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Immune cell therapies and immune cell engineering
Original research
Placental circulating T cells: a novel, allogeneic CAR-T cell platform with preserved T-cell stemness, more favorable cytokine profile, and durable efficacy compared to adult PBMC-derived CAR-T
  1. Natalia Ruggeri Barbaro,
  2. Theodore Drashansky,
  3. Kristina Tess,
  4. Mansour Djedaini,
  5. Robert Hariri,
  6. Shuyang He,
  7. William van der Touw and
  8. Kathy Karasiewicz
  1. Celularity Inc, Florham Park, New Jersey, USA
  1. Correspondence to Kathy Karasiewicz; kathy.karasiewicz{at}celularity.com

Abstract

Background Chimeric antigen receptor (CAR)-T cell quality and stemness are associated with responsiveness, durability, and memory formation, which benefit clinical responses. Autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture. Thus, strategies to develop allogeneic CAR-T platforms including the identification and expansion of T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Here, we compared CAR-T cells generated from healthy adult peripheral blood T cells versus placental circulating T (P-T) cells.

Methods CAR-T cells from healthy adult peripheral blood mononuclear cells (PBMCs) and P-T cells were generated using the same protocol. CAR-T cells were characterized in detail by a combination of multiparameter flow cytometry, functional assays, and RNA sequencing. In vivo antitumor efficacy and persistence of CAR-T cells were evaluated in a Daudi lymphoma xenograft model.

Results P-T cells possess stemness advantages compared with T cells from adult PBMCs. P-T cells are uniformly naïve prior to culture initiation, maintain longer telomeres, resist immune checkpoint upregulation, and resist further differentiation compared with PBMC T cells during CD19 CAR-T manufacture. P-T CD19 CAR-T cells are equally cytotoxic as PBMC-CD19 CAR-T cells but produce less interferon gamma in response to lymphoma. Transcriptome analysis shows P-T CD19 CAR-T cells retain a stem-like gene signature, strongly associate with naïve T cells, an early memory phenotype, and a unique CD4 T cell signature compared with PBMC-CD19 CAR-T cells, which enrich for exhaustion and stimulated memory T cell signatures. Consistent with functional data, P-T CD19 CAR-T cells exhibit attenuated inflammatory cytokine and chemokine gene signatures. In a murine in vivo model, P-T CD19 CAR-T cells eliminate lymphoma beyond 90 days. PBMC-CD19 CAR-T cells provide a non-durable benefit, which only delays disease onset.

Conclusion We identified characteristics of T cell stemness enriched in P-T CD19 CAR-T which are deficient in PBMC-derived products and translate into response durability in vivo. Our findings demonstrate that placental circulating T cells are a valuable cell source for allogeneic CAR-T products. Stemness advantages inherent to P-T cells translate to in vivo persistence advantages and long-term durable activity.

  • Chimeric antigen receptor - CAR
  • T cell
  • Adoptive cell therapy - ACT

Data availability statement

Data are available in a public, open access repository. RNA-Seq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE231429 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231429). For other original data, please contact kathy.karasiewicz{at}celularity.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-008656

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • The manufacturing of autologous chimeric antigen receptor (CAR)-T cells is subject to several limitations, including failure rates, extended manufacturing times, complex logistical challenges, variability in both the starting materials and final products, high costs, and the potential for lower quality CAR-T cells due to the influence of the patient’s disease and prior chemotherapy treatments.

    • Clinical trials and animal studies indicate that optimizing the frequency of naïve and stem cell memory T cells in CAR-T cells leads to better clinical response and persistence.

    • Allogeneic, off-the-shelf CAR-T products may offer an alternative option. However, the source of T cells may introduce heterogeneity among CAR-T cell products. Here, we compare healthy adult peripheral blood T cells to placental circulating T cells for the generation of CAR-T drug products.

    WHAT THIS STUDY ADDS

    • CAR-T derived from placental circulating T cells possess a transcriptomic signature consistent with a less differentiated T cell, which is more resistant to exhaustion than CAR-T derived from healthy, adult peripheral blood mononuclear cells (PBMCs).

    • CAR-T derived from placental circulating T cells have a more favorable cytokine profile with interleukin 2 (IL-2)-skewed naïve helper T cells, that favors IL-2 over interferon gamma secretion, which may lower the potential for toxicity.

    • CAR-T derived from placental circulating T cells demonstrate improved and prolonged in vivo efficacy and persistence compared with healthy, adult PBMC derived CAR-T.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • This study presents a novel, allogeneic T cell platform which enables large scale manufacturing of off-the-shelf CAR-T cells with homogeneous and preserved T-cell stemness to enhance efficacy and improve accessibility of cell therapy to patients.

    Introduction

    Immunotherapy using autologous gene-modified T cells expressing a chimeric antigen receptor (CAR) has changed the lymphoma treatment landscape, achieving complete remissions in heavily pretreated refractory patients.1 However, a number of patients fail to respond or relapse following CAR-T therapy. This may be due to inferior quality of CAR-T cells, variability of starting material and/or T-cell exhaustion from resistant disease.2 Since autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture, strategies to identify and expand the T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Defining T cell specifications that improve successful CAR-T manufacture and maximize clinical efficacy is a shared goal in both the autologous and allogeneic CAR-T spaces.

    T cell stemness, characterized by the ability for self-renewal, multipotency across various T cell lineages, and long-lasting persistence, plays a crucial role in the establishment of durable T cell memory throughout an individual’s lifespan.3 4 Early CAR-T studies revealed that in contrast to T effector (TE) and T effector memory (TEM) populations, CAR-T derived from CD8+ central memory (TCM) T cells were most effective in the presence of naïve (TN) or TCM CD4+ T cell help.5 Within the memory population, a less differentiated T stem cell memory (TSCM) population possesses even greater antitumor activity.4 6 Because TN and TSCM cells in adult peripheral blood mononuclear cells (PBMCs) are less abundant, additional efforts to enrich TSCM or TCM populations during CAR-T manufacture have been performed through selection and/or use of cytokines.7–9 Preclinical models have demonstrated the antitumor benefit of TSCM-enriched CAR-T cells compared with non-enriched cells.9 10 However, the scalability and reproducibility of autologous TSCM expansion across patients and indications can have some limitations.11 Additionally, patients with cancer have a reduced TSCM compartment compared with healthy donors and often have depleted T cell counts due to prior chemotherapy and radiation.12 The remaining T cells manifest higher rates of cell exhaustion, compelling the field to find robust alternatives to autologous therapy. Allogeneic CAR-T approaches can better address product consistency and provide immediate availability for patients as an “off-the-shelf” therapeutic.

    While allogeneic T cells from healthy adult peripheral blood are currently being used, T cells from postpartum placenta/umbilical cord may offer potential phenotypical and functional advantages as a CAR-T cell drug product.13 Cord blood derived T cells have shown promise as the starting material for allogeneic CAR-T cell therapy.14 15 However, a direct comparison of adult peripheral blood T cells to placental circulating T (P-T) cells for CAR-T has not been adequately investigated and is the objective of the present study. In this study, we show that blood from the placenta and umbilical cord are a rich source of T cells whose TN enrichment could prove advantageous to CAR-T manufacture. While CAR-T manufactured from both cell sources demonstrate comparable in vitro CD19-directed cytotoxicity, P-T derived CD19 CAR-T cells (P-T CD19 CAR-T) resist activation-induced maturation and immune checkpoint expression during manufacture. We further show that P-T CD19 CAR-T maintains a CD4 T cell population which favors interleukin 2 (IL-2) over interferon gamma (IFN-γ) secretion. Transcriptome analysis of CAR-T drug product derived from P-T cells and adult PBMCs revealed a diverging CAR-T identity, with cells derived from the placenta displaying some clear phenotypical advantages.16 P-T CD19 CAR-T had attenuated cytokine and chemokine activation, naïve T cell identity, an early memory phenotype, and a unique CD4 T cell signature. Conversely, adult PBMC-CD19 CAR-T cells were enriched for exhaustion and stimulated memory T cell signatures. In line with these phenotypical advantages, P-T CD19 CAR-T cells demonstrated superior and more durable CD19+ lymphoma control in a lymphoma NSG mouse model. Altogether, our findings demonstrate that placental circulating T cells are a promising source for allogeneic CAR-T cell therapy and are enriched for stemness attributes which benefited their in vivo persistence and long-term durable activity.

