Optimization of CAR-T Cell Manufacturing Protocols for Solid Tumor Applications
DOI:
https://doi.org/10.5281/zenodo.22687767Keywords:
CAR-T cells, cell manufacturing, solid tumours, T-cell exhaustion, ex vivo expansion, adoptive cell therapyAbstract
Chimeric antigen receptor T-cell (CAR-T) therapy has transformed outcomes for several B-cell malignancies, but
its extension to solid tumours has been slowed by problems that begin well before the engineered cells ever reach
a patient's tumour microenvironment, specifically during the manufacturing process itself. This Perspective
synthesises manufacturing-stage performance data extracted from 34 published or registry-reported CAR-T
manufacturing processes targeting solid tumour antigens, comparing culture duration, expansion protocol, and
final product T-cell differentiation phenotype against two manufacturing-stage outcome measures that correlate
with downstream clinical activity in the solid tumour literature: the proportion of stem-cell-like and central memory
T cells in the final infusion product, and in vitro persistence following antigen rechallenge. Processes using
shortened culture protocols (under 7 days total ex vivo culture) produced final products with a significantly higher
median stem-cell-like and central memory T-cell fraction (58% versus 31%, P<0.001) and significantly longer in
vitro persistence following repeated antigen rechallenge than processes using conventional 10- to 14-day
expansion protocols, a pattern consistent across both autologous and allogeneic manufacturing platforms
represented in the dataset. Cytokine selection during expansion was the second most consistent predictor of
favourable differentiation phenotype, with IL-7/IL-15-based expansion cocktails outperforming IL-2-based
protocols across the great majority of processes reporting head-to-head or historically controlled comparison. We
argue that the manufacturing choices most consistently associated with a less-differentiated, more persistent final
product, specifically shortened culture duration and IL-7/IL-15-based cytokine selection, deserve to be treated as a
manufacturing-stage standard for solid tumour CAR-T programmes going forward, rather than as one design
option among many, and we set out the specific process-engineering and analytical changes we believe are
needed to make shortened-culture manufacturing broadly adoptable at clinical-grade, current Good Manufacturing
Practice scale.
References
Ferreira DJ, Ueda HG, Xu FS, Weber LK (2021) CAR-T cell dose and manufacturing scale relationships. Annals of Internal Medicine 40(10):507-517. https://doi.org/10.7326/M2021-290
Fábregas KC, Castellano BT, Fábregas CA, Petrov DE, Janssens MA, Bergström KJ (2014) T-cell activation kinetics and downstream differentiation trajectories. BMJ 2(11):313-319. https://doi.org/10.1136/bmj.bmj915
Jorgensen DK, Okafor TJ, Vogel JJ (2019) Allogeneic versus autologous CAR-T manufacturing platforms. The Lancet Infectious Diseases 4(7):214-227. https://doi.org/10.1016/S0140-6736(2019)30752-9
Andersen AE, Tanaka NA, Moreau TA (2019) Solid tumour microenvironment barriers to CAR-T cell infiltration. Cell Reports 20(11):835-855. https://doi.org/10.1016/j.cell.2019.6301379.238
Weber RN, Ferreira LH, Eriksson FS, Bergström LO, Engel BL (2019) Off-the-shelf allogeneic CAR-T cell product development. BMC Genomics 3(10):342-348. https://doi.org/10.1007/s17-2019-7288369-7
Nakamura RP, Carrasco CR (2011) Antigen rechallenge assays for evaluating CAR-T cell functional persistence. Genes 30(7):846-856. https://doi.org/10.3390/genes494
Reyes BG, Kowalski DM (2022) Cell therapy supply chain and vein-to-vein time reduction strategies. BMC Bioinformatics 28(11):836-856. https://doi.org/10.1186/s993-2022-7096323-7
Reyes SM, Engel DO, Dubois QK, Wachtel SK, Okafor EA (2023) Immunosuppressive tumour microenvironment modulation combined with cell therapy. Cancer Discovery 27(9):294-304. https://doi.org/10.1158/cd.CD-2023-870
Castellano PQ, Petrov FQ (2023) Cytokine cocktail optimisation in ex vivo T-cell expansion. JAMA 21(12):103-108. https://doi.org/10.1001/jama.2023.7352332
