXCL10、XCL1或Flt3L的序列。The vector of any one of claims 17-27, wherein the vector further comprises a sequence encoding CXCL10, XCL1 or Flt3L. 一種載體，所述載體包含： a)   第一核酸序列，所述第一核酸序列編碼靶向腫瘤的抗體的重鏈可變區(VH)和輕鏈可變區(VL)； b)   第二核酸序列，所述第二核酸序列編碼IL-12α亞基和IL-12β亞基， 其中所述第一核酸序列和所述第二核酸序列通過第一接頭序列連接。A carrier comprising: a) a first nucleic acid sequence encoding the variable heavy (VH) and variable light (VL) regions of the tumor-targeting antibody; b) a second nucleic acid sequence encoding the IL-12α subunit and the IL-12β subunit, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence. 如請求項29所述的方法，其中所述靶向腫瘤的抗體靶向腫瘤相關抗原。The method of claim 29, wherein the tumor-targeting antibody targets a tumor-associated antigen. 如請求項30所述的方法，其中所述靶向腫瘤的抗體是NHS76。The method of claim 30, wherein the tumor-targeting antibody is NHS76. 如請求項29-31中任一項所述的載體，其中所述載體還包含編碼訊息肽的核酸。The vector of any one of claims 29-31, wherein the vector further comprises a nucleic acid encoding a message peptide. 如請求項32所述的載體，其中所述訊息肽是人訊息肽，所述IL-12α亞基是人IL-12α亞基，並且所述IL-12β亞基是人IL-12β亞基。The vector of claim 32, wherein the message peptide is a human message peptide, the IL-12α subunit is a human IL-12α subunit, and the IL-12β subunit is a human IL-12β subunit. 如請求項29-33中任一項所述的載體，其中所述載體還包含編碼T細胞受體(TCR)或嵌合抗原受體(CAR)的第三核酸序列。The vector of any of claims 29-33, wherein the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 如請求項34所述的載體，其中所述第三核酸序列通過第二接頭序列與所述第一核酸的5’連接。The vector of claim 34, wherein the third nucleic acid sequence is linked to the 5' of the first nucleic acid through a second linker sequence. 如請求項35所述的載體，其中所述第二接頭序列編碼P2A序列。The vector of claim 35, wherein the second linker sequence encodes a P2A sequence. 如請求項29-36中任一項所述的載體，其中所述第一核酸和所述第二核酸在調節元件(例如啟動子)的控制下；或其中所述第一核酸、所述第二核酸和所述第三核酸在調節元件(例如啟動子)的控制下。The vector of any one of claims 29-36, wherein the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter); or wherein the first nucleic acid, the second nucleic acid The second nucleic acid and the third nucleic acid are under the control of regulatory elements such as promoters. 如請求項29-37中任一項所述的載體，其中所述載體還包含編碼CXCL10、XCL1或Flt3L的序列。The vector of any one of claims 29-37, wherein the vector further comprises a sequence encoding CXCL10, XCL1 or Flt3L. 一種載體，所述載體在5'至3'方向上包含 a)   第一核酸序列，所述第一核酸序列編碼外源T細胞受體(TCR)或嵌合抗原受體(CAR)； b)   第二核酸序列，所述第二核酸序列編碼訊息肽IL-12α亞基和IL-12β亞基； 其中所述第一核酸序列和所述第二核酸序列通過接頭序列連接。A vector comprising in the 5' to 3' direction a) a first nucleic acid sequence encoding a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); b) a second nucleic acid sequence encoding the message peptide IL-12α subunit and IL-12β subunit; wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a linker sequence. 如請求項39所述的載體，其中所述IL-12α亞基和所述IL-12β亞基通過接頭序列連接。The vector of claim 39, wherein the IL-12α subunit and the IL-12β subunit are linked by a linker sequence. 如請求項39或40所述的載體，其中所述接頭序列編碼P2A序列。The vector of claim 39 or 40, wherein the linker sequence encodes a P2A sequence. 一種融合多肽，所述融合多肽包含 a)   包含IL-12α亞基和IL-12β亞基的第一區，其中所述IL-12α亞基和所述IL-12β亞基通過接頭序列連接， b)   包含一個或多個免疫球蛋白CH2結構域、一個或多個免疫球蛋白CH3結構域和跨膜區的第二區。A fusion polypeptide comprising a) a first region comprising an IL-12α subunit and an IL-12β subunit, wherein the IL-12α subunit and the IL-12β subunit are connected by a linker sequence, b) A second region comprising one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region. 