    Methods

    T cell isolation

    PBMCs were isolated from consenting healthy donor whole blood using Ficoll gradient centrifugation (donors age 49.3±9.9 years). Placental units (collected within <48 hours postdelivery of normal, healthy, full-term pregnancy) were obtained from consenting mothers. The circulating cells collected from the placental units (placenta and umbilical cord blood) were washed using spinning membrane filtration (Lovo). Mononuclear cells were depleted of monocytes and regulatory T cells (Tregs) using Miltenyi CD14 and CD25 microbeads using CliniMACS (Miltenyi) magnetic bead-based separation or LS columns. Cell fractions were then positively selected for T cells using CD4 and CD8 microbead selection. Eluted cells were frozen using controlled-rate cell freezing, then cryopreserved in liquid nitrogen vapor phase.

    CD19 CAR-T cell generation

    Isolated T cells were activated using 1% TransAct (Miltenyi) and expanded for 3 days in G-Rex plates (Wilson Wolf) containing AIM-V medium (Gibco) supplemented with 5% human serum (Millipore Sigma) and 100 IU/mL of IL-2 (Life Technologies). Transduction with CD19 CAR gene-expressing retrovirus was performed on day 3 in RetroNectin (TaKara Biotech)-coated plates. The CD19 CAR construct contained the anti-CD19 FMC63-based scFv, a hybrid CD8a/CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and the CD3z signaling domain. Transduced cells were expanded from day 4 through day 13. On day 7, T cells underwent CRISPR-Cas9-mediated T-cell Receptor Alpha Chain constant (TRAC) knock-out using the MaxCyte ATx electroporation system, followed by depletion of T-cell receptor (TCR)-expressing cells using TCR α/β microbeads (Miltenyi) on day 12. Cells were cryopreserved on day 13 and stored in liquid nitrogen vapor phase. For in vitro functional experiments, CD19 CAR-T cells were thawed, then recovered for 24 hours in AIM-V complete media at 37°C, 5% CO2.

    Cytotoxicity assay

    Electrical impedance monitoring using xCELLigence (ACEA Biosciences) measured real-time target cell index for determining cytotoxicity. Daudi cells were immobilized to E-Plates (Agilent Technology) coated with anti-CD40 (Agilent Technology). Recovered CAR-T cells were added to Daudi cells at defined effector-to-target (E:T) ratios in RPMI 1640 media with 10% fetal bovine serum incubating at 37°C, 5% CO2. Each donor was analyzed in duplicate, measuring percent cytotoxicity at 4, 8, 12, and 24 hours of coculture time.

    Cytokine release assay

    Recovered CAR-T cells were cocultured with Daudi cells at 1:1 E:T ratio in duplicate in RPMI 1640 media with 10% fetal bovine serum for 24 hours at 37°C, 5% CO2. Collected supernatant was analyzed for IL-2, IFN-γ, and tumor necrosis factor alpha (TNF-α) concentration using the MSD (Meso Scale Diagnostics) platform according to manufacturer’s protocol. Data were analyzed using MSD Discovery Workbench V.4.0 software.

    Telomere length measurement

    Cell telomere length was measured using Telomere PNA Kit (Dako) according to manufacturer’s protocol. The 1301 cell line (Sigma-Aldrich) was used as a control for intra-assay and interassay variability. Quantum Molecules of Equivalent Soluble Fluorochrome (Bangs Laboratories) beads were used for the standardization of fluorescence intensity units to perform absolute fluorescence quantification.

    Flow cytometry

    Refer to online supplemental method for details.

    Supplemental material

    Gating strategies for starting material purity, differentiation and phenotype analysis are shown in online supplemental figure 1. All antibodies are listed in online supplemental table 1.

    RNA-sequencing and bioinformatic analysis

    Refer to online supplemental method for details.

    In vivo model

    To assess efficacy in vivo, P-T and PBMC-derived CD19 CAR-T cells (both without TRAC knockout) were evaluated in a disseminated Daudi (Burkitt’s) lymphoma xenograft model in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory). In vivo studies were conducted at a contract research organization, Invivotek LLC, where fifteen 9-week-old female mice were housed together in cages and acclimated for 2 weeks. Based on previous studies, five experimental mice per group were used to determine statistical significance of treatment effect on group survival. There was no exclusion of experimental mice. Study personnel administering treatments to mice were aware of groups receiving vehicle control versus CAR-T cells but had no prior knowledge of key differences between the cell types administered.

    All mice were preconditioned with a 30 mg/kg dose of busulfan 7 days prior to Daudi inoculation. On day 0, mice received a 3×106 dose of luciferase-labeled Daudi (Daudi-luc+) cells via intravenous administration. Seven days post-tumor cell inoculation, mice were randomized based on body weight and treated with vehicle (n=5; control group), 3×106 PBMC-CD19 CAR+-T (n=5), or 3×106 P-T CD19 CAR-T (n=5) cells by intravenous injection. CAR-T antitumor efficacy was monitored via bioluminescence imaging and survival. On day 122, surviving mice (n=5) were rechallenged with 3×106 Daudi-luc+ cells via intravenous administration. Age-matched NSG mice (n=5) served as controls and were injected with the same dose of Daudi-luc+ cells. Blood, spleen, and bone marrow samples were collected on days 185–215 from the remaining mice (n=4) and analyzed by flow cytometry for the presence and phenotype of persisting CAR-T cells. This study was repeated with additional CAR-T donors (n=5 mice per treatment group). An additional animal study was performed to compare P-T CD19 CAR-KO (with TRAC knockout; n=5) versus P-T CD19 CAR-NT (without TRAC knockout; n=5) with similar conditions as those described earlier with the exception that 6×106 CAR-T cells were infused into mice.

    Statistical analysis

    Statistical analysis was performed using GraphPad Prism V.9.0.2 (GraphPad). Unpaired two-tail t-test calculations were used for comparisons between placental circulating and PBMC-derived T cells. Comparisons of multiple groups were performed using one-way analysis of variance with Dunnett post-hoc test. A log-rank (Mantel-Cox) test was used to compare survival between different groups of mice. Data are represented as mean±SD or SEM as indicated in each figure’s legend. The significance level for all statistical tests was set at p=0.05.

    All reagent details are listed in online supplemental table 2.

