Preparation process and application of cell therapy product

Abstract

Provided herein are methods for preparing populations of cells expressing chimeric antigen receptors (CARs) and compositions, media, or kits useful in the methods. The population of cells has reduced cytotoxicity in vivo.

Description

Technical Field

[0001] The present application relates to the field of preparation of cell therapy products, and in particular to a method for preparing a cell therapy product with reduced in vivo toxicity. Background Art

[0002] The current CAR-T preparation technology includes four steps: (1) T cell isolation, (2) T cell activation, (3) CAR lentiviral transfection, and (4) CAR-T cell expansion. The preparation cycle is about 7-14 days. Among them, T cell activation, CAR gene transfer and in vitro expansion are the key steps in the preparation of CAR-T cells, which affect the final therapeutic effect of CAR-T cells. A good preparation process is the basis for obtaining excellent CAR-T cells. Since resting T cells cannot be genetically transfected, additional additives such as anti-CD3 / CD28 antibodies or magnetic beads coated with anti-CD3 / CD28 antibodies are required for activation. The use of additives not only increases the preparation cost, but also increases the number of steps to remove the additives. The long-term in vitro expansion of CAR-T cells not only requires a long waiting period for patients, but also leads to the differentiation of CAR-T cells, making the infused CAR-T cells less persistent in vivo. Therefore, it is necessary to conduct in-depth research and optimization on the key steps of CAR-T preparation. On the one hand, it is necessary to reduce the in vitro differentiation of T cells and maintain better initial functional characteristics to improve their therapeutic efficacy; on the other hand, it is necessary to shorten the preparation cycle, reduce the number of operating steps, and reduce production costs to quickly promote its clinical industrial transformation. SUMMARY OF THE INVENTION

[0004] The present application provides a method for rapidly preparing a cell colony expressing a chimeric antigen receptor (CAR) without CD28 stimulation, and a composition and use for preparing the cell colony expressing CAR. The cell colony prepared using the method of the present application has better in vivo effectiveness and safety.

[0005] Specifically, this application relates to:

[0006] 1. A method for preparing a cell population expressing a chimeric antigen receptor (CAR), the method comprising:

[0007] Step (i) contacting the cell population with a CD3 / TCR complex agonist;

[0008] Step (ii) contacting the cell population with a nucleic acid molecule encoding the CAR, thereby providing cells comprising the nucleic acid molecule, and

[0009] Step (iii) culture the cell population for a period of time and harvest the cell population.

[0010] 2. A method according to claim 1, wherein the cell population is not contacted with a CD28 agonist during step (i), steps (i) and (ii), steps (i), (ii) and (iii) or the entire preparation process.

[0011] 3. according to the method described in item 1 or 2, wherein the cell colony is not contacted with a cell surface costimulatory molecule agonist in step (i), step (i) and (ii), step (i), (ii) and (iii) or the entire preparation process, and the costimulatory molecule comprises: CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and CD226. In some embodiments, the cell surface costimulatory molecule agonist is an antibody to a surface costimulatory molecule.

[0012] 4. A method according to any one of items 1-3, wherein the CD3 / TCR complex agonist comprises a CD3 antibody. In some embodiments, the CD3 antibody comprises a domain that specifically binds to a CD3ε chain. In some embodiments, the CD3 antibody is a monoclonal antibody or an antigen-binding fragment thereof targeting CD3, a bispecific antibody or a multispecific antibody or an antigen-binding fragment thereof targeting CD3 and other antigen molecules. In some embodiments, the CD3 antibody is an IgG antibody such as a humanized or human IgG antibody, or an antigen-binding fragment thereof.

[0013] 5. A method according to any one of items 1-4, wherein the cell population is contacted with an immune cell activating cytokine in step i), step i) and ii), step i), ii) and iii), or the entire preparation process, wherein the immune cell activating cytokine does not include IL-2.

[0014] 6. The method according to claim 5, wherein the immune cell activating cytokines comprise one or more selected from the group consisting of IL-7, IL-15, IL-21, IL-12, IL-18 and IL-6.

[0015] 7. The method according to item 6, wherein the cytokines comprise: a combination of IL-7 and IL-21.

[0016] 8. A method according to item 6 or 7, wherein the concentration of IL-7 when contacting the cell population is 2.5 to 10 ng / ml, for example, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.1 ng / ml, 5.2 ng / ml, 5.3 ng / ml, 5.4 ng / ml, 5.5 ng / ml, 5.6 ng / ml, 5.7 ng / ml, 5.8 ng / ml, 5.9 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml or 9.5 ng / ml, or any value between 2.5 and 10 ng / ml.

[0017] 9. The method according to any one of items 6 to 8, wherein IL21 is contacted with the cell population at a concentration of 12.5 to 50 ng / ml, for example 13 ng / ml, 13.5 ng / ml, 14 ng / ml, 14.5 ng / ml, 15 ng / ml, 15.5 ng / ml, 16 ng / ml, 16.5 ng / ml, 17 ng / ml, 17.5 ng / ml, 18 ng / ml, 18.5 ng / ml, 19 ng / ml, 19.5 ng / ml, 20 ng / ml, 20.5 ng / ml, 21ng / ml, 21.5ng / ml, 22ng / ml, 22.5ng / ml, 23ng / ml, 23.5ng / ml, 24ng / ml, 24.5ng / ml, 25ng / ml, 25.5ng / ml , 26ng / ml, 26.5ng / ml, 27ng / ml, 27.5ng / ml, 28ng / ml, 28.5ng / ml, 29ng / ml, 29.5ng / ml, 30ng / ml, 30.5ng / ml, 31ng / ml, 31.5ng / ml, 32ng / ml, 32.5ng / ml, 33ng / ml, 33.5ng / ml, 34ng / ml, 34.5ng / ml, 35ng / ml, 35.5ng / ml, 36ng / ml, 36 .5ng / ml, 37ng / ml, 37.5ng / ml, 38ng / ml, 38.5ng / ml, 39ng / ml, 39.5ng / ml, 40ng / ml, 40.5ng / ml, 41ng / ml, 41.5ng / The concentration may be gradually changed, for example, decreased, with the contact time.

[0018] 10. The method according to claim 4, wherein the concentration of the CD3 antibody contacted with the cell population in step i) is ≥ 25 ng / ml, such as 25-200 ng / ml or any value therein, such as 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml,

[0019] 34ng / ml、35ng / ml、36ng / ml、37ng / ml、38ng / ml、39ng / ml、40ng / ml、

[0020] 41ng / ml、42ng / ml、43ng / ml、44ng / ml、45ng / ml、46ng / ml、47ng / ml、

[0021] 48ng / ml、49ng / ml、50ng / ml、51ng / ml、52ng / ml、53ng / ml、54ng / ml、

[0022] 55ng / ml、56ng / ml、57ng / ml、58ng / ml、59ng / ml、60ng / ml、61ng / ml、

[0023] 62ng / ml、63ng / ml、64ng / ml、65ng / ml、66ng / ml、67ng / ml、68ng / ml、

[0024] 69ng / ml、70ng / ml、71ng / ml、72ng / ml、73ng / ml、74ng / ml、75ng / ml、

[0025] 76ng / ml、77ng / ml、78ng / ml、79ng / ml、80ng / ml、81ng / ml、82ng / ml、

[0026] 83ng / ml、84ng / ml、85ng / ml、86ng / ml、87ng / ml、88ng / ml、89ng / ml、

[0027] 90ng / ml、91ng / ml、92ng / ml、93ng / ml、94ng / ml、95ng / ml、96ng / ml、

[0028] 97ng / ml、98ng / ml、99ng / ml、100ng / ml、101ng / ml、102ng / ml、103ng / ml、

[0029] 104ng / ml, 105ng / ml, 106ng / ml, 107ng / ml, 108ng / ml, 109ng / ml, 110ng / ml, 111ng / ml, 112ng / ml, 113ng / ml, 114ng / ml, 115ng / ml, 116ng / ml, 117ng / ml, 118ng / ml, 119ng / ml, 120ng / ml, 121ng / ml, 122ng / ml, 123ng / ml, 124ng / ml, 125ng / ml, 126ng / ml, 127ng / ml, 128ng / ml, 129ng / ml, 130ng / ml, 131ng / ml, 132ng / ml, 133ng / ml, 134ng / ml, 135ng / ml, 136ng / ml, 137ng / ml, 138ng / ml, 139ng / ml, 140ng / ml, 141ng / ml, 142ng / ml, 143ng / ml, 144ng / ml, 145ng / ml, 146ng / ml, 147ng / ml, 148ng / ml, 149ng / ml, 150ng / ml, 151ng / ml, 152ng / ml, 153ng / ml, 154ng / ml, 155ng / ml, 156ng / ml, 157ng / ml, 158ng / ml, 159ng / ml, 160ng / ml, 161ng / ml, 162ng / ml, 163ng / ml, 164ng / ml, 165ng / ml, 166ng / ml, 167ng / ml, 168ng / ml, 169ng / ml, 170ng / ml, 171ng / ml, 172ng / ml, 173ng / ml, 174ng / ml, 175ng / ml, 176ng / ml, 177ng / ml, 178ng / ml, 179ng / ml, 180ng / ml, 181ng / ml, 182ng / ml, 183ng / ml, 184ng / ml, 185ng / ml, 186ng / ml, 187ng / ml, 188ng / ml, 189ng / ml, 190ng / ml, 191ng / ml, 192ng / ml, 193ng / ml, 194ng / ml, 195ng / ml, 196ng / ml, 197ng / ml, 198ng / ml, or 199ng / ml.

[0030] 11. A method according to any one of items 1-3, wherein the cell population is contacted with an immune cell activating cytokine in step i), step i) and ii), step i), ii) and iii), or the entire preparation process, wherein the immune cell activating cytokine includes IL-2.

[0031] 12. The method of claim 11, wherein the IL-2 concentration is 100 IU / ml-500 IU / ml, 150 IU / ml-450 IU / ml, 200 IU / ml-400 IU / ml, or 250 IU / ml-350 IU / ml.

[0032] 13. The method according to any one of items 1-12, wherein the time from step ii) to harvesting the cell population is ≤72 hours or ≤48 hours.

[0033] 14. The method according to any one of items 1-13, wherein the time from step ii) to harvesting the cell colony is ≥6 hours. In some embodiments, the method is ≥6 hours and ≤72 hours from step ii) to harvesting the cell colony, such as 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours. ii). Harvesting the cell population at any time between 6 hours and 72 hours from the start of step ii) is within the scope of the present application.

[0034] 15. A method according to any one of items 1-14, wherein the time from step i) to the start of step ii) is 12 to 24 hours, and the time from the start of step ii) to harvesting the cell population is 14-28 hours.

[0035] 16. A method according to any one of items 1 to 15, wherein the time from step i) to harvesting the cell population is 1 to 4 days, such as 2 days.

[0036] 17. A method according to any one of items 1 to 16, wherein the time from step i) to harvesting the cell population is 1 to 2 days.

[0037] 18. The method according to any one of items 1-17, wherein the cell population is an immune cell, such as a macrophage, a NK cell, a T cell, a B cell, or a dendritic cell. In some embodiments, the cell population is a T cell or a NK cell.

[0038] 19. The method according to item 18, wherein the T cells are derived from PBMC. In some embodiments, the cell colony is peripheral blood mononuclear cells (PBMC). In some embodiments, the cell colony harvested in step (iii) is an immune cell expressing CAR, such as CAR-T or CAR-NK.

[0039] 20. A composition for preparing a cell colony expressing a chimeric antigen receptor (CAR), comprising a CD3 / TCR complex agonist and an immune cell activating cytokine, wherein the immune cell activating cytokine does not contain or contains IL-2. In an embodiment comprising IL-2, the IL-2 concentration is 100IU / ml-500IU / ml, 150IU / ml-450IU / ml, 200IU / ml-400IU / ml, or 250IU / ml-350IU / ml.

[0040] 21. A composition according to item 20, wherein the CD3 / TCR complex agonist comprises a CD3 antibody. In some embodiments, the CD3 antibody comprises a domain that specifically binds to the CD3ε chain. In some embodiments, the CD3 antibody is an IgG that comprises a domain that specifically binds to the CD3ε chain. In some embodiments, the CD3 antibody is an IgG type humanized antibody that comprises a domain that specifically binds to the CD3ε chain.

[0041] 22. The composition according to item 21, wherein the concentration of CD3 antibody is ≥25 ng / ml, such as 25-200 ng / ml or any value therein, such as 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml, 34 ng / ml, 35 ng / ml, 36 ng / ml,

[0042] 37ng / ml, 38ng / ml, 39ng / ml, 40ng / ml, 41ng / ml, 42ng / ml, 43ng / ml,

[0043] 44ng / ml, 45ng / ml, 46ng / ml, 47ng / ml, 48ng / ml, 49ng / ml, 50ng / ml,

[0044] 51ng / ml、52ng / ml、53ng / ml、54ng / ml、55ng / ml、56ng / ml、57ng / ml、

[0045] 58ng / ml、59ng / ml、60ng / ml、61ng / ml、62ng / ml、63ng / ml、64ng / ml、

[0046] 65ng / ml、66ng / ml、67ng / ml、68ng / ml、69ng / ml、70ng / ml、71ng / ml、

[0047] 72ng / ml、73ng / ml、74ng / ml、75ng / ml、76ng / ml、77ng / ml、78ng / ml、

[0048] 79ng / ml、80ng / ml、81ng / ml、82ng / ml、83ng / ml、84ng / ml、85ng / ml、

[0049] 86ng / ml、87ng / ml、88ng / ml、89ng / ml、90ng / ml、91ng / ml、92ng / ml、

[0050] 93ng / ml、94ng / ml、95ng / ml、96ng / ml、97ng / ml、98ng / ml、99ng / ml、

[0051] 100ng / ml、101ng / ml、102ng / ml、103ng / ml、104ng / ml、105ng / ml、

[0052] 106ng / ml, 107ng / ml, 108ng / ml, 109ng / ml, 110ng / ml, 111ng / ml, 112ng / ml, 113ng / ml, 114ng / ml, 115ng / ml, 116ng / ml, 117ng / ml, 118ng / ml, 119ng / ml, 120ng / ml, 121ng / ml, 122ng / ml, 123ng / ml, 124ng / ml, 125ng / ml, 126ng / ml, 127ng / ml, 128ng / ml, 129n g / ml, 130ng / ml, 131ng / ml, 132ng / ml, 133ng / ml, 134ng / ml, 135ng / ml, 136ng / ml, 137ng / ml, 138ng / ml, 139ng / ml, 140ng / ml, 14 1ng / ml, 142ng / ml, 143ng / ml, 144ng / ml, 145ng / ml, 146ng / ml, 147ng / ml, 148ng / ml, 149ng / ml, 150ng / ml, 151ng / ml, 152ng / ml, 153ng / ml, 154ng / ml, 155ng / ml, 156ng / ml, 157ng / ml, 158ng / ml, 159ng / ml, 160ng / ml, 161ng / ml, 162ng / ml, 163ng / ml, 164ng / m l, 165ng / ml, 166ng / ml, 167ng / ml, 168ng / ml, 169ng / ml, 170ng / ml, 171ng / ml, 172ng / ml, 173ng / ml, 174ng / ml, 175ng / ml, 176ng In some embodiments, the CD3 antibody is 50 ng / ml.