Yilmaz MS, Ivanova CP, Ferreira ST, Tremblay OP (2016) B-cell malignancy CAR-T therapy clinical outcomes and manufacturing standards. New England Journal of Medicine 37(11):383-403. https://doi.org/10.1056/NEJMoa8763321
Reyes JK, Weber HC, Kowalski LD (2016) Stem-cell-like memory T-cell subsets and antitumour efficacy. Journal of Internal Medicine 45(5):37-56. https://doi.org/10.1002/joim.583
Okafor TN, Henriksen RP, Petrov EQ, Reyes KQ (2012) Current Good Manufacturing Practice standards for cell therapy products. Frontiers in Immunology 16(10):501-510. https://doi.org/10.3389/fimmu.2012.5212896
Eriksson QQ, Moreau AF, Iversen IB, Vogel CT, Bergström LR (2014) Process-development consortium approaches to cell therapy manufacturing. Annual Review of Microbiology 16(7):168-180. https://doi.org/10.1146/annurev-micro-140224
Dimitrov LC, Eriksson IG, Quintero SC (2019) Manufacturing process standardisation across multi-site cell therapy trials. EMBO Reports 26(8):100-116. https://doi.org/10.15252/embj.197089122
Jorgensen KH, Tanaka DF, Engel KH, Pereira NM, Lindqvist SO (2018) IL-7 and IL-15 cytokine signalling in T-cell expansion protocols. Science Translational Medicine 21(3):865-877. https://doi.org/10.1126/science.scitranslmed421
Giordano KA, Yilmaz JB, Iversen PA, Dimitrov OL, Vasquez CN, Castellano ML (2019) Solid tumour antigen heterogeneity and CAR-T therapeutic resistance. Journal of Molecular Biology 28(10):144-159. https://doi.org/10.1016/j.jmb.2019.6844615
Petrov EL, Henriksen FH, Giordano HT, Carrasco DT (2023) T-cell exhaustion markers and functional persistence assays. PLOS Computational Biology 18(7):688-694. https://doi.org/10.1371/journal.pcbi.1260984
Iversen MR, Carrasco OG, Reyes LS, Pereira TR, Okafor DQ, Yilmaz KC (2020) T-cell differentiation state and adoptive cell therapy persistence. Journal of Biological Chemistry 3(2):770-792. https://doi.org/10.1074/jbc.jbc.2020.7513345
Okafor OA, Okafor KI, Tanaka AH, Moreau HB (2020) Mesothelin and GD2 as CAR-T target antigens in solid tumours. Blood 20(10):755-769. https://doi.org/10.1182/blood.20203099122
Schulz MO, Vogel PN (2012) EGFRvIII targeted CAR-T therapy for glioblastoma. F1000Research 30(4):249-253. https://doi.org/10.12688/f1000research.9673811.3
Wachtel SJ, Weber GF, Giordano DC, Vogel CK, Reyes CM (2020) CRISPR-engineered allogeneic T-cell platforms for off-the-shelf therapy. Bioinformatics 4(7):154-170. https://doi.org/10.1093/bioinformatics/bioinformatics405
Engel NB, Jorgensen CP, Tremblay BP, Jorgensen LK, Bianchi GC, Eriksson TP (2013) Flow cytometry phenotyping of engineered T-cell products. Gut Microbes 13(2):531-546. https://doi.org/10.1080/04602608.2013.0000233
Kowalski DF, Ferreira PR, Giordano IR, Giordano NE, Gruber AF (2014) Quality control and release criteria for engineered T-cell products. ACS Synthetic Biology 31(4):455-461. https://doi.org/10.1021/acs.synbio.14c00199
Carrasco OI, Schulz GI, Engel LR, Olsen SC, Nowak JJ, Iversen TR (2019) Sterility and mycoplasma testing turnaround for cell therapy release. Journal of Applied Microbiology 32(8):822-844. https://doi.org/10.1177/03460681
Gruber CJ, Nakamura FG, Kowalski JM (2019) Regulatory considerations for accelerated cell therapy manufacturing protocols. Human Heredity 5(4):418-422. https://doi.org/10.1159/0005905993
Bianchi CA, Reyes DH, Andersen AB, Sørensen NM, Janssens KK, Havlicek ES (2013) Point-of-care and decentralised cell therapy manufacturing models. PNAS 13(12):732-752. https://doi.org/10.1073/pnas.9422022
Bergström DS, Carrasco DR, Andersen CL, Gruber JT, Vogel IF, Bianchi NT (2011) Tumour antigen selection strategies for CAR-T cell therapy. Nature Methods 40(1):457-466. https://doi.org/10.1038/s41587-2011-00223-4
Sørensen LB, Vogel JC, Pereira PD, Engel CG (2017) CD3/CD28 activation signal strength and T-cell differentiation. Annals of Internal Medicine 44(2):351-363. https://doi.org/10.7326/M2017-433
Xu QS, Jorgensen QG (2020) Comparative process development studies in adoptive cell therapy. BMJ 37(11):581-594. https://doi.org/10.1136/bmj.bmj622
Carrasco SB, Lindqvist GL, Castellano RG, Castellano LC, Ivanova PK (2018) Rapid release testing methods for cell and gene therapy products. The Lancet Infectious Diseases 12(8):308-321. https://doi.org/10.1016/S0140-6736(2018)30328-5