    Results

    Placental circulating T cells are enriched for CD45RA+CCR7+ TN/TSCM

    CD4/CD8 T cell ratios have been reported to impact CAR-T performance5 17 18 leading us to assess any differences in the composition of P-T versus adult PBMC-T cells. P-T and PBMC-T showed an equivalent 2:1 ratio of CD4+ T cells to CD8+ T cells among total CD3+ T cells (figure 1A,B). We next compared the T cell subset composition of adult PBMCs to the post partum placenta. Low percentage (<1.2%) of γδ T cells+ was detected in both PBMC-T and P-T starting materials (online supplemental figure 2). CD45RA, CCR7, and CD27 staining by flow cytometry distinguished effector and memory T cells from naïve and stem cell memory subsets. Using CD45RA and CCR7 to identify TN/TSCM, P-T cells were >90% enriched compared with approximately 40% among PBMC-T cells (figure 1C,D). CD27, a lymphocyte costimulatory molecule, was uniformly expressed at high levels among all P-T cells (figure 1E,F) compared with PBMC-T (86±15.4 vs 99.8±0.17%) consistent with their identity as TN/TSCM as previously reported.19 The CD4:CD8 ratio did not vary between multiple evaluated donors (figure 1B) while the observed distribution of T cell differentiation status between P-T and PBMC-T was statistically significant (figure 1D). PBMC CD4+ T cells were comprised of a mix of TN/TSCM (CD45RA+CCR7+), TCM (CD45RA−CCR7+), TEM (CD45RA−CCR7−) cells, with limited TE (CD45RA+CCR7−) population, while CD8+ T cells contained all four populations. A clear difference was seen in the P-T cells where all cells, whether CD4+ or CD8+ T cells, were TN/TSCM. This distinction within the starting material highlights how differences in T cell subpopulation distribution may influence T cell expansion and phenotype during CAR-T expansion. CD57, a glycan whose expression is associated with differentiated effector memory T cell subsets20 and associated with aging21 was expressed in PBMC-T and no expression was observed in P-T cells (10.6±9.5 vs 0.09±0.05%) (figure 1G,H). Higher expression of exhaustion markers including PD-1 (26.6±4 vs 7.2±3.2) and TIGIT (17.7±11.63 vs 1.2±0.8) was observed in PBMC-T compared with P-T, while no difference was observed in LAG3 and TIM-3 (figure 1I,J).

    Figure 1
    Figure 1

    Placental circulating T cell donor stocks are enriched for naïve/stem cell memory T cells compared with adult derived PBMC T cells. (A) Representative pseudocolor plots of CD4+ and CD8+ T cell distribution among CD56−CD3+ lymphocytes present in P-T and PBMC-T donor stocks. (B) Comparison of CD4+ and CD8+ T cell distribution among different PBMC donors (black, n=6) and P-T donors (green, n=9). (C) Representative pseudocolor plots of CD45RA and CCR7 T cell staining among CD56−CD3+CD4+ lymphocytes present in P-T and PBMC T donor stocks. (D) Comparison of TN/SCM, TCM, TEM, and TE subset distribution among multiple PBMC-T (black) and P-T (green) cell donors based on CD45RA and CCR7 staining. (E) Representative histograms of CD27 expression on total T cells from PBMC-T and P-T donor stocks. (F) Comparison of CD27 expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. (G) Representative pseudocolor plots of CD57 expression on total T cells from PBMC-T and P-T donor stocks. (H) Comparison of CD57 expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. (I) Representative pseudocolor plots of PD-1 and TIM-3 expression and representative histogram of TIGIT expression on total T cells. (J) Comparison of exhaustion markers PD-1, TIM-3, LAG3, and TIGIT expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. Numbers indicate the percentage of cells in each gate. Individual data points shown with mean value±SD. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells.

    CD19 CAR-T generated from placental circulating T cells retain stemness characteristics

    We next evaluated CAR-T cells postexpansion as T cell phenotype may shift following activation, CAR transduction, and cell proliferation. Figure 2 depicts the P-T CD19 CAR-T manufacturing process. The 1:1 ratio of CD4/CD8 CAR-T cells has been shown to confer superior antitumor reactivity in vivo, indicating the synergistic antitumor effects of the two subsets.5 18 22 While P-T CD19 CAR-T maintained a more balanced ratio of CD4+ and CD8+ T cells, adult PBMC-CD19 CAR-T preferentially expanded CD8+ T cells (figure 3A). CD19 CAR transduction efficiency was similar between the two groups (figure 3B). P-T CD19 CAR-T cells retained longer telomeres compared with PBMC-CD19 CAR-T postexpansion (9.1±1.0% vs 15.2±1.3%) (figure 3C), suggesting better T cell proliferative potential and possibly better telomerase activity during T cell activation.23 24 T cell subset (TSCM, TCM, TEM, TE) evaluation of CAR-T products by flow cytometry yielded similar phenotype distribution for both CD4+ and CD8+ T cells in PBMC-CD19 CAR-T and P-T CD19 CAR-T due to IL-2 stimulation and activation-induced CCR7 downregulation (online supplemental figure 3).25 Loss of CD27 has been associated with effector cell differentiation among CD4+ and CD8+ T cells.26 27 Consistent with the profile of P-T starting material, high levels of CD27 costimulatory molecule expression were maintained on P-T CD19 CAR-T (76.8±13%), while CD27 expression was reduced among PBMC-CD19 CAR-T (40.3±16.1%) (figure 3D). CD57 expression was expressed at a significantly lower frequency on P-T CD19 CAR-T cells compared with PBMC-CD19 CAR-T (30.3±11.3% vs 13.1±7.1%) (figure 3E), suggesting that placental T cells better maintain a naïve-like T cell state post-CAR-T expansion. T cell differentiation involving T-bet and EOMES is responsible for effector and memory cell maturation.28 Expression of both transcription factors was significantly diminished in P-T CD19 CAR-T cells compared with PBMC-CD19 CAR-T (EOMES: 44.5±16.9 vs 10.6±12.6 and T-bet: 69.6±6.5 vs 60.1±7.6) (figure 3F). Expression of costimulatory molecule GITR, which supports proliferation and T cell activation by inducing IL2R and IL-2,29 was increased in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T (53.4±12 vs 82.6±8.5) (figure 3G). Immune checkpoint molecules PD-1, TIM3, LAG3, and TIGIT are negative regulators that dampen T cell activation.30 PD-1 and TIM3 were also induced on T cells following TCR-mediated activation and in response to common γ-chain cytokines including IL-2, respectively.30 31 P-T CD19 CAR-T expressed reduced levels of LAG3 (9.7±5.6 vs 0.5±0.2%) and TIGIT (47.4±9.5 vs 13.2±2.7%) compared with PBMC-CD19 CAR-T cells, which were increased in PBMC-CD19 CAR-T (figure 3H,I). While no difference was observed in TIM-3, PD-1 was elevated among P-T CD19 CAR-T (27.1±4.2 vs 49.5±6.0%). These data collectively demonstrate improved maintenance of a less differentiated phenotype among placental circulating T cells following the genetic engineering and expansion of CAR-T cells.

    Figure 2
    Figure 2

    P-T CD19 CAR-T manufacturing scheme. Placental circulating cells are collected at the hospital from normal, healthy, full-term pregnancy then shipped to the manufacturing facility. In upstream manufacturing, ≤48 hours postdelivery placental circulating cells are processed using the Lovo automated system followed by monocyte and regulatory T cell depletion and CD4+CD8+ T cells enrichment. The starting material T cells are then cryopreserved. In downstream manufacturing, isolated placental T cells are activated and expanded for 3 days then transduced with CD19 CAR retrovirus. On day 7, T-cell Receptor Alpha Chain Constant (TRAC) is knocked-out and the cells are allowed to expand for an additional 5 days. On day 12 any residual TCR-expressing cells are depleted, and the cells are left to expand for an additional day. On day 13 P-T CD19 CAR-T cells are cryopreserved and stored in liquid nitrogen vapor phase. CAR, chimeric antigen receptor; P-T, placental circulating T cells; TCR, T-cell receptor.

    Figure 3
    Figure 3

    P-T CD19 CAR-T cells better retain stemness markers than PBMC-CD19 CAR-T cells following culture expansion. (A) CD4+ and CD8+ T cell frequencies in post-thaw PBMC-CD19 CAR-T (black, n=6 donors) and P-T CD19 CAR-T (green, n=9 donors) drug product. (B) Representative pseudocolor plots and bar graphs of CD19 CAR expression on post-thaw PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) drug product. (C) Relative telomere length of total cells post-thaw of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) cell products. (D) Representative histograms and frequencies of CD27+ in total CD5+CAR+ T cells of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (E) Representative pseudocolor plots and frequencies of CD57+ in CAR+ T cells of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (F) Intracellular staining of transcription factors EOMES and T-bet on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green). (G) Surface staining of immunoregulatory receptors GITR and CD25, and from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (H) Representative pseudocolor plots or histograms of exhaustion markers expression PD-1, TIM3, LAG3, and TIGIT on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) (I) Frequencies of exhaustion markers expression PD-1, TIM3, LAG3, and TIGIT on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T. Individual data points shown with mean value±SD. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor.