[0053] 23. A composition according to any one of items 20 to 22, wherein the immune cell activating cytokines comprise one or more selected from the group consisting of IL-7, IL-15, IL-21, IL-12, IL-18 and IL-6.

[0054] 24. A composition according to item 23, wherein the immune cell activating cytokines comprise a combination of IL7 and IL21.

[0055] 25. The composition according to item 24, wherein the concentration of IL-7 is 2.5 to 10 ng / ml, for example 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.1 ng / ml, 5.2 ng / ml, 5.3 ng / ml, 5.4 ng / ml, 5.5 ng / ml, 5.6 ng / ml, 5.7 ng / ml, 5.8 ng / ml, 5.9 ng / ml,

[0056] 6ng / ml, 6.5ng / ml, 7ng / ml, 7.5ng / ml, 8ng / ml, 8.5ng / ml, 9ng / ml or 9.5ng / ml, or any value between 2.5 and 10ng / ml.

[0057] 26. A composition according to item 24 or 25, wherein the concentration of IL21 is 12.5-50 ng / ml, for example 13 ng / ml, 13.5 ng / ml, 14 ng / ml, 14.5 ng / ml, 15 ng / ml, 15.5 ng / ml, 16 ng / ml, 16.5 ng / ml, 17 ng / ml, 17.5 ng / ml, 18 ng / ml, 18.5 ng / ml, 19 ng / ml, 19.5 ng / ml, 20 ng / ml, 20.5 ng / ml, 21ng / ml, 21.5ng / ml, 22ng / ml, 22.5ng / ml, 23ng / ml, 23.5ng / ml, 24ng / ml, 24.5ng / ml, 25ng / ml, 25.5n g / ml, 26ng / ml, 26.5ng / ml, 27ng / ml, 27.5ng / ml, 28ng / ml, 28.5ng / ml, 29ng / ml, 29.5ng / ml, 30ng / ml, 30.5ng / ml, 31ng / ml, 31.5ng / ml, 32ng / ml, 32.5ng / ml, 33ng / ml, 33.5ng / ml, 34ng / ml, 34.5ng / ml, 35ng / ml, 35.5ng / m l, 36ng / ml, 36.5ng / ml, 37ng / ml, 37.5ng / ml, 38ng / ml, 38.5ng / ml, 39ng / ml, 39.5ng / ml, 40ng / ml, 40.5ng / ml, 41ng / ml, 41.5ng / ml, 42ng / ml, 42.5ng / ml, 43ng / ml, 43.5ng / ml, 44ng / ml, 44.5ng / ml, 45ng / ml, 45.5ng / ml, 46ng / ml, 46.5ng / ml, 47ng / ml, 47.5ng / ml, 48ng / ml, 48.5ng / ml, 49ng / ml, or 49.5ng / ml, or any value between 12.5 and 50ng / ml.

[0058] 27. A composition according to any one of items 20 to 26, which does not comprise a CD28 agonist. In some embodiments, the composition does not comprise any cell surface co-stimulatory molecule agonist, the co-stimulatory molecules comprising: CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and CD226.

[0059] 28. A kit comprising the composition according to any one of items 20-27, wherein the kit does not comprise IL-2 and a CD28 agonist.

[0060] 29. A composition according to any one of items 20-27, or use of the kit according to item 28 for preparing a cell population expressing a chimeric antigen receptor (CAR).

[0061] 30. The use according to item 29, wherein the cell population comprises T cells. In some embodiments, the cell population expressing a chimeric antigen receptor (CAR) comprises CAR-T cells.

[0062] 31. A cell culture medium for preparing a cell colony expressing a chimeric antigen receptor (CAR), comprising a CD3 / TCR complex agonist and an immune cell activating cytokine, wherein the immune cell activating cytokine does not comprise IL-2. In some embodiments, the CD3 antibody comprises a domain that specifically binds to a CD3ε chain. In some embodiments, the CD3 antibody is a humanized or human IgG antibody.

[0063] 32. A cell culture medium according to claim 31, wherein the CD3 / TCR complex agonist comprises a CD3 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1A The CAR expression positivity rate of CAR-T cells prepared by different preparation processes is shown.

[0065] Figure 1B Shows the viability detection of CAR-T cells after cryopreservation and recovery using different preparation processes.

[0066] Figure 1C It shows that when the effector-target ratio is 1:2, CAR-T cells prepared with different schemes can accept multiple rounds of stimulation by target cells.

[0067] Figure 1D The CAR-T cell proliferation potential after multiple rounds of stimulation of CAR-T cells prepared with different schemes at an effector-target ratio of 1:2.

[0068] Figure 2A Showing CAR-T activation detection at different activation times.

[0069] Figure 2B CAR expression detection at different activation times is shown.

[0070] Figure 2C Shown are the CAR expression detection at different times of cell harvest after viral transduction.

[0071] Figure 3A Showing the in vivo persistence detection of CAR-T cells using two preparation processes.

[0072] Figure 3BThe in vivo effectiveness of CD19 CAR-T cells prepared by two preparation processes was demonstrated.

[0073] Figure 3C The in vivo effectiveness of CD19 / CD22 CAR-T and CEA CAR-T cells prepared by two processes was demonstrated.

[0074] Figure 4A Shows the viability of CAR-T cells prepared with different stimulation and activation schemes.

[0075] Figure 4B Show the phenotype of CAR-T cells after stimulation with tumor cells prepared with different stimulation and activation schemes.

[0076] Figure 4C Show the in vivo effectiveness verification of CAR-T prepared with different stimulation and activation schemes.

[0077] Figure 4D Show the in vivo safety validation of CAR-T prepared with different stimulation and activation schemes.

[0078] Figure 5 Shows the detection of CAR expression in CAR-T cells under different factor combination conditions.

[0079] Fig. 6A Shows the activity detection of CAR-T cells after cryopreservation and resuscitation under different cytokine combination conditions.

[0080] Figure 6B The CAR-T cell CAR positivity rate is shown under different cytokine combination conditions.

[0081] Fig. 7A Show the in vivo effectiveness verification of CAR-T cells under different cytokine combination conditions.

[0082] Figure 7B Shows the effectiveness results of CD19 / CD22-targeting CAR-T cells prepared by different processes.

[0083] Figure 7C The results of IL-2 secretion by different processes are shown.

[0084] Fig.7D The results of IFN-γ secretion by different processes are shown.

[0085] Fig. 7E The results of TNF-α secretion by different processes are shown.

[0086] Figure 7F The results show the effectiveness of CD19-targeting CAR-T cells prepared using different processes.

[0087] Figure 7GThe cytokine secretion of CD19-targeted CAR-T cells prepared by different processes after killing tumor cells is shown.

[0088] Fig. 8A The percentage of cell activation under different CD3 antibody concentration schemes is shown.

[0089] Figure 8B The CAR positive rate is shown at different CD3 antibody concentrations.

[0090] Figure 8C Showing multiple rounds of stimulation of CAR-T cells prepared under different CD3 antibody concentration schemes when the effector-target ratio was 1:1.

[0091] Fig.8D The results show the effectiveness of CAR-T prepared by P-CAR culture scheme with different CD3 antibody concentrations.

[0092] Fig. 9A The CAR positive rate is shown at different IL7 / IL-21 factor concentrations.

[0093] Fig. 9B Showing multiple rounds of stimulation of CAR-T cells with different factor concentrations at an effector-target ratio of 1:1.

[0094] Fig. 9C Shows the comparison of the killing effects of different short-time preparation schemes on tumor cells.

[0095] Fig.9D The results show the comparison of IL-2 cytokine secretion ability of CAR-T cells prepared by different short-term preparation schemes after tumor cell activation.

[0096] Fig. 10A The killing effect of IL-2 alone, a single factor component of the P-CAR regimen, was verified.

[0097] Fig. 10B The killing effect of IL-7 alone, a factor component of the P-CAR regimen, was verified.

[0098] Fig. 10C The killing effect of IL-21 alone, a factor component of the P-CAR regimen, was verified.

[0099] Fig.11A IL-2 secretion validation showing that the P-CAR regimen factor component is IL-2 alone.

[0100] Fig. 11B IL-2 secretion validation showing that the P-CAR regimen factor component is IL-7 alone

[0101] Fig. 11C Validation of IL-2 secretion showing that the P-CAR regimen factor component is IL-21 alone. DETAILED DESCRIPTION OF THE INVENTION

[0103] Interleukin-2 (IL-2) was first reported in 1976 and has been considered a key molecule for promoting T cell proliferation and differentiation for decades, so it is also called T cell growth factor. However, in recent years, researchers have increasingly found that cell products cultured with IL-2 may have stronger in vivo toxicity and can induce CD8+T cell exhaustion.

[0104] The present application provides a method for preparing engineered immune cells, which does not use CD28 agonists and is safer. Further, the method of the present application can be used without IL-2, and the zero use of IL-2 is successfully achieved by selecting cell stimulating factors. In addition, the preparation method has the characteristics of rapid preparation, that is, the engineered immune cells, such as T cells, can be obtained within 1-4 days. In some embodiments, the method also uses a combination of special cytokines for cell culture during the preparation process. In some embodiments, the method further improves the tumoricidal effect and in vivo safety of the cell population by optimizing the time of each preparation step. In addition, the present application also provides compositions, culture media and kits that can be used for the method.

[0105] definition

[0106] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this application belongs. The following references provide general definitions of many terms used in this application for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al (ed.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them.

[0107] As used herein, "CD3 / TCR complex agonist" includes any antibody, ligand or any small molecule compound that can bind to any one or more subunits of CD3 or TCR, as long as its binding to the CD3 / TCR complex can phosphorylate the tyrosine residues in the ITAM (immunoreceptor tyrosine activation motif) of CD3, thereby causing downstream pathway activation, which is included in the scope of the term "CD3 / TCR complex agonist". In some embodiments, the "CD3 / TCR complex agonist" binds to any one or more chains of CD3. In some embodiments, the "CD3 / TCR complex agonist" binds to one, two or more of the ε chain, γ chain and δ chain of CD3. In some embodiments, the "CD3 / TCR complex agonist" is selected from: one or more of an antibody, a small molecule, a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the "CD3 / TCR complex agonist" is a monoclonal antibody or polyclonal antibody against CD3. In some embodiments, the "CD3 / TCR complex agonist" is a CD3 antibody, i.e., a monospecific antibody or a multispecific antibody against CD3. In some embodiments, the monospecific antibody or the multispecific antibody comprises an antigen binding fragment that specifically binds to the ε chain, the γ chain and / or the δ chain of CD3. In some embodiments, the CD3 antibody is an antibody against human CD3. In some embodiments, the CD3 antibody is a humanized anti-human CD3 antibody. In some embodiments, the CD3 antibody is a humanized anti-human CD3 ε chain antibody.

[0108] As used herein, the term "antibody" has its broadest meaning, specifically including full-length monoclonal antibodies, polyclonal antibodies, and, unless otherwise specified or contradictory to the context, antigen-binding fragments, antibody variants and multispecific molecules thereof, as long as they exhibit the desired biological activity (i.e., the activity of binding to its antigen or its ligand). In general, a "full-length antibody" is a glycoprotein comprising at least two heavy chains and two light chains, interconnected by disulfide bonds. The term antibody refers to an immunoglobulin molecule and an immunologically active fragment of an immunoglobulin molecule, i.e., a molecule containing an antigen binding site. Immunoglobulin molecules can be of any type (such as IgG, IgE, IgM, IgD, IgA and IgY), category (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Therefore, an "antibody" can also be a single variable domain (VHH) on a heavy chain antibody, also known as a single heavy chain antibody (HcAb), which has no light chain and can be naturally produced by camels or sharks. The antigen-binding portion of HcAb consists of a VHH fragment. As used herein, the term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e.g., CD3), including the antigen-binding site or variable region of the intact antibody, and in some cases, excluding the constant heavy chain domains (i.e., CH2, CH3, and / or CH4, depending on the isotype of the antibody) of the Fc region of the intact antibody. Examples of antibody fragments include antibody fragments such as Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; polypeptides consisting of an uninterrupted sequence of continuous amino acid residues in an antibody; and diabodies, etc. The term "antigen-binding fragment" includes, but is not limited to, (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing three CDRs of the light chain variable domain, without a heavy chain molecule, and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing three CDRs of the heavy chain variable region, without a light chain molecule; and multispecific or multivalent structures formed by antibody fragments.

