Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

May combine two claims.

Development of bioreactors and closed system manufacturing platforms mitigates the need for expensive infrastructure and simplifies the multistep CAR-T manufacturing process.

“A paradigm shift enabling POC manufacturing is the development of bioreactors and closed system manufacturing platforms that mitigate the need for expensive infrastructure and simplify the multistep process.2,8,9”

• cites [2] Shah, 2023, Promises and challenges of a decentralized CAR T-cell manufacturing model, Front. Transplant. sourceSupported
• cites [8] Abou-El-Enein, 2021, Scalable manufacturing of CAR T cells for cancer immunotherapy, Blood Cancer Discov. sourcePartly supported

  The sections “Shifting from open to functionally closed systems” and “Replacing manual processing with automation” describe closed bioreactors and systems that reduce product handling and can operate in less stringent cleanrooms (page numbers unavailable). This supports some simplification and reduced cleanroom requirements, but not the unqualified claim that the platforms remove the need for expensive infrastructure: the paper says fill-finish still requires an ISO7 cleanroom and that the systems and reagents can be costly. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [9] Syed, 2022, Chimeric antigen receptor structure and manufacturing of clinical grade CAR engineered cells using different bioreactors, Hematol. Oncol. Stem Cell Ther. sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Early memory T cell subsets (naive, stem cell memory, and central memory T cells) are long-lived, possess high self-renewal ability, and exhibit long-term persistence and superior antitumor response in vivo.

“It is widely accepted that early memory T cell subsets (e.g., naive [TN], stem cell memory [TSCM] and central memory [TCM]T cells), which are long-lived and possess high self-renewal ability, exhibit long-term persistence and superior antitumor response in vivo.17,18,19”

• cites [17] Arcangeli, 2020, Next-generation manufacturing protocols enriching TSCM CAR T cells can overcome disease-specific T cell defects in cancer patients, Front. Immunol. sourcePartly supported

  The Introduction (page unavailable) says that enriching TSCM and TCM CAR T cells can enhance persistence and support a long-lasting antitumor response; the Results also link TSCM frequency to expansion in vivo. The paper does not establish that all three listed subsets, including naive T cells, are long-lived and highly self-renewing, or that each exhibits superior antitumor responses in vivo. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [18] Zhang, 2020, Engineering better chimeric antigen receptor T cells, Exp. Hematol. Oncol. sourcePartly supported

  In “The stage of CAR T cells differentiation” (page not provided), the review says CAR T-cell products rich in naïve, stem cell memory, and central memory T cells have superior antitumor responses and long-term persistence in vivo. It does not state that all three subsets are long-lived and have high self-renewal ability, and its outcome statement concerns CAR T-cell products rich in those subsets rather than each subset individually. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [19] Castella, 2020, Point-of-care CAR T-cell production (ARI-0001) using a closed semi-automatic bioreactor: Experience from an academic phase I clinical trial, Front. Immunol. sourcePartly supported

  The Discussion (page not provided) associates naïve, stem cell memory, and central memory T-cell phenotypes with longer persistence in vivo and higher antitumor efficacy. It does not state that all three subsets are long-lived or have high self-renewal ability, and its association is more qualified than the citing sentence’s categorical wording. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Fresh CAR-T cells were shown earlier to have higher in vitro antitumor activity and lesser microcellular damage than frozen CAR-T cells.

“POC manufacturing enables the delivery of fresh CAR-T cells, which were shown earlier to have higher in vitro antitumor activity and lesser microcellular damage than the frozen,4,5”

• cites [4] Brezinger-Dayan, 2022, Impact of cryopreservation on CAR T production and clinical response, Front. Oncol. sourcePartly supported

  The Discussion reports higher in vitro anti-tumor reactivity in fresh CAR T infusion products than in cryopreserved products. The Results describe differences in cell phenotype, but do not establish that fresh CAR T cells have less microcellular damage. No page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [5] Panch, 2019, Effect of cryopreservation on autologous chimeric antigen receptor T cell characteristics, Mol. Ther. sourcePartly supported

  The Results (page unavailable) report more apoptosis- and cell-cycle-damage-related gene expression in cryo-thawed cells, supporting the comparison of cellular damage. They also report greater expression of pathways associated with T-cell cytolysis in fresh cells, but do not establish higher in vitro antitumor activity; pathway expression is not a direct activity measurement. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

The LVV was manufactured and tested under a quality management system in compliance with GMP guidelines.