    Recent publications have shown that TCR knockout may negatively impact persistence and phenotype of CAR-T cells,32 33 however, we did not observe phenotypic differences between CD19 CAR-T cells with intact TCR (CAR-NT) and TCR knockout cells (CAR-KO) in either PBMC-T or P-T CD19 CAR-T cells including the expression of exhaustion and activation markers (CD57, PD-1, TIM-3, LAG-3, TIGIT, EOMES, CD25) or T cell differentiation status (online supplemental figure 4). Moreover, we used an immunodeficient murine model to compare P-T CD19 CAR-T with intact (CAR-NT) or with deletion of TCR (CAR-KO). NSG mice were preconditioned with Busulfan, followed by Luciferase-expressing Daudi lymphoma inoculation 1 week later. P-T CD19 CAR-T cells with or without TCR deletion were administered intravenously after another week, and mice were monitored for tumor burden using bioluminescence imaging and survival over time. Similar survival was observed between groups indicating that the deletion of TCR does not impact P-T CD19 CAR-T efficacy or CAR-T cells fitness (online supplemental figure 4).

    Transcriptional profiling of CAR-T products identifies distinct differentiation and inflammatory pathways associated with placental starting material

    The significant phenotypic differences that were observed between PBMC-CD19 CAR-T and P-T CD19 CAR-T despite using identical expansion protocols prompted us to analyze the transcriptomic signatures to better capture distinct product characteristics. Full list of differentially expressed genes (DEGs) and normalized counts are shown in online supplemental tables 3 and 4. Next generation RNA sequencing (RNA-seq) data generated from PBMC-CD19 CAR-T (n=6) and P-T CD19 CAR-T (n=7) donors16 clustered in divergent populations corresponding to starting material source following principal component analysis (figure 4A). When comparing DEGs between PBMC-CD19 CAR-T and P-T CD19 CAR-T cohorts, significant differences between many gene targets were observed (figure 4B). Notably, the greatest differences in gene expression were in genes associated with immune function. PBMC-CD19 CAR-T enriched genes associated with the Th17 axis (IL17A, IL17F, IL21, IL22, IL26), inflammation (IFNG, IFNg, IL3, IL5, GZMB, GZMH), chemokines (CCL1, CCL4, CCL3, CXCL2, CXCL8, CXCL10), and immune checkpoints (TIGIT, LAG3) (figure 4B,C, online supplemental figure 5 and table 6). P-T CD19 CAR-T cells were enriched for TLR2, FCER1G, SYK, and IKZF2 (HELIOS) (figure 4B), all genes implicated in T cell activation and maturation state.34–36 P-T CD19 CAR-T also expressed KIT, which is typically expressed in hematopoietic stem cells and progenitor cells but not among adult T cells.37 Unbiased cluster analysis confirmed that DEGs were most strongly associated with starting material source among all donors evaluated (figure 4C, online supplemental figure 5 and table 6). Consistent with reduced IFN-γ levels, STAT1, JAK2, IFN-γ pathways were all significantly reduced in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T donors (figure 4D,E and online supplemental figure 6). Gene signature sets for naïve/stemness were assessed for gene enrichment showing preservation of T-cell stemness in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T cells (online supplemental table 5 and figures 7 and 8). Transcription factors EOMES andTBX21 (T-bet), as well as checkpoint molecules TIGIT and LAG3 were significantly reduced among P-T CD19 CAR-T (figure 4F,G) confirming protein data we previously observed.

    Supplemental material

    Figure 4
    Figure 4

    Transcriptional profiles of CAR-T cellular products identify distinct differentiation and inflammatory pathways associated with neonatal starting material. (A) Principal component analysis of PBMC-CD19 CAR-T (black, n=6 donors) and P-T CD19 CAR-T (green, n=7 donors) and based on differentially expressed genes (DEGs). (B) Volcano plot showing DEGs of P-T CD19 CAR-T versus PBMC-CD19 CAR-T. Genes with p value <0.05 and gene fold change ≥1.5 are marked in black with genes not meeting the cut-off shown in gray. Annotated genes of interest are highlighted in red. (C) Clustering heat map of the top 100 DEGs between PBMC-CD19 CAR-T versus P-T CD19 CAR-T using a cut-off z score=3. Gene list is shown in online supplemental table 6 and online supplemental figure 5. (D) IFN-γ signaling pathway gene counts of IFNG, STAT1, and JAK2 in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (E) Single sample (ss)GSEA score summarizing IFN-γ signaling signatures between PBMC-CD19 CAR-T versus P-T CD19 CAR-T. (F) EOMES and TBX21 (T-bet) gene counts in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (G) Gene counts of exhaustion markers in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (H) Heat map of selected GSEA pathways enriched significantly upregulated or downregulated in PBMC-CD19 CAR-T versus P-T CD19 CAR-T. For each pathway, a single sample enrichment score was calculated and the mean score was calculated per group. The color indicates pathways enriched in induced (red) or repressed (blue) genes. (I) ssGSEA scores from pathways in (H) for each donor replicate between PBMC-CD19 CAR-T and P-T CD19 CAR-T For normalized gene counts, DEG p-adjusted value is shown. Wilcoxon signed-rank test was performed in E and G. *p<0.05, **p<0.01, ***p<0.001. CAR, chimeric antigen receptor; GSEA, gene set enrichment analysis; IFN-γ, interferon gamma; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor.

    A recent study revealed gene signature differences in CAR-T cells from chronic lymphocytic leukemia (CLL) patients classified as responders (complete remission and partial response with transformed disease) versus non-responders38 according to CLL guidelines.39 Single sample (ss) gene set enrichment analysis revealed that P-T CD19 CAR-T product was enriched for genes upregulated in autologous CAR-T drug product from responding patients (figure 4H,I). These P-T CD19 CAR-T enriched gene signatures are involved in early memory formation, naïve, resting CD4+ T cells, and central memory subsets (figure 4H,I, online supplemental figures 7 and 8 and table 5). In contrast, PBMC-CD19 CAR-T product was enriched for gene signatures involved in immune exhaustion, stimulated, and effector memory T cell subsets (figure 4H,I, online supplemental figures 7 and 8 and table 5). These data collectively show the naïve/stem cell memory gene signature of P-T CD19 CAR-T cells and the potentially exhausted and inflammatory nature of PBMC-CD19 CAR-T cells.

    P-T CD19 CAR-T cells are cytotoxic but maintain an IFN-γ-deficient CD4 T cell population