[0109] As used herein, "cell surface co-stimulatory molecule agonist" is a compound or composition that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the "cell surface co-stimulatory molecule agonist" is a CD28 agonist. In some embodiments, the "cell surface co-stimulatory molecule agonist" includes any antibody, ligand or any small molecule compound that can bind to CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and / or CD226, as long as its binding to CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and / or CD226 can produce a co-stimulatory signal of the CD3 / TCR complex, which is included in the scope covered by the term "cell surface co-stimulatory molecule agonist". Similarly, "CD28 agonist" refers to any antibody, ligand or any small molecule compound that can bind to CD28 and produce a co-stimulatory signal of the CD3 / TCR complex by binding to CD28.

[0110] As used herein, "cytokines" can be used to refer to any small molecule protein with a wide range of biological activities synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation. In some embodiments, cytokines regulate cell growth, differentiation and effects by binding to corresponding receptors to regulate immune responses. In some embodiments, cytokines include low molecular weight soluble proteins produced by cells induced by immunogens, mitogens or other stimulants. In some embodiments, cytokines have functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC multipotent cells, and damaged tissue repair. In some embodiments, cytokines cover interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. In some specific embodiments, the "cytokines" of the present application include cytokines required for T cell in vitro culture or maintenance of a good state. As used herein, "immune cell activating cytokine" refers to a cytokine that plays a positive role in the proliferation, differentiation, growth, survival, and maintenance of effector cell (eg, T cell or B cell) responses of immune cells.

[0111] As used herein, "culture medium" refers to any liquid, solid or semi-solid medium containing essential nutrients (including inorganic and organic substances) that can provide cells with in vitro survival. In some embodiments, the culture medium can provide culture conditions that allow cells to survive under serum-free conditions. In some embodiments, the serum-free culture medium is an OpTmizer TM CTSTM (LifeTech), Immunocult TM XF (Stemcelltechnologies), CellGro TM(CellGenix), TexMacsTM (Miltenyi), StemlineTM (Sigma), Xvivo15TM (Lonza), PrimeXV (Irvine Scientific), StemXVivo or (RandD Group). Serum-free culture medium can be supplemented with serum replacement, such as ICSR (immune cell serum replacement) from LifeTech, USA. The addition ratio of serum replacement (such as ICSR) can be, for example, up to 5%, such as about 1%, 2%, 3%, 4%, or 5%. In some embodiments, serum-free culture medium can be supplemented with serum, such as human serum, such as human AB serum. In some embodiments, the serum is human serum that is allowed to coagulate naturally after collection, for example, non-clotting (OTC) serum. In some embodiments, the serum is plasma-derived human serum. Plasma-derived serum can be produced by defibrinating mixed human plasma collected in the presence of an anticoagulant (such as sodium citrate). In some embodiments, the culture medium is a culture medium suitable for T cell culture (e.g., Minimal Essential Media, α-MEM, RPMI 1640 culture medium, AIM-V, DMEM, F-12, or X-vivo15 (Lonza), X-Vivo 20, OpTmizer, and IMDM), and the culture medium may contain factors necessary for cell proliferation and viability. In some embodiments, the culture medium comprises serum (e.g., fetal bovine or human serum), insulin, IFNγ, IL-7, IL-12, IL-15, and TNFα or any other additive known to those skilled in the art for cell growth. And unless otherwise specified, the culture medium and serum of the present application explicitly do not contain interleukin-2 (IL-2). Other additives for cell growth include, but are not limited to, surfactants, human plasma protein products, and reducing agents (e.g., N-acetyl-cysteine ​​and 2-mercaptoethanol). The culture medium may include, but is not limited to, RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, XVivo20, OpTmizer, and IMDM, wherein amino acids, sodium pyruvate, and vitamins are added, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or a certain amount of cytokines sufficient to grow and expand T cells. In some embodiments, the "culture medium" of the present application may further include antibiotics (e.g., penicillin and streptomycin), which are only included in the experimental culture and not in the cell culture to be injected into the subject.

[0112] As used herein, the term "cell colony" is a composition comprising one or more cells. In some embodiments, each cell in the "cell colony" has one or more identical features, such as having the same nuclear typing, having similar functions, having a volume size in a certain same range, having one or more identical cell markers (i.e., the one or more identical cell markers are positive), or not having one or more specific cell markers (i.e., the one or more specific cell markers are negative). Wherein the "not having" or "negative" may not necessarily mean the absolute absence of the marker. Those skilled in the art can easily compare cells with positive and / or negative controls, and / or set a predetermined threshold, and when cells have an expression level lower than a predetermined threshold or have an expression level lower than a predetermined threshold using a conventional detection method (e.g., using flow cytometry), then cells are classified as "not having" or negative for markers. For example, in some embodiments, the cell colony is a T cell colony. In some embodiments, the cell colony is PBMC.

[0113] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of engineered polypeptides or proteins that, when in an immune effector cell, bind to a specific antigen contained on a target cell, and generate an intracellular signal after recognizing the specific antigen, activating the downstream pathway of the cell where the receptor is located, so as to start the killing of the target cell by the immune effector cell. The immune effector cells include but are not limited to NK cells, macrophages, neutrophils, T cells, etc. CAR generally includes at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen binding domain can specifically recognize antigens, and non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that engage their cognate receptors, artificially designed target-specific recognition domains that recognize specific targets, such as fibronectin type III (FN3) domain combinations, target-specific anchor protein repeat proteins (designed ankyrin repeat proteins, DARPins) with recognition of specific targets. In some embodiments, the extracellular antigen binding region may comprise scFv, Fab or natural ligand, and any derivatives thereof. The extracellular antigen binding region may refer to a molecule other than a complete antibody, which may comprise a portion of a complete antibody and may bind to an antigen bound by the complete antibody. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv), ScFv may be ScFv of a mouse antibody or a fully human antibody or a human-mouse chimeric antibody, or may be a single domain antibody such as a shark, alpaca or camel antibody; and multispecific antibodies formed by antibody fragments. The "signal transduction domain" generally comprises an immune-receptor tyrosine-based activation motif (ITAM), the basic composition of which is: YXXL / V. Wherein Y is tyrosine, L / V refers to leucine or valine, and X may be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA connected to it can be phosphorylated under the action of a class of protein tyrosine kinases PTK connected to the cell membrane, thereby recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell. In some embodiments, the "signal transduction domain" uses the intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ. As used herein, the "co-stimulatory domain" is also called the "co-stimulatory signal domain", which is mainly used to provide co-stimulatory signals to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and / or development of memory cells.In some embodiments, the "costimulatory domain" is selected from the intracellular domain of CD28, 4-1BB (CD137), OX40 (CD134), etc. As used herein, the "transmembrane domain", also known as the "transmembrane region", refers to a protein structure region anchored in the cell membrane with thermodynamic stability. The transmembrane domain can be obtained from a natural protein, such as a transmembrane domain derived from a T cell receptor (TCR). In some embodiments, the transmembrane domain is selected from the transmembrane domain of CD4, CD8α, CD28 and CD3ζ.

[0114] In some embodiments, the extracellular antigen binding region of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumors and hematological tumor cells / tissues, and the target molecules include but are not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1) or TAG-72, etc.

[0115] In some embodiments, the structure of "chimeric antigen receptor" or "CAR" comprises: an antigen recognition domain (such as ScFv), a hinge structure (such as 8hinge derived from human CD8), a transmembrane structure (such as CD8TM derived from human CD8 transmembrane) and an intracellular signaling domain, wherein the intracellular signaling domain can be a primary intracellular signaling domain containing only an immunoreceptor tyrosine-based activation motif or ITAM: Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10 and DAP12, etc., and can also include, in addition to the primary intracellular signaling domain, one or two or more costimulatory signaling domains, and the costimulatory signaling domains can be selected from the following Any one or more of the following molecules and functional variants derived therefrom: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc. The derived functional molecule is an amino acid or nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with the above-mentioned molecule.

[0116] "Chimeric antigen receptor" or "CAR" can have a variety of structures, such as containing cytokines and antibody gene sequences that can be secreted or expressed in the membrane; such as containing structures that can be regulated for activation or inactivation, the regulated activation or inactivation structures include: suicide switch such as inducible caspase-9 (iCasp9), thymidine kinase in herpes simplex virus (HSV-TK) and suicide epitope, truncated EGFR (EGFRt), Fas-FasL apoptosis structure; inducible CAR (Inducible CAR) structure: peptide new epitope (PNE), fluorescein (FITC), 10 amino acids (5B9-tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv connected to antibody, streptavidin 2 (mSA2) biotin-binding domain, leucine ZipFv, VIPER CAR induction structure, biotin-targeting immune receptor (biotin-bidingimmune receptor, BBIR) system; "logic gate" regulatory system combined with SynNotch receptor, etc.

[0117] As used herein, the term "C-CAR" means a preparation process in which the entire CAR-T cell preparation cycle is greater than or equal to 7 days, starting from T cell activation, and a CAR-T product prepared using the preparation process.

[0118] As used herein, the term "P-CAR" refers to the CAR-T product prepared using the rapid preparation process described in this application and the process.

[0119] In some embodiments, the structure described in "chimeric antigen receptor" or "CAR" may also include a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain. The extracellular antigen recognition domain of the chimeric fusion protein can be a complete and continuous extracellular segment of the cell membrane of the expressed molecule / polypeptide, or it can be a fusion form of the expressed molecule and other polypeptides such as polypeptides derived from human CD8 and CD4.

[0120] The artificial / engineered TCR receptor described in this article refers to a specific receptor on the surface of a natural or engineered T cell, which mainly relies on affinity-optimized or purely natural TCRs to recognize antigens presented by tumor MHC molecules and transmit stimulation signals to the cell through the TCR-CD3 complex.

[0121] Methods for preparing engineered immune cells

[0122] One aspect of the present application relates to a method for preparing engineered immune cells, the method comprising:

[0123] Step (i) contacting the cell population with a CD3 / TCR complex agonist;

[0124] step (ii) transducing the nucleic acid molecule into the cell population, thereby providing cells comprising the nucleic acid molecule, and

[0125] Step (iii) culturing the cell population for a period of time and harvesting the cell population, where the cell population is the engineered immune cell.

[0126] As used herein, "engineered immune cells" refer to immune cells that have been engineered, such as those into which exogenous nucleic acids or proteins have been introduced or inserted. Among them, immune cells are a general term for cells that participate in the body's immune response, including but not limited to granulocytes, macrophages, monocytes, dendritic (DC) cells, natural killer (NK) cells, T lymphocytes (or T cells), B lymphocytes (or B cells), plasma cells, etc. In this application, unless otherwise specified, the term "immune cells" also includes precursor cells of the above-mentioned immune cells, such as their progenitor cells or stem cells, such as lymphoblasts, lymphoid stem cells, etc.

[0127] In some embodiments, "cell colony" in the present application is a cell colony comprising immune cells and / or precursor immune cells. In some embodiments, the cell colony is from human peripheral blood. In some embodiments, the cell colony comprises T cells. In some embodiments, the T cells are derived from PBMC. In some embodiments, the cell colony is peripheral blood mononuclear cells (PBMC). In some embodiments, the cell colony is a T cell colony, i.e., a cell colony composed of a single or multiple subtype T cells.

[0128] In some embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid molecule encodes a T cell receptor (TCR). In some embodiments, the cell colony comprises T cells, the nucleic acid molecule encodes CAR, and the engineered immune cell is a CAR-T cell. In some embodiments, the cell colony is PBMC, the nucleic acid molecule encodes CAR, and the engineered immune cell is a CAR-T cell. In some embodiments, the cell colony comprises T cells, the nucleic acid molecule encodes TCR, and the engineered immune cell is a TCR-T cell. In some embodiments, the cell colony is PBMC, the nucleic acid molecule encodes TCR, and the engineered immune cell is a CAR-T cell.

[0129] In some embodiments, the cell population is contacted with an immune cell activating cytokine in step i), step ii), and / or step iii), or in the entire preparation process, wherein the immune cell activating cytokine does not include IL-2. The method of the present application can achieve the effect of not using IL-2 in the process of preparing the engineered immune cells, but those skilled in the art should know that when those skilled in the art use the method of the present application to prepare engineered immune cells, adding IL-2 at a certain stage of the preparation process can still prepare the engineered immune cells, but it is still included in the scope of the technical solution of the present application. In some embodiments, the immune cell activating cytokine includes one or more selected from the following: IL-7, IL-15, IL-21, IL-12, IL-18 and IL-6. In some embodiments, the immune cell activating cytokine includes: a combination of IL-7 and IL-21. In some embodiments, the immune cell activating cytokine is a combination of IL-7 and IL-21 without other immune activating cytokines. In some embodiments, when the cell population is contacted with the immune cell activating cytokine, the concentration of IL-7 is 2.5-10 ng / ml, for example, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.1 ng / ml, 5.2 ng / ml, 5.3 ng / ml, 5.4 ng / ml, 5.5 ng / ml, 5.6 ng / ml, 5.7 ng / ml, 5.8 ng / ml, 5.9 ng / ml, 6 ng / ml,

[0130] In some embodiments, the initial concentration of IL-7 when contacting the cell population is 2.5-10 ng / ml, such as 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.1 ng / ml, 5.2 ng / ml, 5.3 ng / ml, 5.4 ng / ml, 5.5 ng / ml, 5.6 ng / ml, 5.7 ng / ml, 5.8 ng / ml, 5.9 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml,