“The LVV was manufactured and tested under a quality management system in compliance with GMP guidelines and was previously used in numerous clinical studies.26,27,28”

• cites [26] Jackson, 2020, Automated manufacture of autologous CD19 CAR-T cells for treatment of non-hodgkin lymphoma, Front. Immunol. sourceCould not verify

  The Materials and Methods section (“Lentiviral Vector”; page unavailable) identifies the LVV used and its provider, but does not describe how it was manufactured or tested. The Introduction and Discussion describe GMP compliance for the CAR-T cell manufacturing system, not a quality management system for the LVV. Searches for vector production, quality control, and GMP testing found no passage addressing the claim. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [27] Maschan, 2021, Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients, Nat. Commun. sourcePartly supported

  The Methods section (page unavailable) says lentiviral vectors were manufactured. The Discussion (page unavailable) describes the CAR-T cell manufacturing system as GMP-compliant, but does not say the lentiviral vector itself was manufactured and tested under a GMP-compliant quality management system. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [28] Caimi, 2021, Prophylactic tocilizumab prior to anti-CD19 CAR-T cell therapy for non-Hodgkin lymphoma, Front. Immunol. sourceCould not verify

  The Methods section identifies a lentiviral vector used to manufacture CAR-T cells, but does not describe how the vector itself was manufactured or tested. Searches for GMP compliance and a quality management system found no passage addressing those claims; page numbers are unavailable in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

May combine two claims.

In cases 2, 5, and 6, where starting patient cells had very low CD4/CD8 ratios less than one-third (0.22, 0.29, and 0.20), there was an extremely low CD3+ T cell number (29.51 ± 6.28 × 10^6 cells) and a large amount of cell debris on day 5.

“However, we observed an extremely low CD3+ T cell number (29.51 ± 6.28 × 106 cells) and a large amount of cell debris on day 5 in cases 2, 5, and 6, where a starting patient’s cells (post-enrichment) had very low CD4/CD8 ratios of less than one-third (0.22, 0.29, and 0.20)”

May combine two claims.

The pre-enriched product contained 9.92% ± 4.55% CD4+ T cells and 22.69% ± 12.31% CD8+ T cells; post-column recovery was 31.72% ± 15.81% for CD4+ T cells and 55.12% ± 18.52% for CD8+ T cells, with an unchanged CD4/CD8 ratio (p = 0.418).

“The T cell composition of the pre-enriched product was 9.92% ± 4.55% in CD4+ T cells and 22.69% ± 12.31% in CD8+ T cells, and the post-column recovery of CD4+ T cells was 31.72% ± 15.81% and that of CD8+ T cells was 55.12% ± 18.52%, with an unchanged CD4/CD8 ratio (Figure 2C; p = 0.418).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The total number of enriched CD3+ T cells was 1.94 ± 0.77 × 10^9 cells, but only 100-150 × 10^6 cells were seeded in the culture chamber.

“The total number of enriched CD3+ T cells was 1.94 ± 0.77 × 109 cells, but only 100–150 × 106 cells were seeded in the culture chamber for the cultivation process”

May combine two claims.

At the time of manuscript submission, nine patients had been enrolled, two of whom (case nos. 3 and 7) received allogeneic CAR-T cells from their stem cell transplantation donors.

“At the time of submitting this manuscript, nine patients have been enrolled, two of whom (case nos. 3 and 7) received allogeneic CAR-T cells from their stem cell transplantation donors”

May combine two claims.