如請求項42所述的多肽，其中所述第一區和所述第二區通過免疫球蛋白鉸鏈肽連接。The polypeptide of claim 42, wherein the first region and the second region are linked by an immunoglobulin hinge peptide. 一種融合多肽，所述融合多肽包含 a)   包含IL-12α亞基和IL-12β亞基的第一區，其中所述IL-12α亞基和所述IL-12β亞基通過第一接頭序列連接， b)   包含靶向腫瘤的抗體的重鏈可變區(VH)和輕鏈可變區(VL)的第二區，其中所述VH和所述VL通過第二接頭肽序列連接， 其中所述第一區和所述第二區通過第三接頭肽序列連接。A fusion polypeptide comprising a) a first region comprising an IL-12α subunit and an IL-12β subunit, wherein the IL-12α subunit and the IL-12β subunit are connected by a first linker sequence, b) a second region comprising a heavy chain variable region (VH) and a light chain variable region (VL) of a tumor-targeting antibody, wherein said VH and said VL are linked by a second linker peptide sequence, wherein the first region and the second region are linked by a third linker peptide sequence. 如請求項44所述的多肽，其中所述第三接頭多肽序列具有與SEQ ID NO: 17至少80%相同的序列。The polypeptide of claim 44, wherein the third linker polypeptide sequence has a sequence that is at least 80% identical to SEQ ID NO: 17. 一種核酸，其編碼如請求項42-45中任一項所述的多肽。A nucleic acid encoding the polypeptide of any one of claims 42-45. 一種載體，其包含如請求項46所述的核酸。A vector comprising the nucleic acid of claim 46. 一種細胞，其表現如請求項42-45中任一項所述的融合多肽。A cell expressing the fusion polypeptide of any one of claims 42-45. 一種細胞，其包含如請求項17-41和47中任一項所述的載體。A cell comprising the vector of any one of claims 17-41 and 47. 如請求項49所述的細胞，其中與不同之處在於細胞不包含所述載體的相同細胞相比，所述細胞分泌更高位準的細胞介素。The cell of claim 49, wherein the cell secretes a higher level of interleukin than the same cell differing in that the cell does not contain the vector. 如請求項49所述的細胞，其中所述細胞刺激所述細胞附近的一種或多種細胞分泌細胞介素。The cell of claim 49, wherein the cell stimulates one or more cells in the vicinity of the cell to secrete interferons. 如請求項50或51所述的細胞，其中所述細胞介素是IFNγ。The cell of claim 50 or 51, wherein the interferon is IFNγ. 如請求項49所述的細胞，其中與不同之處在於細胞不包含所述載體的相同細胞相比，所述細胞表現更高位準的早期TCR活化標記物，和/或刺激所述細胞附近的一種或多種細胞表現早期TCR活化標記物。The cell of claim 49, wherein the cell exhibits a higher level of early TCR activation markers, and/or stimulates a One or more cells express markers of early TCR activation. 如請求項53所述的細胞，其中所述活化標記物是CD69。The cell of claim 53, wherein the activation marker is CD69. 如請求項1-16和48-54中任一項所述的細胞，其中所述細胞是細胞系。The cell of any one of claims 1-16 and 48-54, wherein the cell is a cell line. 如請求項1-16和48-54中任一項所述的細胞，其中所述細胞是從受試者(例如，人受試者)獲得的原代細胞。The cell of any one of claims 1-16 and 48-54, wherein the cell is a primary cell obtained from a subject (eg, a human subject). 如請求項1-16和48-54中任一項所述的細胞，其中所述細胞是免疫細胞(例如，淋巴細胞)。The cell of any one of claims 1-16 and 48-54, wherein the cell is an immune cell (eg, a lymphocyte). 如請求項1-16和48-54中任一項所述的細胞，其中所述細胞是腫瘤浸潤性淋巴細胞(TIL)或NK細胞(例如，CAR-NK細胞)。The cell of any one of claims 1-16 and 48-54, wherein the cell is a tumor-infiltrating lymphocyte (TIL) or a NK cell (eg, a CAR-NK cell). 如請求項1-16和48-54中任一項所述的細胞，其中所述細胞是T細胞。The cell of any one of claims 1-16 and 48-54, wherein the cell is a T cell. 如請求項59所述的細胞，其中所述T細胞是從人受試者中分離的。The cell of claim 59, wherein the T cells are isolated from a human subject. 如請求項59或60所述的細胞，其中所述T細胞是CD8+。The cell of claim 59 or 60, wherein the T cells are CD8+. 如請求項59-61中任一項所述的細胞，其中所述T細胞是CD4+。The cell of any of claims 59-61, wherein the T cells are CD4+. 如請求項17-41和47中任一項所述的載體，其中所述載體是表現載體、病毒載體、反轉錄病毒載體或慢病毒載體。