[0131] 8ng / ml, 8.5ng / ml, 9ng / ml or 9.5ng / ml, or any value in the range of 2.5 to 10ng / ml, and the concentration may gradually change, for example, decrease, over the contact time. In some embodiments, when the cell population is contacted with the immune cell activating cytokine, the concentration of IL21 is 12.5 to 50ng / ml, for example, 13ng / ml, 13.5ng / ml, 14ng / ml, 14.5ng / ml, 15ng / ml, 15.5ng / ml, 16ng / ml, 16.5ng / ml, 17ng / ml, 17.5ng / ml, 18ng / ml, 18.5ng / ml, 19ng / ml, 19.5ng / ml, 20ng / ml, 20.5ng / ml, 21ng / ml, 21.5ng / ml, 22ng / ml, 22.5ng / ml, 23ng / ml, 23.5ng / ml, 24ng / ml, 24.5ng / ml, 25ng / ml, 25.5ng / ml, 26ng / ml, 26.5ng / ml, 27ng / ml, 27.5ng / ml, 28ng / ml, 28.5ng / ml, 29ng / ml, 29.5ng / ml, 30ng / ml, 30 .5ng / ml, 31ng / ml, 31.5ng / ml, 32ng / ml, 32.5ng / ml, 33ng / ml, 33.5ng / ml, 34ng / ml, 34.5ng / ml, 35ng / ml, 35.5 ng / ml, 36ng / ml, 36.5ng / ml, 37ng / ml, 37.5ng / ml, 38ng / ml, 38.5ng / ml, 39ng / ml, 39.5ng / ml, 40ng / ml, 40.5ng / ml, 41ng / ml, 41.5ng / ml, 42ng / ml, 42.5ng / ml, 43ng / ml, 43.5ng / ml, 44ng / ml, 44.5ng / ml, 45ng / ml, 45.5ng / ml, 46ng / ml, 46.5ng / ml, 47ng / ml, 47.5ng / ml, 48ng / ml, 48.5ng / ml, 49ng / ml, or 49.5ng / ml, or any value between 12.5 and 50ng / ml.In some embodiments, the initial concentration of IL21 contacted with the cell population is 12.5-50 ng / ml, for example, 13 ng / ml, 13.5 ng / ml, 14 ng / ml, 14.5 ng / ml, 15 ng / ml, 15.5 ng / ml, 16 ng / ml, 16.5 ng / ml, 17 ng / ml, 17.5 ng / ml, 18 ng / ml, 18.5 ng / ml, 19 ng / ml, 19.5 ng / ml, 20 ng / ml, 20.5 ng / ml. , 21ng / ml, 21.5ng / ml, 22ng / ml, 22.5ng / ml, 23ng / ml, 23.5ng / ml, 24ng / ml, 24.5ng / ml, 25ng / ml, 25.5ng / ml, 26n g / ml, 26.5ng / ml, 27ng / ml, 27.5ng / ml, 28ng / ml, 28.5ng / ml, 29ng / ml, 29.5ng / ml, 30ng / ml, 30.5ng / ml, 31ng / ml, 31.5ng / ml, 32ng / ml, 32.5ng / ml, 33ng / ml, 33.5ng / ml, 34ng / ml, 34.5ng / ml, 35ng / ml, 35.5ng / ml, 36ng / ml, 36.5 ng / ml, 37ng / ml, 37.5ng / ml, 38ng / ml, 38.5ng / ml, 39ng / ml, 39.5ng / ml, 40ng / ml, 40.5ng / ml, 41ng / ml, 41.5ng / ml , 42ng / ml, 42.5ng / ml, 43ng / ml, 43.5ng / ml, 44ng / ml, 44.5ng / ml, 45ng / ml, 45.5ng / ml, 46ng / ml, 46.5ng / ml, 47ng / ml, 47.5ng / ml, 48ng / ml, 48.5ng / ml, 49ng / ml, or 49.5ng / ml, or any value between 12.5 and 50ng / ml. The concentration may gradually change, for example, decrease, with contact time.

[0132] In some embodiments, the cell population is contacted with an immune cell activating cytokine in step i), step ii), and / or step iii), or during the entire preparation process, wherein the immune cell activating cytokine comprises IL-2. In some embodiments, the IL-2 concentration is 100 IU / ml-500 IU / ml, 150 IU / ml-450 IU / ml, 200 IU / ml-400 IU / ml, or 250 IU / ml-350 IU / ml.

[0133] In some embodiments, the cell colony is not contacted with CD28 agonists in step i), step i) and ii), step i), ii) and iii) or the whole preparation process.In some embodiments, the cell colony is not contacted with cell surface costimulatory molecule agonists in step i), step i) and ii), step i), ii) and ii) or the whole preparation process, and the costimulatory molecule includes: CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and CD226.

[0134] As used herein, the term "preparation process" refers to the process of preparing the engineered immune cells using the method for preparing engineered immune cells, including all steps of the method for preparing engineered immune cells, and other necessary steps for preparing engineered immune cells. The steps of preparing engineered immune cells, such as CAR-T or TCR-T, are known in the art, such as those described in CN106459917B, WO2003095663A3 and US7446190B2. In some embodiments, "the whole preparation process" refers to the whole process of harvesting the cell colony from the moment when the cell colony is contacted with the CD3 / TCR complex agonist to step iii). In some embodiments, "the whole preparation process" refers to the whole process of harvesting the cell colony from an organism (e.g., a mammal, such as a human being) separated from the cell colony to step iii).

[0135] In some embodiments, the CD3 / TCR complex agonist comprises a CD3 antibody. The CD3 antibody can be coated on beads, such as magnetic beads, or dispersed in a liquid. In some embodiments, the concentration of the CD3 antibody contacted with the cell population in step i) is ≥25 ng / ml, such as 25 to 200 ng / ml or any value therein, such as 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml, 34 ng / ml, 35 ng / ml, 36 ng / ml, 37 ng / ml, 38 ng / ml, 39 ng / ml, 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47ng / ml, 48ng / ml, 49ng / ml, 50ng / ml, 51ng / ml, 52ng / ml, 53ng / ml, 54ng / ml, 55ng / ml, 56ng / ml, 57ng / ml, 58ng / ml, 59ng / ml, 60ng / ml, 61ng / ml, 62ng / ml, 63ng / ml, 64ng / ml, 65ng / ml, 66ng / ml, 67ng / ml, 68ng / ml, 69ng / ml, 70ng / ml, 71ng / ml, 72ng / ml, 73ng / ml, 74ng / ml, 75ng / ml, 76 ng / ml, 77ng / ml, 78ng / ml, 79ng / ml, 80ng / ml, 81ng / ml, 82ng / ml, 83ng / ml, 84ng / ml, 85ng / ml, 86ng / ml, 87ng / ml, 88ng / ml, 89ng / ml, 90ng / ml , 91ng / ml, 92ng / ml, 93ng / ml, 94ng / ml, 95ng / ml, 96ng / ml, 97ng / ml, 98ng / ml, 99ng / ml, 100ng / ml, 101ng / ml, 102ng / ml, 103ng / ml, 104ng / ml ,105ng / ml, 106ng / ml, 107ng / ml, 108ng / ml, 109ng / ml, 110ng / ml, 111ng / ml, 112ng / ml, 113ng / ml, 114ng / ml, 115ng / ml, 116ng / ml, 117ng / ml ,118ng / ml, 119ng / ml, 120ng / ml, 121ng / ml, 122ng / ml, 123ng / ml, 124ng / ml, 125ng / ml, 126ng / ml, 127ng / ml, 128ng / ml, 129ng / ml, 130ng / ml,131ng / ml, 132ng / ml, 133ng / ml, 134ng / ml, 135ng / ml, 136ng / ml, 137ng / ml, 138ng / ml, 139ng / ml, 140ng / ml, 141ng / ml, 142ng / ml, 143ng / ml, 144ng / ml, 145ng / ml, 146ng / ml, 147ng / ml, 14 8ng / ml, 149ng / ml, 150ng / ml, 151ng / ml, 152ng / ml, 153ng / ml, 154ng / ml, 155ng / ml, 156ng / ml ,157ng / ml, 158ng / ml, 159ng / ml, 160ng / ml, 161ng / ml, 162ng / ml, 163ng / ml, 164ng / ml, 165ng / ml, 166ng / ml, 167ng / ml, 168ng / ml, 169ng / ml, 170ng / ml, 171ng / ml, 172ng / ml, 173ng / ml, 1 74ng / ml, 175ng / ml, 176ng / ml, 177ng / ml, 178ng / ml, 179ng / ml, 180ng / ml, 181ng / ml, 182ng / m l, 183ng / ml, 184ng / ml, 185ng / ml, 186ng / ml, 187ng / ml, 188ng / ml, 189ng / ml, 190ng / ml, 191n g / ml, 192ng / ml, 193ng / ml, 194ng / ml, 195ng / ml, 196ng / ml, 197ng / ml, 198ng / ml, or 199ng / ml. In some embodiments, the initial concentration of the CD3 antibody in contact with the cell population is ≥25 ng / ml, such as 25-200 ng / ml or any value therein, such as 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml, 34 ng / ml, 35 ng / ml, 36 ng / ml, 37 ng / ml, 38 ng / ml, 39 ng / ml, 40 ng / ml, 41 ng / ml. ml, 42ng / ml, 43ng / ml, 44ng / ml, 45ng / ml, 46ng / ml, 47ng / ml, 48ng / ml, 49ng / ml, 50ng / ml, 51ng / ml, 52ng / ml, 53 ng / ml, 54ng / ml, 55ng / ml, 56ng / ml, 57ng / ml, 58ng / ml, 59ng / ml, 60ng / ml, 61ng / ml, 62ng / ml, 63ng / ml, 64ng / ml,65ng / ml、66ng / ml、67ng / ml、68ng / ml、69ng / ml、70ng / ml、71ng / ml、72ng / ml、73ng / ml、74ng / ml、75ng / ml、76ng / ml、77ng / ml、78ng / ml、79ng / ml、80ng / ml、81ng / ml、82ng / ml、83ng / ml、84ng / ml、85ng / ml、86ng / ml、87ng / ml、88ng / ml、89ng / ml、90ng / ml、91ng / ml、92ng / ml、93ng / ml、94ng / ml、95ng / ml、96ng / ml、97ng / ml、98ng / ml、99ng / ml、100ng / ml、101ng / ml、102ng / ml、103ng / ml、104ng / ml、105ng / ml、106ng / ml、107ng / ml、108ng / ml、109ng / ml、110ng / ml、111ng / ml、112ng / ml、113ng / ml、114ng / ml、115ng / ml、116ng / ml、117ng / ml、118ng / ml、119ng / ml、120ng / ml、121ng / ml、122ng / ml、123ng / ml、124ng / ml、125ng / ml、126ng / ml、127ng / ml、128ng / ml、129ng / ml、130ng / ml、131ng / ml、132ng / ml、133ng / ml、134ng / ml、135ng / ml、136ng / ml、137ng / ml、138ng / ml、139ng / ml、140ng / ml、141ng / ml、142ng / ml、143ng / ml、144ng / ml、145ng / ml、146ng / ml、147ng / ml、148ng / ml、149ng / ml、150ng / ml、151ng / ml、152ng / ml、153ng / ml、154ng / ml、155ng / ml、156ng / ml、157ng / ml、158ng / ml、159ng / ml、160ng / ml、161ng / ml、162ng / ml、163ng / ml、164ng / ml、165ng / ml、166ng / ml、167ng / ml、168ng / ml、169ng / ml、170ng / ml、171ng / ml、172ng / ml、173ng / ml、174ng / ml、175ng / ml、176ng / ml、177ng / ml、178ng / ml、179ng / ml、180ng / ml, 181ng / ml, 182ng / ml, 183ng / ml, 184ng / ml, 185ng / ml, 186ng / ml, 187ng / ml, 188ng / ml, 189ng / ml, 190ng / ml, 191ng / ml, 192ng / ml, 193ng / ml, 194ng / ml, 195ng / ml, 196ng / ml, 197ng / ml, 198ng / ml, or 199ng / ml, and the concentration may gradually change, for example, decrease, over the contact time.

[0136] As mentioned above, all references to the "initial concentration of a protein in contact with the cell population" or "concentration in contact with the cell population" refer to the environment in which the cell population is located, such as the concentration of the protein in the culture medium in which the cell population is located, such as the protein concentration of anti-CD3 antibodies, IL21, or IL7. The "concentration of the protein in the culture medium" refers to the ratio of the mass of the protein contained in the culture medium to the volume of the culture medium. Due to reasons such as evaporation of the culture medium or protein degradation, the concentration may change as the culture time increases. As long as it remains within the above concentration range or can maintain the above concentration for a period of time, it still falls within the scope of this application.

[0137] The method for preparing engineered immune cells of the present application can make the preparation of the engineered immune cells complete in a short time. For example, in some embodiments, the time from step i) to harvesting the cell colony is only 1 to 4 days. In some embodiments, the time from step i) to harvesting the cell colony is only 1 to 2 days. That is, the method for preparing engineered immune cells of the present application can make the preparation of the engineered immune cells complete within 4 days, or even within 1-2 days. This time range should not be understood as a limitation of the present application, because those skilled in the art know that if the motif is cultured to the cell colony, for example, the time of steps i), ii) and / or iii) is changed, so that the time from step i) to harvesting the cell colony exceeds 4 days, the engineered immune cells can still be obtained. Therefore, the optional time from step i) to harvesting the cell colony is still included in the scope of the present application. The time of steps i), ii) and iii) provided in the present application and the time from step i) to harvesting the cell colony are only one, which is an optional or preferred scheme in terms of time, economic cost or cell colony quality. In some embodiments, steps i), ii) and / or iii) may overlap partially or completely, or may not overlap at all. For example, in some embodiments, the CD3 / TCR complex agonist in step i) may be present in the entire preparation process starting from step i), and the contact between the CD3 / TCR complex agonist and the cell population may be stopped at any time period in the entire preparation process (e.g., before, at or after the start of step ii). In some embodiments, step ii) may start before, after or at the same time as step i). In some embodiments, step ii) starts earlier than step i). That is, regardless of whether step (i) and step (ii) overlap in time, or the order of step (i) and step (ii), they are all covered within the scope of the present application.

[0138] In some embodiments, the duration of step i) is 12 to 24 hours, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. In some embodiments, the duration of step i) is greater than 12 to 24 hours, such as 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, or 23.5 hours.