    Due to the diverging phenotype of PBMC-CD19 CAR-T versus P-T CD19 CAR-T products, CD19-specific cytotoxicity and cytokine effector potential required evaluation. Following product expansion and cryopreservation, thaw-recovered P-T CD19 CAR-T cells demonstrated comparable cytotoxicity kinetics and activity as PBMC-CD19 CAR-T against CD19+ Daudi lymphoma (figure 5A). Minimal background killing was observed using controls generated in the absence of CD19 CAR transduction indicating CAR-directed antigen specificity. Following coculture with Daudi lymphoma, IFN-γ and TNF-α levels were significantly elevated in cultures containing PBMC-CD19 CAR-T cells when compared with cocultures with P-T CD19 CAR-T cells (IFN-γ: 261.2±78.4 vs 114.2±66.0 ng/mL and TNF-α: 4.45±2.0 vs 2.6±0.86 ng/mL) (figure 5B). Both P-T CD19 CAR-T and PBMC-CD19 CAR-T secreted similar levels of IL-2 (figure 5B). To determine if differential cytokine production is isolated to CD4+ or CD8+ T cells, CAR-T cells were stimulated with PMA-ionomycin in the presence of Golgi transport inhibition (figure 5C–E). Intracellular cytokine staining showed that TNF-α was universally produced by CD4+ and CD8+ T cells, indicating similar activation between P-T CD19 CAR-T and PBMC-CD19 CAR-T. CD4+ and CD8+ PBMC-CD19 CAR-T cells strongly coexpressed IFN-γ, while IFN-γ was largely absent among CD4+ P-T CD19 CAR-T (29.7±12.3% vs 4.2±1.5%) but instead favored IL-2 production (73±8.1% vs 91.2±4.5%) (figure 5C–E). These dynamics were not observed among the CD8+ subset. Across multiple donors, the differences in the CD4+ helper T cell population were consistent and significant (figure 5D). The differences among the CD4+ T cell populations are in accordance with the transcriptome data describing altered CD4+ T cell subsets and IFN-γ pathways previously discussed. To understand whether our observed differences in IFN-γ directly affect PBMC-CD19 CAR-T or P-T CD19 CAR-T cytotoxicity, we included anti-IFN-γ blocking antibodies during Daudi lymphoma cell coculture (figure 5F). Compared with isotype controls, IFN-γ blockade had no impact on CD19-mediated cytotoxicity of CAR-T cells (figure 5G). Our data are consistent with others demonstrating IFN-γ does not directly impact CAR-directed cytotoxicity in hematology.40 Overall, these data demonstrate that P-T CD19 CAR-T cells have similar acute effector function to PBMC-CD19 CAR-T cells but produce limited IFN-γ, which does not negatively impact CAR-T cytotoxicity.

    Figure 5
    Figure 5

    P-T CD19 CAR-T have similar cytotoxicity to PBMC-CD19 CAR-T but maintain an IFN-γ-attenuated helper T cell population. (A) Cytotoxicity of PBMC-CD19 CAR-T and P-CD19 CAR-T products against Daudi lymphoma cells at effector-to-target (E:T) ratio of 2.5:1. Non-transduced (NT) PBMC T cells (n=6) and P-T cells (n=9) were included as negative control. (B) Secreted IL-2 (left), IFN-γ (middle), and TNF-α (right) measured from supernatant of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) cocultured for 24 hours with Daudi cancer lines at E:T ratio of 1:1. (C) Representative pseudocolor plots of CD4 and CD8 cell subsets for IL-2, TNF-α, IFN-γ intracellular cytokine staining (ICCS) from 4-hour stimulated CAR-T cells. Lymphocytes were gated on SSC-A versus FSC-A for debris exclusion followed by doublet discrimination on FSC-> doublet discrimination on SSC ->gating on live cells (Aqua-) ->CD5+ ->CD4+ or CD8+ cells. (D) Frequencies of IL-2, IFN-γ, and TNF-α positive populations by ICCS among CD4+ and CD8+ PBMC-CD19 CAR-T (black) or P-T CD19 CAR-T (green) for individual donors tested from (C). (E) Pie charts illustrating differences in IL-2 and IFN-γ distribution in P-T CD19 CAR-T and PBMC CAR-T CD4+ and CD8+ T cells.(F) IFN-γ secretion from PBMC-CD19 CAR-T (n=3) and P-T CD19 CAR-T (n=3) cells against Daudi cancer lines at E:T ratio of 1:1 post-24-hour coculture after treatment with IFN-γ antagonist antibody or isotype control. (G) Cytotoxicity of CAR-T products against Daudi cancer line at E:T ratio of 2.5:1 after IFN-γ blockade. Mean±SEM is presented. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GSEA, gene set enrichment analysis; IFN-γ, interferon gamma; IL-2, interleukin 2; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor; TNF-α, tumor necrosis factor alpha.

    P-T CD19 CAR-T cells clear lymphoma and respond to re-challenge in vivo

    We have reported differences in the stemness and functional phenotype of P-T CD19 CAR-T and PBMC-CD19 CAR-T cells in acute settings. To evaluate how in vitro activity translates to efficacy for targeting lymphoma, we used an immunodeficient murine model. NSG mice were preconditioned with Busulfan, followed by Luciferase-expressing Daudi lymphoma inoculation 1 week later. CD19 CAR-T cells were administered intravenously after another week, and mice were monitored for tumor burden using bioluminescence imaging and survival over time (figure 6A). In the absence of any CD19 CAR-T administration, Daudi lymphoma was detected systemically with tumor burden intensity increasing until all mice succumbed to disease burden by day 35 (31.2±4.5 days). Administration of PBMC-CD19 CAR-T delayed the onset of detectable lymphoma by about 14–21 days, but mice progressed with advanced disease by day 67 (58.3±10.3 days) (figure 6B,C,E). Only the mice dosed with P-T CD19 CAR-T cells exhibited 100% survival and cleared lymphoma with no detectable systemic bioluminescence signal observed during the 100-day observation period (figure 6B,C and E). All P-T CD19 CAR-T-treated mice, exhibiting long-term lymphoma control beyond 120 days, were given a second inoculation of Daudi lymphoma on day 122 to evaluate any recall response and antitumor activity of persisting P-T CD19 CAR-T from the original infusion. An additional group of age-matched NSG mice was also infused with Daudi lymphoma cells as vehicle control. Of note, one mouse from the P-T CD19 CAR-T-treated re-challenge group died on day 133 due to significant body weight drop not associated with tumor burden or tumor-related clinical symptoms (hunching, hind limb paralysis, closed, opaque, or sunken eyes). Re-challenged mice demonstrated a delay in tumor onset (figure 6D) and resulted in a significant survival benefit (186.4±33.1 days) compared with the vehicle control arm (151.8±1.1 days) (figure 6E). Prior to Daudi rechallenge, persistent P-T CD19 CAR-T cells were detected in the peripheral blood at low levels (<0.1%, n=4) at day 115 and subsequently expanded post-Daudi re-challenge at day 122, with significantly higher levels present at day 129 (0.69±0.27%) and day 136 (1.78±2.13%) (figure 6F). Four mice were evaluated for CAR-T persistence and phenotype at end-of-life assessment between days 185 and day 215. Human CD45+CD3+CD19CAR+ cells were found in the blood, spleen, and bone marrow of all mice averaging 4.24±5.74%, 21.6±16.9%, and 2.22±2.1% of total nucleated cells, respectively (figure 6G). Among persisting CAR+ T cells detected at the end of the study, the majority of the cells were TEM cells (68.9%–94.8%), indicating memory formation (figure 6H), and continued to retain high expression of CD27 (86.1%–99.9%) and low expression of CD57 (6.74%–28.7%) (figure 6I), comparable to that of the infused product (figure 3D,E). Taken together, these data suggest that P-T CD19 CAR-T cells have the capacity to persist longer in vivo than PBMC-CD19 CAR-T cells while controlling CD19+ lymphoma cell proliferation.

    Figure 6
    Figure 6

    P-T CD19 CAR-T cells clear lymphoma and exhibit long-term persistence and recall response in vivo. (A) Schema of the Daudi-Luc in vivo NSG mouse model using PBMC-CD19 CAR-T and P-T CD19 CAR-T product (n=5 mice per cohort). (B) Bioluminescence images (BLI) of mice treated with vehicle control, PBMC-CD19 CAR-T cells, and P-T CD19 CAR-T. (C) Weekly BLI measurements of individual mice from vehicle control treated (gray open circle), PBMC-CD19 CAR-T (black), and P-T CD19 CAR-T (green) during the first 100 study days. (D) Weekly BLI measurements of individual mice during the re-challenge period from vehicle control treated (black open circle) and P-T CD19 CAR-T (green) study day 122 to 215. (E) Kaplan-Meier survival curve of mice treated with vehicle (gray), PBMC-CD19 CAR-T (black), and P-T CD19 CAR-T cells (green), and re-challenge vehicle control (black open circle) for the entire in vivo study period. (F) Frequency of P-T CD19 CAR-T detected in blood mononuclear cells prior to Daudi rechallenge, and at 1 and 2 weeks post re-challenge. (G) End of study analysis of relative frequency of human P-T CD19 CAR-T cells in blood, spleen, and bone marrow from surviving mice (n=4 mice left after re-challenge). (H) Differentiation status of persistent P-T CD19 CAR-T cells based on CCR7 and CD45RA staining. (I) CD27+ (left) and CD57+ (right) expression on persisting P-T CD19 CAR-T cells at end of study day 215. An unpaired Student’s t-test was performed for statistical analyses in (F). **p<0.01. Kaplan-Meier curve analysis (Log rank Mantel-Cox test) was performed for statistical analysis of mouse survival. CAR, chimeric antigen receptor; GSEA, gene set enrichment analysis; IL-2, interleukin 2; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor.