The total number of manufactured CAR+ CD3+ T cells in the no HS and 3% HS groups was 1.57 ± 0.32 × 10^9 cells, far exceeding the dosing need, and at least three bags of double doses were cryopreserved.

“The total number of manufactured CAR+ CD3+ T cells in no HS and 3% HS groups was 1.57 ± 0.32 × 109 cells (Figure 4D), far exceeding the dosing need (Figure 4E), and at least three bags of double doses were cryopreserved.”

May combine two claims.

For case 6, supplementing extra 3% HS as early as day 5 led to full recovery on day 11 and harvest on day 18.

“For case 6, an extra 3% HS was supplemented as early as day 5, and the cells fully recovered on day 11 and were harvested on day 18.”

May combine two claims.

The mean vector copy number was 1.97 ± 0.35 copies per CAR-T cell, and all products met the requirement of <5 copies per cell.

“with a mean VCN of 1.97 ± 0.35 copies per CAR-T cell (all of which met the requirement of authorities at <5 copies per cell).16”

• cites [16] Chen, 2020, Vector copy number quality control testing for CAR T-cells: critical validation parameters, Cytotherapy sourceNot checked — no full text of the source

May combine two claims.

The enrichment process did not significantly affect cell viability and yielded high T cell purity (range 78.67%-97.77%).

“The enrichment process did not significantly affect cell viability (Figure 2A) and yielded high T cell purity (range 78.67%–97.77%).”

May combine two claims.

The minimum target dose of 1 × 10^6 CAR-T cells per kilogram was not reached in case 5, and the patient received an adjusted dose of 0.14 × 10^6 CAR-T cells per kilogram.

“As the minimum target dose of 1 × 106 CAR-T cells per kilogram was not reached in case 5, the patient received an adjusted dose of 0.14 × 106 CAR-T cells per kilogram.”

May combine two claims.

For case 5, adding extra 5% HSA on day 11 failed to rescue the cells; they were harvested on day 14 as they could no longer proliferate.

“For case 5, we tried to rescue the cells by adding an extra 5% HS albumin (HSA) to the media on day 11, when we found out that the cells failed to self-recover, and harvested the cells on day 14 as they were no longer able to proliferate.”

May combine two claims.

Although SiCF-019 cells induced both CD19+ and CD19− tumor cell death in a dose-dependent manner, they had a more profound effect toward CD19+ cells.

“The results show that although SiCF-019 cells induced both CD19+ and CD19− tumor cell death in a dose-dependent manner, they had a more profound effect toward CD19+ cells.”

May combine two claims.

In the final products, median contaminating B cells were 0.00% (range 0.00-0.04), monocytes 0.00% (0.00-0.01), neutrophils 0.01% (0.00-0.46), and eosinophils 0.04% (0.00-0.29).

“The median contaminating B cells in the final products were 0.00% (range: 0.00%–0.04%), monocytes were 0.00% (range: 0.00%–0.01%), neutrophils were 0.01% (range: 0.00%–0.46%), and eosinophils were 0.04% (range: 0.00%–0.29%)”

May combine two claims.

Nine POC cell products designated SiCF-019 have been manufactured and used; seven were generated from heavily pretreated patients' own cells and two from healthy donors.

“To date, nine POC cell products, designated as SiCF-019 cells, have been manufactured and used—seven products were generated from heavily pretreated patients’ own cells, and two products were from healthy donors.”

May combine two claims.

Patient case 5 achieved complete response at 1 month and remained in complete response at 6 months despite receiving an adjusted dose.

“Despite this, patient case 5 achieved complete response (CR) at 1 month and remained in CR at 6 months.”

May combine two claims.

The transduction efficiency of the final products was 40.66% ± 16.23%, similar to that analyzed on day 5 of culture.

“The transduction efficiency of our final products was 40.66% ± 16.23%, similar to those analyzed on day 5 of culture”

May combine two claims.

A decent amount of NKT cells were detected (median 5.84%, range 4.41-10.69%), while there was a very minimal number of NK cells (median 0.12%, range 0.02-3.17%).