[0139] In some embodiments, the time from step ii) to harvesting the cell population is ≤72 hours or ≤48 hours. In some embodiments, the time from step ii) to harvesting the cell population is ≥6 hours. In some embodiments, the time from step ii) to harvesting the cell population is ≥6 hours and ≤72 hours, such as 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours. ii). Harvesting the cell population at any time between 6 hours and 72 hours from the start of step ii) is within the scope of the present application.

[0140] In some embodiments, the time from step i) to the start of step ii) is 12 to 24 hours, and the time from step ii) to harvesting the cell population is 14-28 hours; that is, step i) starts before step ii) and continues until step ii) starts or the inhibition continues until a time after the start of step ii), and the time from step i) to the start of step ii) is 12 to 24 hours, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, and the time from step ii) to harvesting the cell population is 14-28 hours, such as 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours or 27 hours. In some embodiments, the time from step i) to the start of step ii) is 12 to 24 hours, and the time from the start of step ii) to the harvesting of the cell population is 14-28 hours, and the time from the start of step i) to the harvesting of the cell population is 1 to 4 days, such as 2 days, 3 days.

[0141] Composition

[0142] In a second aspect of the present application, a composition for preparing an engineered immune cell is provided. In some embodiments, the engineered immune cell is a T cell or a precursor cell thereof. In some embodiments, the engineered immune cell is a CAR-T cell or a TCR-T cell.

[0143] The composition comprises a CD3 / TCR complex agonist and an immune cell activating cytokine, wherein the immune cell activating cytokine does not comprise or comprises IL-2. In some embodiments, the CD3 / TCR complex agonist comprises a CD3 antibody. In some embodiments, the concentration of the CD3 antibody is ≥25 ng / ml, for example, 25 to 200 ng / ml or any value therein, for example, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml, 34 ng / ml, 35 ng / ml, 36 ng / ml, 37 ng / ml, 38 ng / ml, 39 ng / ml, 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml ml, 48ng / ml, 49ng / ml, 50ng / ml, 51ng / ml, 52ng / ml, 53ng / ml, 54ng / ml, 55ng / ml, 56ng / ml, 57ng / ml, 58ng / ml, 59ng / ml, 60ng / ml, 61ng / ml l, 62ng / ml, 63ng / ml, 64ng / ml, 65ng / ml, 66ng / ml, 67ng / ml, 68ng / ml, 69ng / ml, 70ng / ml, 71ng / ml, 72ng / ml, 73ng / ml, 74ng / ml, 75ng / ml, 76ng / ml, 77ng / ml, 78ng / ml, 79ng / ml, 80ng / ml, 81ng / ml, 82ng / ml, 83ng / ml, 84ng / ml, 85ng / ml, 86ng / ml, 87ng / ml, 88ng / ml, 89ng / ml, 9 0ng / ml, 91ng / ml, 92ng / ml, 93ng / ml, 94ng / ml, 95ng / ml, 96ng / ml, 97ng / ml, 98ng / ml, 99ng / ml, 100ng / ml, 101ng / ml, 102ng / ml, 103ng / m l, 104ng / ml, 105ng / ml, 106ng / ml, 107ng / ml, 108ng / ml, 109ng / ml, 110ng / ml, 111ng / ml, 112ng / ml, 113ng / ml, 114ng / ml, 115ng / ml, 116 ng / ml, 117ng / ml, 118ng / ml, 119ng / ml, 120ng / ml, 121ng / ml, 122ng / ml, 123ng / ml, 124ng / ml, 125ng / ml, 126ng / ml, 127ng / ml, 128ng / ml,129 ng / ml, 130 ng / ml, 131 ng / ml, 132 ng / ml, 133 ng / ml, 134 ng / ml, 135 ng / ml, 136 ng / ml, 137 ng / ml, 138 ng / ml, 139 ng / ml, 140 ng / ml, 141 ng / ml, 142 ng / ml, 143 ng / ml, 144 ng / ml, 145 ng / ml, 146 ng / ml, 147 ng / ml, 148 ng / ml, 149 ng / ml, 150 ng / ml, 151 ng / ml, 152 ng / ml, 153 ng / ml, 154 ng / ml, 155 ng / ml, 156 ng / ml, 157 ng / ml, 158 ng / ml, 159 ng / ml, 160 ng / ml, 161 ng / ml, 162 ng / ml, 163 ng / ml, 164 ng / ml, 165 ng / ml, 166 ng / ml, 167 ng / ml, 168 ng / ml, 169 ng / ml, 170 ng / ml, 171 ng / ml, 172 ng / ml, 173 ng / ml, 174 ng / ml, 175 ng / ml, 176 ng / ml, 177 ng / ml, 178 ng / ml, 179 ng / ml, 180 ng / ml, 181 ng / ml, 182 ng / ml, 183 ng / ml, 184 ng / ml, 185 ng / ml, 186 ng / ml, 187 ng / ml, 188 ng / ml, 189 ng / ml, 190 ng / ml, 191 ng / ml, 192 ng / ml, 193 ng / ml, 194 ng / ml, 195 ng / ml, 196 ng / ml, 197 ng / ml, 198 ng / ml, or 199 ng / ml.

[0144] In some embodiments, the immune cell activating cytokines include one or more selected from the following: IL-7, IL-15, IL-21, IL-12, IL-18 and IL-6. In some embodiments, the immune cell activating cytokines include a combination of IL7 and IL21. In some embodiments, the immune cell activating cytokines are a combination of IL7 and IL21 without other immune cell activating cytokines. In some embodiments, the concentration of IL-7 in the composition is 2.5-10 ng / ml, for example, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.1 ng / ml, 5.2 ng / ml, 5.3 ng / ml, 5.4 ng / ml, 5.5 ng / ml, 5.6 ng / ml, 5.7 ng / ml, 5.8 ng / ml, 5.9 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml or 9.5 ng / ml, or any value between 2.5 and 10 ng / ml.In some embodiments, the concentration of IL21 in the composition is 12.5-50 ng / ml, for example 13 ng / ml, 13.5 ng / ml, 14 ng / ml, 14.5 ng / ml, 15 ng / ml, 15.5 ng / ml, 16 ng / ml, 16.5 ng / ml, 17 ng / ml, 17.5 ng / ml, 18 ng / ml, 18.5 ng / ml, 19 ng / ml, 19.5 ng / ml, 20 ng / ml, 20.5 ng / ml , 21ng / ml, 21.5ng / ml, 22ng / ml, 22.5ng / ml, 23ng / ml, 23.5ng / ml, 24ng / ml, 24.5ng / ml, 25ng / ml, 25.5ng / ml , 26ng / ml, 26.5ng / ml, 27ng / ml, 27.5ng / ml, 28ng / ml, 28.5ng / ml, 29ng / ml, 29.5ng / ml, 30ng / ml, 30.5ng / ml, 31ng / ml, 31.5ng / ml, 32ng / ml, 32.5ng / ml, 33ng / ml, 33.5ng / ml, 34ng / ml, 34.5ng / ml, 35ng / ml, 35.5ng / ml, 36ng / ml, 36.5ng / ml, 37ng / ml, 37.5ng / ml, 38ng / ml, 38.5ng / ml, 39ng / ml, 39.5ng / ml, 40ng / ml, 40.5ng / ml, 4 1ng / ml, 41.5ng / ml, 42ng / ml, 42.5ng / ml, 43ng / ml, 43.5ng / ml, 44ng / ml, 44.5ng / ml, 45ng / ml, 45.5ng / ml, 46ng / ml, 46.5ng / ml, 47ng / ml, 47.5ng / ml, 48ng / ml, 48.5ng / ml, 49ng / ml, or 49.5ng / ml, or any value between 12.5 and 50ng / ml.

[0145] In some embodiments, the composition does not include a CD28 agonist. In some embodiments, the composition does not include any cell surface co-stimulatory molecule agonist. In some embodiments, the cell surface co-stimulatory molecules include: CD28, ICOS, CD27, HVEM, LIGH T, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and CD226.

[0146] In some embodiments, the composition is a culture medium, which, in addition to CD3 / TCR complex agonists and immune cell activating cytokines, further comprises essential nutrients (including inorganic and organic substances) that can provide cells with in vitro survival, such as water, amino acids, sodium pyruvate, and vitamins. In some embodiments, the essential nutrients also include nutrients (such as ICSR) that can be used for cell survival under serum-free conditions, such as serum replacement. In some embodiments, the essential nutrients include serum, such as human serum or fetal bovine serum. In some embodiments, the composition is a commercial culture medium to which CD3 / TCR complex agonists and immune cell activating cytokines have been added, and the commercial culture medium can be selected from, for example, OpTmizer TM CTS TM 、Immunocult TM XF、CellGro TM (CellGenix), TexMacs TM 、Stemline TM 、Xvivo15 TM , PrimeXV, StemXVivo, α-MEM, RPMI 1640, AIM-V, DMEM, F-12, X-vivo15, X-Vivo 20, OpTmizer, and IMDM, etc. In some embodiments, the culture medium further comprises one or more selected from the following: insulin, IFNγ, IL-7, IL-12, IL-15, and TNFα, a surfactant, a human plasma protein product, and a reducing agent (such as N-acetyl-cysteine ​​and 2-mercaptoethanol), etc.

[0147] In addition, the present application also relates to a kit comprising the above composition, wherein the kit does not contain IL-2 and CD28 agonist.

[0148] The present application also relates to the use of the above-mentioned composition and kit. In some embodiments, the use relates to the preparation of engineered immune cells. In some embodiments, the engineered immune cells include T cells. In some embodiments, the engineered immune cells are derived from PBMC. In some embodiments, the engineered immune cells are CAR-T cells or TCR-T cells.

[0149] The beneficial effects of this application are:

[0150] 1) CAR-T cell preparation can be completed within 48 hours, which is faster than traditional methods.

[0151] 2) The prepared CAR-T cells have better in vivo effectiveness and sustainability.

[0152] 3) The entire preparation process does not require any CD28 agonists, and the prepared CAR-T cells are safer than the currently disclosed preparation methods.

[0153] 4) The scheme described in this application can also be done without adding IL-2, further improving the safety of CAR-T products.

[0154] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art to which the present application belongs. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments. Example

[0155] The exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding, which should be considered to be merely exemplary. Therefore, it should be appreciated by those of ordinary skill in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and brevity, descriptions of well-known features and structures are omitted in the following description.

[0156] The relevant amino acid sequences of the CAR structures used in the following examples are shown in the sequence table at the end of the text.

[0157] Example 1: New CAR-T cell preparation process

[0158] The traditional preparation of CAR-T cells is divided into three processes: cell activation, viral transduction, and cell harvesting. In the rapid preparation scheme, it is divided into two stages: cell activation-viral transduction and viral transduction-cell harvesting. Cell activation-viral transduction refers to the time period from the addition of culture reagents to viral transduction of cells. Viral transduction-cell harvesting refers to the time from the addition of viruses to the activated T cell culture for viral transduction until cell harvesting. The activation reagent is no longer removed between the two processes, and the virus is no longer removed from viral transduction to cell harvesting. Whether the culture time of the above two stages will affect the final function of CAR-T cells, there is currently no existing technology that can provide a hint, which requires our creative exploration.

[0159] 1.1 Confirmation of a more effective short-term CAR-T preparation regimen

[0160] Mononuclear cells were obtained by separation or T cells generated by sorting mononuclear cells (the cells can also be recovered after freezing the above cells), activated with 50ng / ml CD3 antibody (Recombinant anti-Human CD3mAb, GMP-A018), cultured in a system containing 5ng / ml IL-7 and 25ng / ml IL-21 cytokines (SCGM serum-free medium, CellGenix, 20802-0500), transduced with lentivirus after different activation times (such as Table 1 Cell activation-viral transduction), and cells were harvested at different times after viral transduction (such as Table 1 Viral transduction-cell harvesting). The following four schemes were designed for different activation times (cell activation-viral transduction) and viral transduction CAR-T preparation times (viral transduction-cell harvesting) to explore the optimal rapid cell preparation scheme:

[0161] Table 1 CAR-T cell preparation scheme (time optimization)

[0162] Grouping Cell activation-viral transduction Viral transduction-cell harvest Total preparation time Solution 1 0h 24h 1day Solution 2 6h 18h 1day Solution 3 12-24h 14-28h 2days Solution 4 24h 72h 4days

[0163] According to the above preparation scheme, four kinds of rapidly prepared CAR-T cells of schemes 1-4 were prepared, and the CAR positivity of cells prepared by several schemes and the survival rate of cells after cryopreservation and resuscitation were compared. The harvested CAR-T cells of different preparation schemes were resuspended in PBS solution containing 1% fetal bovine serum by volume, and the collected cells were divided into 100 μl per tube, and 5 μl of PE-PL reagent was added to each tube of cells (Protein-L can recognize the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L, so Protein-L can be used to detect the CAR positivity rate), incubated at 4°C for 30 minutes, washed twice with PBS solution, and detected by flow cytometer. The results of CAR expression positivity rate detection are as follows: Figure 1A As shown, Figure 1A The horizontal axis represents CAR-T cells prepared in different schemes, and the vertical axis represents the CAR expression positive rate. The results show that scheme 3 obtains the best CAR positive cell ratio compared with schemes 1 and 2.

[0164] Furthermore, considering that the application scenarios of CAR-T cells often involve the need for cryopreservation, the prepared CAR-T cells need to be revived before being reinfused into patients. The viability of CAR-T cells will affect their actual dose calculation and in vivo effectiveness. Therefore, the cryoprotective properties of CAR-T cells prepared by different culture schemes are also important for the function of CAR-T cells. The above-mentioned CAR-T cells are prepared using commercial cryopreservation agents ( CS10) was frozen for 48 hours. After the frozen CAR-T cells were quickly revived in a 37°C water bath, a certain amount of cells were taken for cell viability detection using AOPI. The test results were as follows Figure 1B As shown, the horizontal axis represents the CAR-T cells of different preparation schemes, and the vertical axis represents the proportion of live cells of CAR-T cells. The results showed that scheme 4 prolonged the preparation cycle of CAR-T cells, and the survival rate of cells after cryopreservation and recovery was significantly reduced.