    To confirm the in vivo effector function of PT-CD19 CAR-T cells against Daudi lymphoma, an additional animal study was performed using a different donor (online supplemental figure 9). Of note, in this study the dissemination of tumor in mice occurred at a faster rate than in the previous study resulting in the vehicle group perishing sooner. Without CD19 CAR-T administration, all mice in the vehicle group succumbed to disease burden by day 26 (22.29±5.06 days), compared with 35 days in the previous study (figure 6). Administration of PBMC-CD19 CAR-T delayed the onset of detectable lymphoma; however, mice progressed with advanced disease by day 53 (34.40±14.67 days), while the mice dosed with P-T CD19 CAR-T cells exhibited reduced tumor burden and longer survival up to 91 days (71.6±11.59 days) (online supplemental figure 9). Both studies demonstrate the functional superiority of P-T CD19 CAR-T cells over PBMC D19 CAR-T by enhanced antitumor activity and extended animal lifespan.

    Discussion

    Autologous CAR-T therapy has transformed the treatment landscape in a number of disease areas. However, current autologous options still have patients who do not receive CAR-T cells due to unsuccessful drug product manufacture or fail to respond in spite of the expensive, patient-specific manufacturing processes. Allogeneic CAR-T approaches offer scalable, more cost-effective solutions to these current challenges while ensuring consistent product performance. There are multiple potential sources of donor material but the phenotypic and functional differences in T cell starting material have consequential effects on CAR-T cell manufacture and potency. CAR-T cell efficacy has not been associated with tumor burden, age, or disease specific factors. However, drug product features including proliferation potential, memory status, and a less exhausted profile directly correlate with clinical outcomes.38 41 This is important for allogeneic approaches where the donor and origin of T cells can be scrutinized. Potency and durability advantages of TSCM and the importance of stemness have emerged from preclinical CAR-T studies.5 10 There are clear advantages of using healthy donor-derived cells as opposed to patient-derived cells as a CAR-T starting material as it addresses T cell fitness and exhaustion issues related to prior therapy.42 We further demonstrate additional functional advantages of using placental circulating allogeneic T cells over healthy adult PBMCs as a starting material for CAR-T cell manufacture which translate to durable responses in vivo.

    Premanufactured P-T cells are almost entirely TN and TSCM subsets (CD45RA+CCR7+) with uniformly high expression of CD27 and lower or absent expression of exhaustion markers and differentiation markers (PD-1, TIM-3, LAG3, TIGIT, and CD57). A recent study showed that enrichment of TN prior to manufacture can generate TSCM-like CAR-T cells resulting in superior persistence and survival in animal studies.9 Strategies in autologous CAR-T cell manufacturing to preserve TSCM have been explored showing higher expansion when a preserved T cell phenotype is infused in both animal and human studies.43 Moreover, translational studies have demonstrated a correlation between higher frequency of TN and TSCM (CD45RO−CD27+CD8+ T cells) in autologous BCMA-targeted CAR-T and improved patient responses.17 In the current study, following CAR-T expansion, P-T CD19 CAR-T maintained higher CD27 expression and resisted expression of TEM associated transcription factors when compared with PBMC-CD19 CAR-T. In line with that, P-T CD19 CAR-T have longer telomeres than PBMC-CD19 CAR-T, which confer greater proliferative potential and may offer longer functional capability. P-T CD19 CAR-T similarly maintained features of naïve-memory phenotype while PBMC-CD19 CAR-T had increased expression of activation and terminal differentiation (CD57) and exhaustion (TIGIT and LAG3) markers. The increased expression of PD-1 observed in P-T CD19 CAR-T, was not accompanied by a corresponding increase in CD57, LAG3, or TIGIT, indicating that PD-1 on P-T CD19 CAR-T was driven by T cell activation rather than exhaustion. Our data are consistent with reports suggesting that cord blood derived T cells revert to a naïve status after differentiation while naïve T cells from peripheral blood possess comparatively limited plasticity.25 Taken together, we present a novel allogeneic T cell platform which enables large-scale manufacturing of CAR-T cells with a homogeneous and preserved stem cell memory T-cell phenotype. Our current manufacturing and process development efforts are optimizing the harvesting procedures of the placental cell starting material derived from both the cord blood and the perfusate of the placenta to further maximize the yield, quality, and desired phenotype of cells for use in CAR-T manufacture.

    The importance of CAR-T cell phenotype has been recently demonstrated by translational studies from CLL patients treated with CD19 CAR-T therapy. Patient clinical response was correlated with CAR-T drug product containing memory-related genes, where patients achieving a complete response showed enrichment of CD45RO−CD27+ CD8 T cells (consistent with TN and TSCM) in their dosed CAR-T product when compared with non-responders.38 A separate study identified the same T cell population in the starting material for BCMA-targeted CAR-T products for multiple myeloma correlating with improved patient response.17 Another study using patient’s cells in an autologous setting showed that a reduction in culture time during manufacturing enriched for the TSCM population from 17% to 45%.43 P-T CD19 CAR-T cells have on average >45% TSCM at the end of 13 days manufacturing allowing high CAR-T yield while maintaining stemness. Transcriptome analysis of CAR-T drug products has proven to be a strong predictor of clinical outcome linking the enrichment of non-exhausted memory cell gene signatures with complete remission.41 RNA-seq analyses confirmed that P-T CD19 CAR-T retained a stem-like gene signature. Additionally, the observed transcriptional signatures for P-T CD19 CAR-T cells are consistent with those reported for autologous CAR-T drug product from patients who achieved complete response.35 Conversely, we observed exhausted and stimulated gene signatures relatively enriched in PBMC-CD19 CAR-T, consistent with drug product signatures of non-responder patients.38 41 In vivo testing confirmed the durability advantage of P-T CD19 CAR-T cells demonstrating lymphoma control beyond 100 days, functional recall response, and persistence up until day 215. P-T CD19 CAR-T cells offer an advantageous phenotype and homogeneous drug product which may contribute to better clinical outcomes.

    These phenotypic differences may also confer P-T CD19 CAR-T with potential safety advantages. P-T CD19 CAR-T secretes lower IFN-γ and TNF-α, and our data demonstrate CD4+ P-T CD19 CAR-T favor IL-2 production over IFN-γ. We observed similar findings in solid tumor models comparing P-T CD19 CAR-T to PBMC-CD19 CAR-T.44 Other studies demonstrated that TN/SCM versus bulk T cells have enhanced antitumor responses while also reducing the risk of severe cytokine release syndrome (CRS) in CD19-targeted murine models.45 Lower IFN-γ levels may help mitigate toxicity without compromising antitumor activity46 as IFN-γ blockade reduces macrophage-mediated cytokines associated with CRS without impacting CAR-T efficacy or persistence.40 47 IFN-γ antagonism has successfully treated CRS associated with CD19 CAR-T and the related macrophage activation syndrome in a patient’s case study.47 In our study, attenuated IFN-γ did not affect the in vitro killing activity of P-T CD19 CAR-T, nor did additional IFN-γ blockade. Reduced IFN-γ from P-T CD19 CAR-T is also expected to provide additional benefits. CAR-T skewing away from IFN-γ and towards IL-2 may favor the proliferative and cytotoxic potential of allogeneic P-T CD19 CAR-T cells while lowering the potential for toxicity.