“A decent amount of natural killer T (NKT) cells were detected (median: 5.84%, range: 4.41%–10.69%), while there was a very minimal number of NK cells (median: 0.12%, range: 0.02%–3.17%).”

May combine two claims.

Seven leukapheresis products from heavily pretreated patients with NHL or ALL had low CD4/CD8 ratios (median 0.352, range 0.196-0.657), and two products from healthy HLA-matched sibling donors had normal CD4/CD8 ratios (median 1.163, values 0.784 and 1.541).

“Seven leukapheresis products were obtained from heavily pretreated patients with non-Hodgkin lymphoma (NHL) or acute lymphoblastic leukemia (ALL), most of which had a low CD4/CD8 ratio (median 0.352, range 0.196–0.657), and two products were from healthy, HLA-matched sibling donors (case IDs H3 and H7) with a normal CD4/CD8 ratio (median 1.163, values of 0.784 and 1.541).”

May combine two claims.

No significant differences were observed in transduction efficiency on day 5, total CAR-T cells at end of culture, and in vitro antitumor response between culture conditions with and without HS at end of culture.

“We did not observe the significant differences in transduction efficiency on day 5 of culture (p = 0.07), the number of total CAR-T cells at the end of culture (p = 0.21), and in vitro antitumor response (p > 0.05) between the culture conditions with and without HS at the end of culture (Figures 4 and S2).”

May combine two claims.

In this pilot study, enrolled patients had received multiple prior lines of therapy, and their T cells had relatively low fitness as evaluated by CD4/CD8 ratio (median 0.352).

“In the current pilot study, only patients who had previously received multiple lines of therapies were eligible; hence, their T cells had a relatively low fitness, as evaluated by the CD4/CD8 ratio (median 0.352).”

May combine two claims.

At an effector-to-target ratio of 5:1, death rates were 61.65% ± 13.90% in CD19+ Z-138 cells and 44.88% ± 20.38% in CD19+ REH cells versus 22.31% ± 10.12% in CD19− K562 cells (p < 0.001 and p < 0.05, respectively).

“For example, the death rates at the E:T ratio of 5:1 were 61.65% ± 13.90% in CD19+ Z-138 cells and 44.88% ± 20.38% in CD19+ REH cells versus 22.31% ± 10.12% in CD19− K562 cells (p < 0.001 and p < 0.05, respectively)”

May combine two claims.

Despite low T cell fitness, manufacturing yielded CAR-T cell products far exceeding dosing need from most patients' materials (median 13 doses, range 7-20), except rescued cases with CD4/CD8 ratio <1/3 (median 0.21).

“Despite this, we were able to manufacture CAR-T cell products that far exceeded the dosing need from most patients’ materials (median: 13 doses, range: 7−20 doses), except for the rescued cases, in whom the CD4/CD8 ratio was less than one-third (median: 0.21).”

May combine two claims.

SiCF-019 cells exposed to Z-138 cells produced a significant amount of TNF-α but had a substantially greater IFN-γ level than untransduced T cells even without stimulation with target tumor cells.

“Figure 6B shows that SiCF-019 cells exposed to Z-138 cells produced a significant amount of TNF-α but had a substantially greater IFN-γ level than untransduced T cells even without stimulation with target tumor cells.”

May combine two claims.

In rescued cases 5 and 6, the CD3+ T cell number dropped tremendously after transduction, evaluated on days 5, 7, or 8 of culture, concomitant with a remarkable increase in TIM3 and PD1.

“In both cases, the CD3+ T cell number dropped tremendously after transduction, as evaluated on days 5, 7, or 8 of culture (Figure 7A), concomitant with a remarkable increase in TIM3 and PD1 (Figure 7B).”

May combine two claims.

All patients except case 7, who died from severe cytokine release syndrome, achieved complete response within 1 month, and lymphoma responses were sustained, with the majority of patients having complete response maintained for at least 6 months.

“we observed that all patients—except for case 7, who died from severe cytokine release syndrome (CRS)—achieved CR within 1 month and that lymphoma responses were sustained, with the majority of patients having CR maintained for at least 6 months”

May combine two claims.