[0165] Furthermore, the CD19-targeted CAR-T cells (CD19-targeted CAR-T cells such as patent 201710613317.6) prepared using the above four schemes were co-cultured with Nalm6-Luc-GFP tumor cells for multiple repeated tumor killing tests. The main implementation process was to co-culture the four groups of CAR-T cells with Nalm6-Luc-GFP cells at a 1:2 effector-target ratio, and detect the proportion of GFP+ tumor cells in the cell population by flow cytometry. When the proportion of GFP+ cells was lower than 10%, the cell count was collected, and the tumor cells were replenished again at a 1:2 effector-target ratio for repeated stimulation and killing. When the proportion of GFP+ tumor cells in the cell population exceeded 90%, it was used as the killing test endpoint. The stimulation rounds of tumor cells in each test group and the proliferation multiples of CAR-T cells during the stimulation process were calculated, and the function and persistence of CAR-T cells under different preparation schemes were reflected by the CAR-T cells' ability to stimulate multiple rounds of target cells and the proliferation after stimulation. The results are shown in Figure 1C As shown, the horizontal axis represents the number of days of continuous proliferation of CAR-T cells in vitro, the vertical axis represents different preparation methods, and different column colors represent different stimulation rounds. Figure 1D The horizontal axis represents the number of days of sustained proliferation of CAR-T cells in vitro, and the vertical axis represents the proliferation of cells prepared in different ways. The results showed that scheme 3 showed the most advantageous anti-tumor function, and showed sustained anti-tumor function at a low efficiency target ratio of 1:8 (CAR-T:Nalm6).

[0166] Based on the above results, the preferred CAR-T cell preparation cycle is Scheme 3, namely:

[0167] Mononuclear cells are obtained by separation or T cells generated by sorting mononuclear cells (or cells recovered after freezing the above cells) are activated and cultured in a system containing 5ng / ml IL-7 and 25ng / ml IL-21 cytokines using 50ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018). After activation for 12-24 hours, a lentivirus containing a CD19 CAR gene is added for CAR gene transduction. After adding the virus, the cells are cultured in a system containing 5ng / ml IL-7 and 25ng / ml IL-21 cytokines for 14-28 hours to harvest the CAR-T cells. The entire preparation cycle is 26 hours to 52 hours. The CAR-T cells are CAR-T cells targeting CD19, and the specific structure is an anti-CD19ScFv-8h-8TM-BBZ structure (CD19 CAR), and the sequence is shown in the sequence table.

[0168] 1.2 Study on the optimal time range for cell activation and viral transduction

[0169] T cells were activated with 50ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018), cultured in a system containing 5ng / ml IL-7 and 25ng / ml IL-21 cytokines, and transduced with lentivirus after 12h, 16h, 20h and 24h of activation, respectively. Cells were harvested 18-28h after viral transduction. The activation time range of CAR-T cells was studied. Here, the activation intensity of CAR-T indirectly reflects the functionality of CAR-T. CD25 is the α chain of the IL-2 receptor, which is upregulated within 24 hours after T cell activation and remains elevated for several days. CD25 can be used to reflect the degree of activation of CAR-T cells. The harvested CAR-T cells were labeled with anti-CD25 antibody (CD25 BV421, Biolegend, 302630) and flow cytometry was performed. The results are as follows: Figure 2A As shown, Figure 2A The horizontal axis represents different activation times of cells, and the vertical axis represents the positive expression rate of CAR-T cell CD25. The results show that with the extension of activation time, the cell activation indicator CD25 shows an upward expression trend, reaching the optimal activation intensity at 20h.

[0170] Short-term preparation: Since the CAR-T cell culture time is short and the T cell-virus contact time is short, the CAR positivity rate is often low in the short-term preparation process. The CAR positivity rate of CAR-T prepared at different time points is also one of the better cell activation and transduction time ranges. Figure 2B and 2C As shown, Figure 2BThe CAR expression positive rate of CAR-T cells prepared at different activation time points, the horizontal axis is the different activation time of cells, and the vertical axis is the CAR expression positive rate. The results of the CAR positive rate of cells showed that the CAR positive rate increased with the extension of activation time, and the 24-hour activation group maintained a higher proportion of CAR+ cells; Figure 2C The activation time is 12-24 hours, and the CAR expression of cells harvested at different times after viral transduction. The horizontal axis is the different times of cell harvest after viral transduction, and the vertical axis is the CAR expression positive rate. The results showed that with the extension of activation time, the CAR positive rate showed an upward trend, reaching the highest positive rate at 20h~28h and maintaining stability.

[0171] Based on the above results, the optimal time for T cell activation is 20h~24h, and the optimal time for cell harvesting after virus transduction is 20h~28h.

[0172] Example 2: Verification of the anti-tumor effects of CAR-T cells prepared by the preparation process of the present application and CAR-T cells prepared by traditional processes

[0173] Traditional preparation process CAR-T preparation: C-CAR

[0174] T cells in donor PBMCs were activated with anti-CD3 / CD28 antibodies or magnetic beads at a bead:cell ratio of 1:1 (anti-CD3 / CD28 Dynabeads 40203D), and transduced with CD19-targeted CAR lentiviral vector 12 to 24 hours after activation. They were expanded and cultured for 8 to 11 days in a culture system containing only 5 ng / ml IL-7 and 25 ng / ml IL-21 cytokines (SCGM serum-free medium, CellGenix, 20802-0500), and CAR-T cells were harvested and frozen.

[0175] CAR-T cell preparation in this application process: P-CAR

[0176] The T cells of the donor's PBMC were activated with 50ng / ml CD3 antibody (T cells were not exposed to CD28 antibody throughout the process) (Recombinant anti-Human CD3 mAb, GMP-A018), and transduced with CD19-targeted CAR lentiviral vectors 12 to 24 hours after activation. Only two cytokines, 5ng / ml IL-7 and 25ng / ml IL-21, and culture medium (SCGM serum-free medium, CellGenix, 20802-0500) were added to the culture system. CAR-T cells were collected and frozen 14 to 28 hours after viral transduction. CAR-T cells are CAR-T cells targeting CD19. For specific sequences, see the sequence table.

[0177] Control-T cells were prepared by the same process and were not transduced with virus as a control.

[0178] 6-8 week old NOG female mice were intravenously loaded with Nalm6-Luc-GFP tumor cells at 1.00E+06 / mouse for 3 days. CAR-T cells prepared by the two schemes were intravenously infused at 1.00E+06CAR+ / mouse, and the anti-tumor effect of CAR-T cells in vivo was compared by weekly in vivo imaging. At the same time, CAR-T cells prepared by the two preparation processes were co-cultured with Nalm6-Luc-GFP cells at a 1:1 effector-target ratio. Tumor cells were repeatedly added for stimulation every 3 days at the same effector-target ratio, and the proliferation of CAR-T cells was calculated by cell counting. The results of the persistence of CAR-T cells prepared by different processes in tumor-bearing mice are shown in the figure. Figure 3A As shown, Figure 3A The horizontal axis is the time point of CAR-T cell detection, and the vertical axis is the CAR-T cell proliferation multiple. The results show that the P-CAR rapid preparation process has a more advantageous cell proliferation ability than the traditional C-CAR after multiple rounds of tumor antigen stimulation. The effectiveness results of CAR-T cells prepared by the two preparation processes on tumor-bearing mice are as follows Figure 3B As shown, the results of in vivo animal experiments showed that the anti-tumor function of P-CAR-T cells in vivo was significantly better than that of CAR-T cells prepared by traditional processes.

[0179] To further confirm the feasibility of the CAR-T cell production process of the present application in multiple target CAR-T cells, P-CAR and C-CAR were prepared according to the above scheme, and bispecific lentivirus targeting CD19 / CD22 and lentiviral vector targeting CEA were transduced. When comparing the in vivo functions of CD19 / CD22 P-CAR and C-CAR, 6-8 week old NOG female mice were intravenously loaded with Nalm6-Luc-GFP tumor cells at 1.00E+06 / mouse for 3 days, and CAR-T cells prepared by the two schemes were intravenously infused at 1.00E+06CAR+ / mouse, and weekly in vivo imaging was used to compare the anti-tumor effects of CAR-T cells in vivo. When comparing the in vivo functions of P-CAR and C-CAR of CEA, 6-8 week old NCG female mice were subcutaneously (sc) inoculated with tumor cells overexpressing CEA at a dose of 1.00E+06CAR+ / mouse to establish a subcutaneous solid tumor model. After two weeks of tumor bearing, CAR-T cells prepared by the two schemes were intravenously infused at a dose of 5.00E+05CAR+ / mouse, and weekly in vivo imaging was used to compare the anti-tumor effects of CAR-T cells in vivo. The results of the effectiveness of CAR-T cells targeting CD19 / CD22 and CEA on tumor-bearing mice are shown in Figure 2. Figure 3CAs shown, the results of in vivo animal experiments showed that the anti-tumor function of P-CAR-T cells of CD19 / CD22 and CEA was significantly better than that of CAR-T cells prepared by traditional methods. The CAR-T cells of the present application process showed in vivo anti-tumor effects superior to those of traditional methods in tumor models of multiple targets. The sequences of CAR targeting CD19 / CD22 and CAE are shown in the sequence table respectively.

[0180] Example 3 Comparison of CD3 antibody activation scheme and anti-CD3 / CD28 magnetic bead activation scheme

[0181] The CAR-T cells prepared by the process of the present application are prepared according to the P-CAR-T scheme in Example 2 and are represented by CD3; anti-CD3 / CD28 magnetic beads are used to activate T cells according to bead:cell=1:1 (anti-CD3 / CD28Dynabeads, 40203D) and lentivirus is transduced after 12 to 24 hours, and cultured in a system containing different cytokines such as 5ng / ml IL-7 and 25ng / ml IL-21 (SCGM serum-free medium, CellGenix, 20802-0500). Cells are harvested 14 to 28 hours after viral transduction to obtain CAR-T cells activated by anti-CD3 / CD28 magnetic beads, represented by CD3 / CD28. CAR-T cells are CAR-T cells targeting CD19. For specific sequences, see the sequence table. The viability of the cells after activation and after cryopreservation and recovery of CAR-T cells was detected, and the results are as follows Figure 4A As shown, the horizontal axis represents two activation schemes, and the vertical axis represents the viability of CAR-T cells. CD3 antibody activation maintains a higher viability of cells and a higher viability stability of CAR-T cells after cryopreservation and recovery.

[0182] The CAR-T cells prepared by the two activation schemes were co-cultured with Nalm6-Luc-GFP cells at a 1:1 (CAR-T:Nalm6) effector-target ratio, and the phenotype of the CAR-T cells was detected after three repeated tumor stimulations. Figure 4B As shown, the horizontal axis represents two activation schemes, and the vertical axis represents different phenotypes of CAR-T cells. CAR-T cells prepared by the CD3 antibody activation scheme showed a higher Tscm stem memory cell phenotype, and a lower proportion of TIM-3 and LAG-3 immunosuppressive phenotypes, suggesting that CD3 antibody activation has the effect of maintaining cell memory phenotype and reducing cell exhaustion.

[0183] To further verify the effectiveness of the CD3 antibody activation scheme and the anti-CD3 / CD28 magnetic bead activation scheme in vivo, 8-10 week old NOG female mice were intravenously loaded with Nalm6-Luc-GFP tumor cells at 1.00E+06 / mouse for 3 days. CAR-T cells prepared by the two schemes were intravenously infused at 1.00E+06CAR+ / mouse. The anti-tumor effects of CAR-T cells in vivo were compared, and the survival of mice with the two schemes was evaluated. Figure 4C As shown, mice died 41 days after the CD3 / CD28 activation CAR-T cell infusion, and no superior anti-tumor effect was shown in mice. The short-term CAR-T preparation scheme using anti-CD3 antibody activation alone in this application has higher safety and better in vivo effectiveness.

[0184] Furthermore, the in vivo efficacy and safety of the P-CAR regimen described in this application were evaluated compared with the short-term regimen (CD3 / CD28) activated by anti-CD3 / CD28 magnetic beads used in the art, and the CAR-T cells targeting the CD70 target prepared by the traditional C-CAR process (the structure of CAR introduces the sequence of CD70 CAR of patent 202111239047.X). SKOV3-Luc-GFP tumor cells 1.00E+06 / mouse were used for intraperitoneal tumor bearing for 7 days, and CAR-T cells prepared by the three regimens were intraperitoneally infused at 3.00E+05CAR+ / mouse. The in vivo anti-tumor effects of CAR-T cells were compared, and the survival of mice of the three regimens was evaluated. The results are as follows Figure 4D As shown, although the CAR-T cells of the CD3 / CD28 scheme are superior to the CAR-T cells of the C-CAR scheme, the mice died on the 28th day due to excessive immune response in the body. The P-CAR scheme described in this application not only has better in vivo efficacy than C-CAR and is not inferior to the CD3 / CD28 scheme, but is also significantly better than the CD3 / CD28 scheme in terms of safety. The short-term CAR-T preparation scheme using the anti-CD3 antibody activation alone of this application has higher safety and better in vivo effectiveness.

[0185] Example 4 Application of different cytokines or their combination to the short-term preparation scheme of CAR-T

[0186] T cells were activated with 50ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018) for 12 to 24 hours before transduction with lentivirus. The cells were cultured in a system containing both 5ng / ml IL-7 and 25ng / ml IL-21 or 5ng / ml IL-7 and 25ng / ml IL-21 and no cytokines (SCGM serum-free medium, CellGenix, 20802-0500). Cells were harvested 14 to 28 hours after viral transduction. The cell harvest was completed the day after viral transduction. Comparison of different cytokines was a strategy to compare the transduction CAR positivity of cells in the absence of cytokines (no-cytokine). The CAR positivity rate was detected by flow cytometry after being labeled with PE-PL. The results are shown in the figure. Figure 5 As shown, the horizontal axis is different factor schemes, and the vertical axis is the CAR expression positive rate. The results show that the short-term preparation of the present application has the effect of increasing the CAR positive rate by adding the combination of cytokines IL7 and IL-21 compared with factor-free culture or single factor.