    The efficacy of CAR-T cell products has been related to proliferative capability in vivo. Insufficient CAR-T cells ratio to tumor burden due to poor expansion capacity or low dose may affect efficacy of CAR-T cells.38 41 Effector cells differentiate toward a non-proliferative, exhausted state with limited expansion capability. RNA-seq analysis revealed that PBMC-CD19 CAR-T have enhanced exhaustion and activation gene signatures including EOMES, TBX21, GZMB, GZMH, CCL4, IFNG, CTLA4, TIGIT, and LAG3. Consistent with transcriptome analysis, surface expression of immune checkpoints LAG3 and TIGIT were also increased in PBMC-CD19 CAR-T. A clinical study of CAR-T cell patients who did not respond showed high expression of TIGIT and poor expansion of CAR-T, which could be rescued by TIGIT blockage.48 In line with expression of activation and exhaustion markers, P-T CD19 CAR-T persisted three times longer than CAR-T PBMCs in our lymphoma animal model. Similarly, efficacy and persistence of CAR-T cells derived from umbilical cord blood has been shown by other groups in different animal studies.49 The transient durability of PBMC-derived CD19 CAR-T cells in our study is consistent with other studies using lymphoma animal models.50–52

    CAR-T cell therapy has shown remarkable effectiveness for lymphoma patients. However, autologous therapies are difficult to access. Current options are costly with complicated logistics, creating barriers for widespread use in the clinical setting. Bespoke manufacturing of autologous CAR-T causes a delay for dosing patients, creates risks in successful GMP manufacturing, may produce inferior quality CAR-T cells due to disease and/or prior chemotherapy treatment, and adds variability in potential product efficacy. Allogeneic approaches offer solutions to these bottlenecks. While healthy adult T cells are one option, and in vivo studies demonstrate that CAR-T from healthy donors are superior over cancer patient-derived T cells,11 38 50 53 here we demonstrate that placental circulating T cells possess unique characteristics which translate into additional product advantages over healthy adult blood-derived CAR-T.

    Additional studies are required to evaluate potential allorejection by host immune cells and the usage of different CAR constructs for comparative analyses between PBMC-T and P-Ts. As allogeneic CAR-T cell persistence encompasses both improved T cell fitness and circumvention of allorejection by host immune cells, the use of P-T cells has demonstrated an advantage with the former. Further development on the P-T platform is planned and will incorporate stealth modifications via gene editing to prevent rejection by host immune cells. Finally, our study focused on showing differences of starting material source to generate CAR-T cells. It is known that intracellular costimulatory domains and vector choice can influence the phenotype and effector function of CAR-T cells. Previous study has shown that CAR-T cells containing the CD28 costimulatory domain preferentially differentiate into central and effector memory phenotype, while those containing 4-1BB promotes central memory expansion.54 However, herein we show that using a CD28 costimulatory CAR, P-T cells maintained their stemness profile compared with PBMCs CAR-T and demonstrate better efficacy and persistence in vivo. Follow-up studies should examine differences between PBMCs and P-T derived CAR-T using different antigen targets, vectors, and intracellular co-domains. In summary, given the superior T-cell fitness profile of P-T cells compared with healthy PBMCs as source material, these modifications can potentially further improve on future P-T CAR-T cells drug products.

    Supplemental material

    Data availability statement

    Data are available in a public, open access repository. RNA-Seq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE231429 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231429). For other original data, please contact kathy.karasiewicz{at}celularity.

    Ethics statements

    Patient consent for publication

    Ethics approval

    All procedures involving animal subjects described in this manuscript were performed at Invivotek LLC and were approved by the institutional Animal Care and Use Committee (IACUC) under institutional animal use protocols (AUP) 19-1M and 19-10M.

    Acknowledgments

    We would like to thank Jonathan Fu, Carissa Claudio, Adriana Freitas, Brenda Calalpa, Matthew Hingle, and Sharon Kalsi for the processing and isolation of T cells from placental blood units, and Bhavani Stout for processing and isolating T cells from adult donor whole blood. We extend our gratitude to Michael Curto for assistance with the processing of large-scale P-T CD19 CAR-T cell cultures to support our studies. We recognize John Fitzgerald for his technical support toward multiple in vitro studies. In addition, we thank Joseph Gleason, Dr Weifang Ling, and Dr Kevin Jhun for their assistance and contributions to the animal studies and acknowledge Invivotek LLC for the execution of these studies. We gratefully acknowledge Sorrento Therapeutics Inc for providing the CD19 CAR retrovirus vector. Finally, we thank Dr Adrian Kilcoyne for the thorough review of the manuscript.