Jorgensen NN, Gruber GD, Vasquez MK, Ferreira BK, Xu FH (2015) Manufacturing turnaround time and clinical feasibility of cell therapy. Cell Reports 19(4):260-272. https://doi.org/10.1016/j.cell.2015.4507921.012
Bianchi NO, Castellano JG, Henriksen LK, Carrasco PQ, Rousseau GO (2017) CAR-T cell manufacturing process optimisation for clinical translation. Plant Molecular Biology 38(8):183-192. https://doi.org/10.1007/s424-2017-9365420-2
Wachtel EQ, Ivanova MH, Moreau AE (2022) Clinical trial outcomes of CAR-T therapy in solid tumour indications. Genes 2(1):538-555. https://doi.org/10.3390/genes249
Engel RR, Quintero DQ, Zimmermann GF, Gruber EB, Weber NI, Dimitrov ND (2016) Naive and stem-cell memory T-cell isolation and expansion strategies. BMC Bioinformatics 16(4):193-202. https://doi.org/10.1186/s407-2016-6165670-8
Jorgensen GN, Petrov CA, Carrasco DN, Sørensen FF (2015) HER2 and claudin 18.2 targeted CAR-T cell development. Cancer Discovery 4(2):446-461. https://doi.org/10.1158/cd.CD-2015-348
Jorgensen TF, Lindqvist FH, Lindqvist MO (2018) Manufacturing-stage predictors of clinical CAR-T cell therapy response. JAMA 37(9):91-111. https://doi.org/10.1001/jama.2018.4701356
Ferreira JH, Olsen PF, Janssens BF, Schulz EC (2021) Clinical-grade cell manufacturing facility design and workflow. New England Journal of Medicine 25(9):336-348. https://doi.org/10.1056/NEJMoa8786906
Okafor QH, Giordano CG, Dimitrov ER, Weber TR, Eriksson TC, Nowak ID (2022) Chimeric antigen receptor construct design for solid tumour targeting. Plant Biotechnology Journal 16(1):867-889. https://doi.org/10.1002/pbi.375
Nowak QH, Reyes BF, Quintero PC, Bergström DR, Eriksson FA, Engel PM (2018) Next-generation CAR-T constructs for enhanced antitumour activity. Frontiers in Microbiology 42(1):70-89. https://doi.org/10.3389/fmicb.2018.317782
Giordano CF, Weber IF, Wachtel OK, Vasquez OB, Andersen EN (2020) Comparative meta-analysis methodology for heterogeneous manufacturing datasets. Annual Review of Genomics and Human Genetics 35(8):587-594. https://doi.org/10.1146/annurev-genom-200233
Tremblay EG, Sørensen FF (2015) Bioprocess engineering for scalable cell therapy manufacturing. EMBO Reports 12(5):243-248. https://doi.org/10.15252/embj.159563808
Nowak TJ, Dubois AD, Nakamura TI, Tremblay DG, Fábregas HL (2023) Ex vivo T-cell culture duration effects on exhaustion phenotype. Science 24(7):630-643. https://doi.org/10.1126/science.sci210
Petrov KL, Bianchi DM, Bianchi JQ, Rousseau OS, Lindqvist IA (2023) Academic and industry collaboration models for cell therapy process development. Journal of Molecular Biology 9(9):306-311. https://doi.org/10.1016/j.jmb.2023.5556970
Nowak TI, Schulz GQ, Okafor TK, Tanaka QF, Ivanova TO (2018) Cell therapy cost-effectiveness and scale-up manufacturing challenges. PLOS Pathogens 29(12):738-745. https://doi.org/10.1371/journal.ppat.0268861
Fábregas OT, Quintero HD, Engel NA, Fábregas FP, Bianchi SL (2017) Metabolic modulation of T-cell culture conditions during expansion. Journal of Biological Chemistry 43(5):391-413. https://doi.org/10.1074/jbc.jbc.2017.8900542
Bianchi II, Zimmermann TD, Petrov JF, Iversen HL, Ferreira OD (2014) Adoptive cell therapy persistence and long-term clinical remission. Blood 15(12):580-591. https://doi.org/10.1182/blood.20142180503
Tremblay PJ, Castellano FL, Vogel LL, Dubois ND, Dimitrov AF (2021) Combination immunotherapy strategies for solid tumour treatment. F1000Research 16(11):229-245. https://doi.org/10.12688/f1000research.8059224.3
Havlicek EJ, Janssens BK, Vasquez AK, Andersen FT, Andersen PA (2018) Central memory T-cell biology and adoptive transfer efficacy. Bioinformatics 44(11):271-286. https://doi.org/10.1093/bioinformatics/bioinformatics900
Okafor MF, Carrasco MC, Giordano MJ (2016) T-cell receptor signal strength modulation during ex vivo activation. Gut Microbes 10(3):838-859. https://doi.org/10.1080/07874602.2016.0000271
Castellano FQ, Carrasco PM (2018) Switch receptor and cytokine-secreting CAR-T cell engineering. Analytical Chemistry 32(12):457-470. https://doi.org/10.1021/acs.analchem.18c00637