[0187] Furthermore, the following four groups of schemes were designed for different cytokines:

[0188] Protocol 1: 50 ng / ml CD3 antibody activation, 5 ng / ml IL-7 and 25 ng / ml IL-21 cytokine culture;

[0189] Scheme 2: 50 ng / ml CD3 antibody activation, 500 IU / ml IL-2 cytokine culture;

[0190] Scheme 3: 50 ng / ml CD3 antibody activation, 20 ng / ml IL-15 cytokine culture;

[0191] Scheme 4: 50 ng / ml CD3 antibody activation, 50 ng / ml IL-18 cytokine culture.

[0192] The P-CAR preparation scheme of Example 2 was adopted, and the "culture system (SCGM serum-free medium, CellGenix, 20802-0500) was replaced with the cytokine scheme of the above schemes 1-4, and the prepared CAR-T cells were expressed as IL-7 / IL-21, IL-2, IL-15, and IL-18, respectively. After harvesting, the prepared cells were frozen in a commercial cryopreservation solution.

[0193] 4.1 Viability detection of the above-mentioned frozen CAR-T cells after recovery

[0194] After the frozen CAR-T cells were rapidly revived in a 37°C water bath, a certain amount of cells were taken for cell viability detection using AOPI. The test results were as follows: Fig. 6A As shown, the horizontal axis represents CAR-T cells prepared in different schemes, and the vertical axis represents the live cell ratio of CAR-T cells; the resuscitated CAR-T cells are further subjected to CAR expression detection using the scheme of Example 1, as shown in Figure 6B As shown, the results showed that the cytokine combination of IL-7+IL-21 maintained a higher CAR positivity rate and cell viability after recovery.

[0195] 4.2 In vivo effectiveness verification

[0196] 8-10 week old NOG female mice were intravenously loaded with Nalm6-Luc-GFP tumor cells at 1.00E+06 / mouse for 3 days, and CAR-T cells prepared by 4 schemes were intravenously infused at 1.00E+06CAR+ / mouse to compare the anti-tumor effects of CAR-T cells in vivo and evaluate the survival of mice in the two schemes. CAR-T cells are CAR-T cells targeting CD19 / CD22, and the specific sequences are shown in the sequence table.

[0197] The results are as follows Fig. 7A The results showed that the IL-7+IL-21 culture regimen showed a more superior anti-tumor effect in mice compared with the IL-15 and IL-18 regimens, and had the same in vivo effectiveness as the IL-2 regimen. However, the IL-2 and IL-15 culture regimens began to monitor mouse deaths 34 days after CAR-T cell infusion, and the safety-related risks were higher than those of the IL-7+IL-21 regimen.

[0198] Furthermore, for Scheme 1, Scheme 2, Scheme 4, and different component combinations of 50 ng / ml CD3 antibody activation, 5 ng / ml IL-7; 50 ng / ml CD3 antibody activation, 25 ng / ml IL-21, we used the P-CAR process and the existing CD3 / CD28 short-time preparation scheme in the field in Example 3 to compare the safety.

[0199] In the existing scheme, the CD3 / CD28 short-term preparation scheme uses anti-CD3 / CD28 magnetic beads to activate T cells and then transduce lentivirus, and the scheme is cultured in an IL-2 system containing 100IU / ml-500IU / ml, which we mark as CD3CD28+IL-2 scheme. In the following example verification, we respectively use the specific existing CD3 / CD28 short-term preparation scheme in Example 3, which we mark as CD3CD28, or use the scheme with IL-2 as the factor component, CD3CD28+IL-2 scheme, and the IL-2 concentration is 500IU / ml.

[0200] Cytokine release syndrome (CRS) is an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction, which is associated with chimeric antigen receptor (CAR) T-cell therapy, therapeutic antibodies and haploidentical allogeneic transplantation. Based on the clinical literature of the article "Side-effect management of chimeric antigen receptor (CAR) T-cell therapy" published in 2020, the article summarizes the clinical side effects after CAR-T therapy. In the "Cytokine release syndrome" section of the main text, it is summarized that "As an asupraphysiologic inflammatory state, CRS (Figure 1) is triggered by inflammatory cytokines and chemokines released by CAR T cells, such as interferon (IFN) γ, tumor necrosis factor (TNF) α, granulocyte macrophage colonystimulating factor (GM-CSF), interleukin (IL)-2, IL-8 and IL-10, after engaging with the corresponding target Antigen." (CRS is a physiological inflammatory state, which is caused by the release of inflammatory cytokines and chemokines after CAR-T cells bind to the corresponding target antigen, such as interferon (IFN) γ, tumor necrosis factor (TNF) α, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL) -2, IL-8 and IL-10). It can be seen that in CAR-T cell therapy, CRS is mainly caused by excessive or uncontrolled release of proinflammatory cytokines such as IL-2, TNF-α and IFN-γ after CAR-T cells are activated by tumor cells. These proinflammatory cytokines are hallmarks of CAR-T function, but they need to be maintained in an appropriate concentration range and cannot be excessively released or uncontrolled. Therefore, under the premise of tumor cell killing verification, the release of IL-2, TNF-α and IFN-γ is lower and safer.

[0201] Therefore, the safety of P-CAR process and CD3 / CD28 short-time preparation process (CD3CD28+IL-2 scheme, CD3CD28 scheme) was tested by in vitro killing and cytokine secretion detection. CAR-T cells were the aforementioned CAR-T targeting CD19 and CAR-T cells targeting CD19 / CD22. The tumor target cells were Nalm6-Luc-GFP. The effector-target ratio was 1:1. Tumor cells and CAR-T cells were co-incubated for 24 hours to analyze the killing ability of CAR-T cells on tumor cells. The results are shown in the figure. Figure 7B and 7F As shown, Figure 7B The effectiveness results of CAR-T cells targeting CD19 / CD22 prepared by different processes. Figure 7F The results show the effectiveness of CAR-T cells targeting CD19 prepared by different processes. Both the P-CAR process and the CD3 / CD 28 short-time preparation process have the ability to effectively kill Nalm6-Luc-GFP, and the function of CAR-T cells prepared by the P-CAR process is not weaker than that of the CD3 / CD 28 short-time preparation process (or equivalent to it). In some targets, the effectiveness of the P-CAR process is better than that of the CD3 / CD 28 short-time preparation process.

[0202] The killing supernatant was further collected and the secretion of IL-2 (invitrogen, catalog number 88-7025-88), TNF-α (Biolegend, catalog number 430201) and IFN-γ (BD, catalog number 555142) was detected by ELISA (enzyme-linked immunosorbent assay). The specific detection method was performed according to the instructions. The cytokine secretion results are shown in Figure 2. Figure 7C-7G As shown, Figure 7C-7E The cytokine secretion of CD19 / CD22-targeted CAR-T cells prepared by different processes after killing tumor cells: 7C is the secretion result of IL-2 by different processes, Fig.7D The results of IFN-γ secretion in different processes are shown in Figure 2. Fig. 7E The results of TNF-α secretion in different processes. Figure 7G The cytokine secretion of CD19-targeted CAR-T cells prepared by different processes after killing tumor cells. The results showed that the secretion of IL-2, TNF-α and IFN-γ by P-CAR was greater than 500pg / ml combined with the in vitro killing ability, indicating that the CAR-T of the P-CAR process has the ability to effectively kill tumor cells, and the secretion of IL-2, TNF-α and IFN-γ by P-CAR is significantly lower than that of the CD3 / CD 28 short-time preparation process, indicating that the safety of CAR-T prepared by the P-CAR process is significantly better than that of the CD3 / CD 28 short-time preparation process.

[0203] Figure 7B The corresponding specific kill value percentages are shown in Table 2 below:

[0204] Table 2

[0205] CD3+IL2 CD3+IL7+IL21 CD3CD28+IL2 CD3CD28 41.37 66.08 83.19 86 22.9 46.08 64.36 65.43 29.71 52.95 79.58 82.21 71.31 71.4 56.09 61.59 47.97 49.36 30.27 30.81 59.99 60.27 53.46 52.06

[0206] Figure 7C The corresponding specific IL-2 factor secretion values ​​pg / ml are shown in Table 3 below:

[0207] Table 3

[0208] CD3+IL2 CD3+IL7+IL21 CD3CD28+IL2 CD3CD28 3438.787 3704.097 29754.965 33680.212 3255.645 3116.233 19871.141 19756.75 7469.542 7744.703 36672.248 34070.886

[0209] Fig.7D The corresponding specific IFN-γ factor secretion values ​​pg / ml are shown in Table 4 below:

[0210] Table 4

[0211] CD3+IL2 CD3+IL7+IL21 CD3CD28+IL2 CD3CD28 43665.676 57316.831 110462.29 112990.093 55931.144 69555.139 114885.37 115941.599 69403.195 83740.451 133135.215 128360.003

[0212] Fig. 7E The corresponding specific TNF-α factor secretion values ​​pg / ml are shown in Table 5 below:

[0213] Table 5

[0214] CD3+IL2 CD3+IL7+IL21 CD3CD28+IL2 CD3CD28 1335.03 2152.444 4978.401 6846.206 1583.939 2291.187 4848.312 6261.967 2753.558 3820.563 7441.971 7347.821

[0215] Figure 7F The corresponding specific kill value percentages are shown in Table 6 below:

[0216] Table 6

[0217] CD3+IL2 CD3+IL7 CD3+IL21 CD3+IL7+IL21 CD3+IL18 CD3CD28+IL2 67 68.37 81.68 64.24 88.63 42.66 87.24 90.34 84.93 82.83 82.46 64.38 88.37 93.27 86.92 88.37 75.81 56.92

[0218] Figure 7G The corresponding specific IL-2 factor secretion values ​​pg / ml are shown in Table 7 below:

[0219] Table 7

[0220] CD3+IL2 CD3+IL7 CD3+IL21 CD3+IL7+IL21 CD3+IL18 CD3CD28+IL2 18818.203 19344.739 15244.384 14773.989 2074.481 49047.152 15887.119 22440.545 15833.996 19314.074 1747.662 70638.768 9868.311 14666.943 7235.675 11466.374 557.109 46652.491

[0221] The results showed that the activation scheme using only CD3 antibodies was better than the activation scheme using anti-CD3 / CD28 antibodies or magnetic beads coated with anti-CD3 / CD28 antibodies. The T cell activation signal provided by CD3 antibodies can maintain the anti-tumor function of short-term CAR-T cells while reducing the risk of side effects. The CD3 activation scheme was combined with cytokines such as IL7, IL-21, IL7+IL-21, IL2, IL15 and IL-18. The results showed that the factor combination scheme of IL-7+IL-21 can maintain a more stable and higher CAR positive rate, while showing better therapeutic effects in animal models and maintaining the survival of mice. In summary, CD3 antibody activation combined with IL-7+IL-21 cytokine combination has more outstanding application feasibility on the short-term CAR-T cell platform.

[0222] Example 5: Safety of CAR-T cells prepared in a short time in this application

[0223] To further confirm the clinical safety and effectiveness of the CAR-T cell P-CAR-T prepared by the short-term preparation scheme of this application, we activated T cells based on 50ng / mlCD3 antibody, used 5ng / ml IL-7+25ng / ml IL-21 cytokines for culture, and used SCGM serum-free medium (CellGenix, 20802-0500) as the culture medium. The cells were activated for 12 to 24 hours and transduced with CD19-targeting lentiviral vectors. The cells were harvested 14 to 28 hours after viral transduction to prepare the produced CD19-P-CAR-T cells, and the treatment and efficacy evaluation of 19 patients with acute lymphoblastic leukemia and lymphoma were completed. Comparative literature "Novel CD19 chimeric antigen receptor T cells manufactured next-day for acute lymphoblastic leukemia" (Literature 1) and "Next-day manufacture of a novel anti-CD19 CAR-T therapy for B-cell acute lymphoblastic leukemia: first-in-human clinical study" (Literature 2) use Dynabeads coated with anti-CD3 / CD28 antibodies and IL-2 to produce CD19-Fast CAR-T cells within two days after PBMC isolation for the treatment of acute lymphoblastic leukemia clinical results. The results are shown in Table 8 below:

[0224] Table 8 Comparison of in vivo efficacy and safety of short-term CAR-T cells prepared by different regimens

[0225]

[0226] The short-term CAR-T cells targeting CD19 prepared by the anti-CD3 antibody preparation scheme described in this application are used to treat acute lymphoblastic leukemia and lymphoma. Compared with the cells prepared by Dynabeads and IL-2 schemes coated with anti-CD3 / CD28 antibodies, they show a higher clinical complete remission rate (CR), lower CRS and iCANS side effect rates, and the proportion of severe CRS and iCANS above grade 3 is significantly lower. This suggests that the use of anti-CD3 antibody + IL7 / IL-21 to prepare rapid cell products has better clinical effects and lower side effect rates than the anti-CD3 / CD28 antibody + IL-2 scheme, and its clinical application value is better.

[0227] Example 6: CD3 antibody concentration test

[0228] T cells were activated with 25-200 ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018), cultured in a system containing 5 ng / ml IL-7 and 25 ng / ml IL-21 cytokines (SCGM serum-free medium, CellGenix, 20802-0500), and transduced with lentivirus after 12-24 hours of activation, and cells were harvested 18-24 hours after viral transduction. The effect of different concentrations of CD3 antibody on the preparation process was explored. The appropriate concentration of CD3 antibody was investigated by detecting the cell activation indicators CD25 / CD69 and the proportion of CAR+ cells. The results are as follows: Fig. 8A and Figure 8B As shown, the horizontal axis represents the culture system with different CD3 antibody concentrations at 5ng / ml IL-7 and 25ng / ml IL-21 cytokine concentrations, and the vertical axis represents the cell activation percentage and CAR expression positive rate. The results show that the activation of CAR-T cells and CAR gene transduction ability can be well achieved at the concentration of 25ng / ml to 200ng / ml.