The majority of circulating CD4+ CAR-T cells were TCM, while the majority of circulating CD8+ CAR-T cells were either TCM or TEM.

“Figure 8D shows that the majority of circulating CD4+ CAR-T cells were TCM, while the majority of circulating CD8+ CAR-T cells were either TCM or TEM”

May combine two claims.

CAR-T cells peaked in most patients in the first week (day 7) and, in all cases, within the first month after infusion.

“CAR-T cells peaked in most patients in the first week (day 7) and, in all cases, within the first month after infusion”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

The POC manufacturing and quality control settings could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.

“The POC manufacturing and quality control settings herein could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.”

• AssumptionRests on an assumption: Access to centralized, industrial CAR-T cells is not economically feasible for most local Thai patients.
• AssumptionRests on an assumption: Point-of-care cell products are considerably less costly, abstracting from intellectual property rights.

May combine two claims.

The phenotypic and functional characteristics of the manufactured cell products qualified them for clinical application, supporting the feasibility of POC manufacturing of CAR-T cells using a GMP-compliant closed system in Thailand.

“Here, we demonstrated the phenotypic and functional characteristics of the cell products obtained that qualified them for clinical application, supporting the feasibility of POC manufacturing of CAR-T cells using a GMP-compliant closed system in Thailand.”

Classified as probably not a substantial claim.

Decentralized or point-of-care manufacturing of CAR-T cells at academic medical centers could reduce the acquisition cost and complexity of the manufacturing process, including cryopreservation and cold chain logistics.

“Alternatively, decentralized or point-of-care (POC) manufacturing of CAR-T cells at academic medical centers could reduce the acquisition cost and complexity of the manufacturing process (i.e., cryopreservation and cold chain logistics).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The research was conducted in the absence of any commercial, proprietary, or financial relationships that could be construed as a potential conflict of interest.

“All authors declare no competing interests. The research was conducted in the absence of any commercial, proprietary, or financial relationships that could be construed as a potential conflict of interest.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Mycoplasma testing for IPC and product release was determined in-house by a MycoAlert PLUS Mycoplasma Detection Kit and subsequently validated by molecular testing by qPCR.

“Mycoplasma testing for IPC and product release was determined in-house by a MycoAlert PLUS Mycoplasma Detection Kit (Lonza), which is a rapid, selective biochemical assay based on the activity of mycoplasmal enzymes, due to time constraints, and subsequently validated by molecular testing by qPCR at the Research Division, Faculty of Medicine Siriraj Hospital.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Products were released when IPC samples passed the generally employed 6- or 7-day incubation periods of the blood culture system and cautiously used while awaiting the release of final sterility reports.

“Products were released when IPC samples passed the generally employed 6- or 7-day incubation periods of the blood culture system and cautiously used while awaiting the release of final sterility reports.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

This study was approved by the Siriraj institutional review board (COA no. Si 966/2020; SIRB Protocol no. 472/2562(EC1)) and conducted in accordance with the 1975 Declaration of Helsinki.

“This study was approved by the Siriraj institutional review board (COA no. Si 966/2020; SIRB Protocol no. 472/2562(EC1)) and conducted in accordance with the 1975 Declaration of Helsinki.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The trial is registered on the ISRCTN registry (ISRCTN17901467).

“The trial is registered on the ISRCTN registry (ISRCTN17901467, https://www.isrctn.com/ISRCTN17901467).”

Classified as probably not a substantial claim.

All participating patients and healthy donors gave written informed consent.

“All participating patients and healthy donors gave written informed consent.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All authors declare no competing interests.

“All authors declare no competing interests. The research was conducted in the absence of any commercial, proprietary, or financial relationships that could be construed as a potential conflict of interest.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All reagents used were GMP products purchased from Miltenyi Biotec, unless otherwise specified.

“All reagents used were GMP products purchased from Miltenyi Biotec, unless otherwise specified.”