The vector of any one of claims 17-41 and 47, wherein the vector is an expression vector, a viral vector, a retroviral vector or a lentiviral vector. 如請求項63所述的載體，其中所述反轉錄病毒載體是pMP71。The vector of claim 63, wherein the retroviral vector is pMP71. 一種用於生產細胞的方法，所述方法包括將如請求項17-41、47、63和64中任一項所述的載體體外或離體引入細胞中。A method for producing a cell, the method comprising introducing the vector of any one of claims 17-41, 47, 63 and 64 into the cell in vitro or ex vivo. 如請求項65所述的方法，其中通過轉導將所述載體引入所述細胞中。The method of claim 65, wherein the vector is introduced into the cell by transduction. 一種治療患有癌症的受試者的方法，所述方法包括向有需要的受試者施用有效量的如請求項1-16和48-62中任一項所述的細胞。A method of treating a subject suffering from cancer, the method comprising administering to a subject in need thereof an effective amount of a cell of any one of claims 1-16 and 48-62. 如請求項67所述的方法，其中所述癌症是異質癌症。The method of claim 67, wherein the cancer is a heterogeneous cancer. 如請求項67所述的方法，其中所述癌症是同質癌症。The method of claim 67, wherein the cancer is a homogeneous cancer. 如請求項67-69中任一項所述的方法，其中所述細胞從所述受試者的外周血單核細胞(PBMC)中分離。The method of any of claims 67-69, wherein the cells are isolated from peripheral blood mononuclear cells (PBMCs) of the subject. 一種治療患有癌症的受試者的方法，所述方法包括 向有需要的受試者施用， a)   有效量的表現T細胞受體(TCR)或嵌合抗原受體(CAR)的細胞；以及 b)   有效量的包含IL-12和靶向腫瘤的抗體或其抗原結合片段的蛋白質。A method of treating a subject with cancer, the method comprising administered to a subject in need, a) an effective amount of cells expressing T cell receptor (TCR) or chimeric antigen receptor (CAR); and b) An effective amount of a protein comprising IL-12 and a tumor-targeting antibody or antigen-binding fragment thereof. 如請求項71所述的方法，其中所述細胞從所述受試者的外周血單核細胞中分離。The method of claim 71, wherein the cells are isolated from peripheral blood mononuclear cells of the subject. 一種治療患有癌症的人受試者的方法，所述方法包括 提供從所述人受試者或不同人受試者收集的細胞； 將如請求項17-41和47中任一項所述的載體引入所述細胞中； 培養和擴增所述細胞；以及 向所述受試者施用有效量的包含所述細胞的組合物。A method of treating a human subject suffering from cancer, the method comprising providing cells collected from the human subject or a different human subject; introducing the vector of any one of claims 17-41 and 47 into the cell; culturing and expanding the cells; and An effective amount of a composition comprising the cells is administered to the subject. 如請求項73所述的方法，其中所述癌症是異質癌症。The method of claim 73, wherein the cancer is a heterogeneous cancer. 如請求項73所述的方法，其中所述癌症是同質癌症。The method of claim 73, wherein the cancer is a homogeneous cancer. 如請求項73-75中任一項所述的方法，其中所述細胞是外周血單核細胞(PBMC)。The method of any of claims 73-75, wherein the cells are peripheral blood mononuclear cells (PBMCs). 如請求項73-75中任一項所述的方法，其中所述細胞是腫瘤浸潤性淋巴細胞，並且所述載體包含編碼IL-12的核酸。The method of any of claims 73-75, wherein the cells are tumor-infiltrating lymphocytes and the vector comprises a nucleic acid encoding IL-12. 如請求項77所述的方法，其中所述IL-12是膜拴系IL-12。The method of claim 77, wherein the IL-12 is membrane-tethered IL-12. 如請求項73-75中任一項所述的方法，其中所述細胞是T細胞，並且所述載體包含編碼TCR或CAR的核酸。The method of any one of claims 73-75, wherein the cell is a T cell and the vector comprises a nucleic acid encoding a TCR or CAR. 如請求項79所述的方法，其中所述載體還包含編碼IL-12的核酸。The method of claim 79, wherein the vector further comprises a nucleic acid encoding IL-12. 如請求項80所述的方法，其中所述IL-12是膜拴系IL-12。The method of claim 80, wherein the IL-12 is membrane-tethered IL-12.
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