[0229] At the same time, the in vitro anti-tumor function of CAR-T cells prepared by the process of this application was also confirmed by continuous multiple rounds of in vitro tumor cell stimulation experiments at the concentrations of 0ng / ml, 25ng / ml, 50ng / ml, 200ng / ml, and 500ng / ml of CD3 antibodies. CD19 lentiviral vectors were transduced after activating T cells for 12 to 24 hours using 0ng / ml, 25ng / ml, 50ng / ml, 200ng / ml, and 500ng / ml CD3 antibodies (Recombinant anti-Human CD3 mAb, GMP-A018), and cultured in a system containing 5ng / ml IL-7 and 25ng / ml IL-21 (SCGM serum-free medium, CellGenix, 20802-0500), and cell harvesting was completed after 14 to 28 hours of viral transduction. The CD19-targeted CAR-T cells prepared using the above scheme (CAR-T cells targeting CD19 such as patent 201710613317.6) were co-cultured with Nalm6-Luc-GFP tumor cells at an effector-target ratio of 1:2, and repeated tumor killing tests were performed. The proportion of GFP+ tumor cells in the cell population was detected by flow cytometry. When the proportion of GFP+ cells was lower than 10%, the cell count was collected, and tumor cells were replenished again at an effector-target ratio of 1:2 for repeated stimulation and killing. When the proportion of GFP+ tumor cells in the cell population exceeded 90%, the killing test endpoint was used. The stimulation rounds of tumor cells in each test group and the proliferation multiples of CAR-T cells during the stimulation process were calculated. The function and persistence of CAR-T cells under different preparation schemes were reflected by the CAR-T cells' ability to respond to multiple rounds of target cell stimulation and the proliferation after stimulation. The results are shown in Figure 8C As shown, in the absence of CD3 antibody, that is, CD3 antibody at 0 ng / ml, CAR-T cells cannot be stimulated by target cells to activate and proliferate. Therefore, CD3 antibody at a concentration of 25 ng / ml to 200 ng / ml can be well used in the short-term CAR-T cell preparation process described in this application.

[0230] Furthermore, CD3 antibodies (Recombinant anti-Human CD3 mAb, GMP-A018) of 0 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 200 ng / ml, and 500 ng / ml were used to activate T cells for 12 to 24 hours, and then CD19 lentiviral vectors were transduced. The cells were cultured in a system containing 5 ng / ml IL-7 and 25 ng / ml IL-21 cytokines (SCGM serum-free medium, CellGenix, 20802-0500), and the cells were harvested 14 to 28 hours after viral transduction. The CD19-targeting CAR-T cells prepared using the above scheme (CAR-T cells targeting CD19 such as patent 201710613317.6) were co-cultured with Nalm6-Luc-GFP tumor cells at an effector-target ratio of 1:1 to confirm the killing ability of the prepared CAR-T cells. The results are as follows Fig.8D As shown, under the culture schemes of 12.5ng / ml, 25ng / ml, 50ng / ml, 200ng / ml and 500ng / ml CD3 antibodies, they all had significant killing ability, and the killing percentages are shown in Table 9 below.

[0231] Table 9 Killing percentage of tumor cells by CAR-T cells prepared by P-CAR process with different CD3 concentrations

[0232] 0ng / ml 12.5ng / ml 25ng / ml 50ng / ml 200ng / ml 500ng / ml -8.16 78.99 85.8 88.08 87.79 87.48 -7.9 51.92 71.65 73.89 77.88 67.6

[0233] The results showed that the short-term CAR-T cell preparation process described in this application can be well used when the concentration of CD3 antibody is between 12.5 ng / ml and 200 ng / ml.

[0234] Example 7 IL-7 / IL-21 cytokine concentration test

[0235] During the preparation of P-CAR-T cells, the virus was transduced after T cell activation for 12 to 24 hours, and the cells were harvested 14 to 28 hours after virus transduction. 50 ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018) was used to activate T cells, and different concentrations of IL-7 and IL-21 cytokine combinations were used for cell culture. The culture medium used was SCGM serum-free culture medium (CellGenix, 20802-0500). The effect of different cytokine concentrations on maintaining cell viability and viral transduction efficiency was evaluated.

[0236] Regimen 1: 2.5 ng / ml IL-7 and 12.5 ng / ml IL-21

[0237] Regimen 2: 5 ng / ml IL-7 and 25 ng / ml IL-21

[0238] Regimen 3: 10 ng / ml IL-7 and 50 ng / ml IL-21

[0239] Regimen 4: 2.5 ng / ml IL-7 and 50 ng / ml IL-21

[0240] Regimen 5: 10 ng / ml IL-7 and 12.5 ng / ml IL-21

[0241] The selection of different factor concentrations was investigated by measuring the proportion of CAR+ cells. The results are as follows Fig. 9A As shown, the combination of 2.5-10 ng / ml IL-7 and 12.5-50 ng / ml IL-21 cytokines can well complete the CAR gene transduction ability of CAR-T cells.

[0242] Further, the combination of 0.05ng / ml IL-7 and 2.5ng / ml IL-21, 0.5ng / ml IL-7 and 2.5ng / ml IL-21, 5ng / ml IL-7 and 25ng / ml IL-21, 50ng / ml IL-7 and 250ng / ml IL-21, 250ng / ml IL-7 and 1250ng / ml IL-21 was used to verify the comparison of killing and cytokines using the P-CAR short-term preparation scheme and the CD3 / CD28 short-term preparation scheme of the prior art. Among them, the CD3 / CD28 short-term preparation scheme adopts the scheme of Example 3, and the results are as follows Fig. 9C and Fig.9D shown. Fig. 9C The results show that the combination of IL-7 concentration range of 0.05-250ng / ml and IL21 concentration range of 0.25-1250ng / ml can exert a good killing ability on tumor cells, and the killing ability is not weaker than the existing CD3 / CD28 short-time preparation scheme. The specific data are shown in Table 10. Fig.9DThe IL-2 cytokine secretion capacity of CAR-T cells prepared by the P-CAR short-time preparation scheme and the CD3 / CD28 short-time preparation scheme of the prior art after tumor cell activation is compared. The ordinate is the concentration of IL-2 secretion, and the abscissa is the combination grouping of IL-7 and IL-21 factors of different concentrations. The cytokine secretion concentration is shown in the following Table 11. The results show that the IL-2 cytokine secretion of CAR-T cells prepared by the P-CAR short-time preparation scheme is greater than 500pg / ml after tumor cell activation, and is significantly less than that of CAR-T prepared by the CD3 / CD28 short-time preparation scheme of the prior art.

[0243] Table 10 IL-2 cytokine secretion after activation of CAR-T cells prepared by P-CAR process with different concentrations of IL-7+IL21, pg / ml

[0244]

[0245] Table 11 IL-7+IL21 different concentration combinations P-CAR process to prepare CAR-T cell killing function

[0246]

[0247] Combination Fig. 9A , Fig. 9C and Fig.9D In the short-term preparation scheme of P-CAR described in this application, the concentration of IL-7 cytokine is 0.05-250 ng / ml, and the concentration range of IL21 is 0.25-1250 ng / ml.

[0248] At the same time, the in vitro anti-tumor function of the CAR-T cells prepared by the process of this application was also confirmed by continuous multiple rounds of in vitro tumor cell stimulation experiments. The cytokine IL-7 was 0ng / ml, 2.5ng / ml, 5ng / ml, 10ng / ml, 50ng / ml, and IL-21 was 0ng / ml, 12.5ng / ml, 25ng / ml, 50ng / ml, and 250ng / ml. After activating T cells with 50ng / ml CD3 antibody (Recombinantanti-Human CD3 mAb, GMP-A018) for 12 to 24 hours, CD19 lentiviral vectors were transduced, and cultured in a system containing different concentrations of IL-7 and IL-21 cytokines, and the cells were harvested 14 to 28 hours after viral transduction. The CD19-targeted CAR-T cells prepared using the above scheme (CAR-T cells targeting CD19 such as patent 201710613317.6) were co-cultured with Nalm6-Luc-GFP tumor cells at an effector-target ratio of 1:2, and repeated tumor killing tests were performed. The proportion of GFP+ tumor cells in the cell population was detected by flow cytometry. When the proportion of GFP+ cells was lower than 10%, the cell count was collected, and tumor cells were replenished again at an effector-target ratio of 1:2 for repeated stimulation and killing. When the proportion of GFP+ tumor cells in the cell population exceeded 90%, the killing test endpoint was used. The stimulation rounds of tumor cells in each test group and the proliferation multiples of CAR-T cells during the stimulation process were calculated. The function and persistence of CAR-T cells under different preparation schemes were reflected by the CAR-T cells' ability to respond to multiple rounds of target cell stimulation and the proliferation after stimulation. The results are shown in Fig. 9B As shown, only the CAR-T cells prepared using the preparation scheme described in this application with a combination of 2.5-10 ng / ml IL-7 and 12.5-50 ng / ml IL-21 cytokines have the potential for persistence and proliferation.

[0249] The P-CAR scheme described in this application: CD3 antibodies are used to activate T cells. On this basis, the cytokine combination used can also be IL-2 alone (Scheme 2 of Example 3); it can also be a combination of IL-7 alone, that is, IL-21 with a concentration of 0 ng / ml; it can also be a combination of IL-21 alone, that is, IL-7 with a concentration of 0 ng / ml. In order to verify the feasibility and advantages of the above combination, and to confirm the concentration range of different factor components, a short-term preparation scheme of P-CAR was used, 50 ng / ml CD3 antibody (Recombinant anti-Human CD3 mAb, GMP-A018) was used to activate T cells and CD3CD28+IL-2 (see Example 4 for specific scheme) short-term preparation schemes of the prior art were used for killing and cytokine secretion.

[0250] The results are as follows Figures 10A-10C , Table 12 and Figures 11A-11C , as shown in Table 13, where Fig. 10A To verify the killing effect of IL-2 as a single factor component, Fig. 10B To verify the killing effect of IL-7 as a single factor component, Fig. 10C To verify the killing effect of IL-21 as a single factor component, Fig.11A For the IL-2 secretion verification of the factor component being IL-2 alone, Fig. 11B To verify the IL-2 secretion of IL-7 as a single factor component, Fig. 11C The IL-2 secretion of IL-21 was verified as a single factor component.

[0251] Table 12 Killing percentage of different cytokine components P-CAR process

[0252]

[0253]

[0254] Table 13 IL-2 cytokine secretion after activation of CAR-T cells by P-CAR process with different cytokine components, pg / ml

[0255]

[0256] In the above table, only IL2, IL-7, and IL21 are marked, which means that CAR-T cells are prepared using the P-CAR preparation process, and the cytokine components in the process are IL2, IL-7, or IL21, respectively.

[0257] The results show that the P-CAR scheme described in this application, based on the activation of CD3 antibodies, can also be a single IL-2 (Scheme 2 of Example 3) with a concentration range of 5-12500IU / ml; it can also be a single IL-7, that is, a combination of IL-7 with a concentration range of 0.5-250ng / ml and IL-21 with a concentration of 0ng / ml; it can also be a single IL-21, that is, a combination of IL-21 with a concentration range of 0.25-1250ng / ml and IL-7 with a concentration of 0ng / ml. The CAR-T cells prepared by the P-CAR process in the above combination have excellent tumor-killing activity and are safer than the existing CD3 / CD28 process.

[0258] The amino acid sequence of CAR used in the above examples of the present application is shown in the following sequence table. It should be understood that the following sequence is only an exemplary sequence of the implementation scheme of the present application, and not any limitation to the implementation scheme of the present application.

[0259] Sequence Listing:

[0260]

[0261]

Claims

1. A method for preparing a cell population expressing a chimeric antigen receptor (CAR), the method comprising: Step (i) contacting the cell population with a CD3 / TCR complex agonist; Step (ii) contacting the cell population with a nucleic acid molecule encoding the CAR, thereby providing cells comprising the nucleic acid molecule, and Step (iii) culture the cell population for a period of time and harvest the cell population.

2. The method of claim 1, wherein the cell population is not contacted with a CD28 agonist during step (i), steps (i) and (ii), steps (i), (ii) and (iii), or the entire preparation process.

3. The method of claim 1 or 2, wherein the CD3 / TCR complex agonist comprises a CD3 antibody.

4. The method according to any one of claims 1 to 3, wherein the cell population is contacted with an immune cell activating cytokine in step (i), steps (i) and (ii), steps (i), (ii) and (iii), or during the entire preparation process, wherein the immune cell activating cytokine does not include IL-2. 5 . The method of claim 4 , wherein the immune cell activating cytokines comprise one or more selected from the group consisting of IL-7, IL-15, IL-21, IL-12, IL-18 and IL-6. The method according to claim 5 , wherein the cytokines comprise: a combination of IL-7 and IL-21.

7. The method according to claim 5 or 6, wherein the concentration of IL-7 when contacting the cell population is 2.5 to 10 ng / ml.

8. The method according to any one of claims 5 to 7, wherein the concentration of IL21 when contacting the cell population is 12.5 to 50 ng / ml.

9. The method according to claim 3, wherein the concentration of the CD3 antibody contacted with the cell population in step i) is ≥ 25 ng / ml, such as 25-200 ng / ml.

10. The method according to any one of claims 1-3, wherein the cell population is contacted with an immune cell activating cytokine in step i), steps i) and ii), steps i), ii) and iii), or the entire preparation process, wherein the immune cell activating cytokine comprises IL-2.

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