    References

    1. ↵
      1. Chavez JC,
      2. Bachmeier C,
      3. Kharfan-Dabaja MA
      . CAR T-cell therapy for B-cell lymphomas: clinical trial results of available products. Ther Adv Hematol 2019;10:2040620719841581. doi:10.1177/2040620719841581
    2. ↵
      1. Shah NN,
      2. Fry TJ
      . Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol 2019;16:372–85. doi:10.1038/s41571-019-0184-6
      OpenUrlCrossRefPubMed
    3. ↵
      1. Lugli E,
      2. Dominguez MH,
      3. Gattinoni L, et al
      . Superior T memory stem cell persistence supports long-lived T cell memory. J Clin Invest 2013;123:594–9. doi:10.1172/JCI66327
      OpenUrlCrossRefPubMedWeb of Science
    4. ↵
      1. Gattinoni L,
      2. Lugli E,
      3. Ji Y, et al
      . A human memory T cell subset with stem cell-like properties. Nat Med 2011;17:1290–7. doi:10.1038/nm.2446
      OpenUrlCrossRefPubMed
    5. ↵
      1. Sommermeyer D,
      2. Hudecek M,
      3. Kosasih PL, et al
      . Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 2016;30:492–500. doi:10.1038/leu.2015.247
      OpenUrlCrossRefPubMed
    6. ↵
      1. Gattinoni L,
      2. Zhong X-S,
      3. Palmer DC, et al
      . Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med 2009;15:808–13. doi:10.1038/nm.1982
      OpenUrlCrossRefPubMedWeb of Science
    7. ↵
      1. Alvarez-Fernández C,
      2. Escribà-Garcia L,
      3. Vidal S, et al
      . A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy. J Transl Med 2016;14:214. doi:10.1186/s12967-016-0973-y
    8. ↵
      1. Hinrichs CS,
      2. Spolski R,
      3. Paulos CM, et al
      . IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood 2008;111:5326–33. doi:10.1182/blood-2007-09-113050
      OpenUrlAbstract/FREE Full Text
    9. ↵
      1. Meyran D,
      2. Zhu JJ,
      3. Butler J, et al
      . T(STEM)-Like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models. Sci Transl Med 2023;15:eabk1900. doi:10.1126/scitranslmed.abk1900
    10. ↵
      1. Sabatino M,
      2. Hu J,
      3. Sommariva M, et al
      . Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood 2016;128:519–28. doi:10.1182/blood-2015-11-683847
      OpenUrlAbstract/FREE Full Text
    11. ↵
      1. Arcangeli S,
      2. Falcone L,
      3. Camisa B, et al
      . Next-generation manufacturing protocols enriching TSCM CAR T cells can overcome disease-specific T cell defects in cancer patients. Front Immunol 2020;11:1217. doi:10.3389/fimmu.2020.01217
    12. ↵
      1. Depil S,
      2. Duchateau P,
      3. Grupp SA, et al
      . 'Off-the-shelf' allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 2020;19:185–99. doi:10.1038/s41573-019-0051-2
      OpenUrlPubMed
    13. ↵
      1. Karasiewicz K,
      2. He S,
      3. Ng M, et al
      . Preclinical evaluation of human placental-derived allogeneic CD19 CAR-T cells against B cell malignancies. Blood 2019;134:3222. doi:10.1182/blood-2019-130782
    14. ↵
      1. Olbrich H,
      2. Theobald SJ,
      3. Slabik C, et al
      . Adult and cord blood-derived high-affinity gB-CAR-T cells effectively react against human cytomegalovirus infections. Hum Gene Ther 2020;31:423–39. doi:10.1089/hum.2019.149
      OpenUrl
    15. ↵
      1. Ma Q,
      2. Garber HR,
      3. Lu S, et al
      . A novel TCR-like CAR with specificity for PR1/HLA-A2 effectively targets myeloid leukemia in vitro when expressed in human adult peripheral blood and cord blood T cells. Cytotherapy 2016;18:985–94. doi:10.1016/j.jcyt.2016.05.001
      OpenUrlPubMed
    16. ↵
      1. Karasiewicz K,
      2. van der Touw W,
      3. Ruggeri Barbaro N
      . Placental T cells retain enhanced stemness and altered cytokine response favoring durable efficacy of CD19 CAR-T. Gene Expression Omnibus; 2023. Available: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231429
    17. ↵
      1. Cohen AD,
      2. Garfall AL,
      3. Stadtmauer EA, et al
      . B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–21. doi:10.1172/JCI126397
      OpenUrlCrossRefPubMed
    18. ↵
      1. Gardner RA,
      2. Finney O,
      3. Annesley C, et al
      . Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood 2017;129:3322–31. doi:10.1182/blood-2017-02-769208
      OpenUrlAbstract/FREE Full Text
    19. ↵
      1. Gattinoni L,
      2. Speiser DE,
      3. Lichterfeld M, et al
      . T memory stem cells in health and disease. Nat Med 2017;23:18–27. doi:10.1038/nm.4241
      OpenUrlCrossRefPubMed
    20. ↵
      1. Nociari MM,
      2. Telford W,
      3. Russo C
      . Postthymic development of CD28-CD8+ T cell subset: age-associated expansion and shift from memory to naive phenotype. J Immunol 1999;162:3327–35.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Merino J,
      2. Martínez-González MA,
      3. Rubio M, et al
      . Progressive decrease of CD8High+ CD28+ CD57- cells with ageing. Clin Exp Immunol 1998;112:48–51. doi:10.1046/j.1365-2249.1998.00551.x
      OpenUrlCrossRefPubMed
    22. ↵
      1. Turtle CJ,
      2. Hanafi L-A,
      3. Berger C, et al
      . CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016;126:2123–38. doi:10.1172/JCI85309
      OpenUrlCrossRefPubMed
    23. ↵
      1. Weng NP,
      2. Levine BL,
      3. June CH, et al
      . Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proc Natl Acad Sci U S A 1995;92:11091–4. doi:10.1073/pnas.92.24.11091
      OpenUrlAbstract/FREE Full Text
    24. ↵
      1. Roth A,
      2. Yssel H,
      3. Pene J, et al
      . Telomerase levels control the lifespan of human T lymphocytes. Blood 2003;102:849–57. doi:10.1182/blood-2002-07-2015
      OpenUrlAbstract/FREE Full Text
    25. ↵
      1. Frumento G,
      2. Verma K,
      3. Croft W, et al
      . Homeostatic cytokines drive epigenetic reprogramming of activated T cells into a "naive-memory" phenotype. iScience 2020;23:100989. doi:10.1016/j.isci.2020.100989
    26. ↵
      1. De Jong R,
      2. Brouwer M,
      3. Hooibrink B, et al
      . The CD27- subset of peripheral blood memory CD4+ lymphocytes contains functionally differentiated T lymphocytes that develop by persistent antigenic stimulation in vivo. Eur J Immunol 1992;22:993–9. doi:10.1002/eji.1830220418
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Hamann D,
      2. Baars PA,
      3. Rep MHG, et al
      . Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med 1997;186:1407–18. doi:10.1084/jem.186.9.1407
      OpenUrlAbstract/FREE Full Text
    28. ↵
      1. Intlekofer AM,
      2. Takemoto N,
      3. Wherry EJ, et al
      . Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol 2005;6:1236–44. doi:10.1038/ni1268
      OpenUrlCrossRefPubMedWeb of Science
    29. ↵
      1. Ronchetti S,
      2. Nocentini G,
      3. Bianchini R, et al
      . Glucocorticoid-induced TNFR-related protein LOWERS the threshold of CD28 Costimulation in CD8+ T cells. J Immunol 2007;179:5916–26. doi:10.4049/jimmunol.179.9.5916
      OpenUrlAbstract/FREE Full Text
    30. ↵
      1. Qin S,
      2. Xu L,
      3. Yi M, et al
      . Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4. Mol Cancer 2019;18:155. doi:10.1186/s12943-019-1091-2
      OpenUrlCrossRefPubMed
    31. ↵
      1. Mujib S,
      2. Jones RB,
      3. Lo C, et al
      . Antigen-independent induction of Tim-3 expression on human T cells by the common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway. J Immunol 2012;188:3745–56. doi:10.4049/jimmunol.1102609
      OpenUrlAbstract/FREE Full Text
    32. ↵
      1. Yang Y,
      2. Kohler ME,
      3. Chien CD, et al
      . TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance. Sci Transl Med 2017;9:eaag1209. doi:10.1126/scitranslmed.aag1209
    33. ↵
      1. Stenger D,
      2. Stief TA,
      3. Kaeuferle T, et al
      . Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR. Blood 2020;136:1407–18. doi:10.1182/blood.2020005185
      OpenUrlPubMed
    34. ↵
      1. Akimova T,
      2. Beier UH,
      3. Wang L, et al
      . Helios expression is a marker of T cell activation and proliferation. PLoS One 2011;6:e24226. doi:10.1371/journal.pone.0024226
    35. ↵
      1. Reynolds JM,
      2. Dong C
      . Toll-like receptor regulation of effector T lymphocyte function. Trends Immunol 2013;34:511–9. doi:10.1016/j.it.2013.06.003
      OpenUrlCrossRefPubMed
    36. ↵
      1. Chu DH,
      2. Morita CT,
      3. Weiss A
      . The syk family of protein tyrosine Kinases in T-cell activation and development. Immunol Rev 1998;165:167–80. doi:10.1111/j.1600-065x.1998.tb01238.x
      OpenUrlCrossRefPubMedWeb of Science
    37. ↵
      1. Lennartsson J,
      2. Rönnstrand L
      . Stem cell factor receptor/C-kit: from basic science to clinical implications. Physiol Rev 2012;92:1619–49. doi:10.1152/physrev.00046.2011
      OpenUrlCrossRefPubMed
    38. ↵
      1. Fraietta JA,
      2. Lacey SF,
      3. Orlando EJ, et al
      . Determinants of response and resistance to CD19 Chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 2018;24:563–71. doi:10.1038/s41591-018-0010-1
      OpenUrlCrossRefPubMed
    39. ↵
      1. Hallek M,
      2. Cheson BD,
      3. Catovsky D, et al
      . Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the international workshop on chronic lymphocytic leukemia updating the National cancer institute-working group 1996 guidelines. Blood 2008;111:5446–56. doi:10.1182/blood-2007-06-093906
      OpenUrlAbstract/FREE Full Text
    40. ↵
      1. Bailey SR,
      2. Vatsa S,
      3. Larson RC, et al
      . Blockade or deletion of Ifngamma reduces macrophage activation without compromising CAR T-cell function in hematologic malignancies. Blood Cancer Discovery 2022;3:136–53. doi:10.1158/2643-3230.BCD-21-0181
      OpenUrlPubMed
    41. ↵
      1. Kirouac DC,
      2. Zmurchok C,
      3. Deyati A, et al
      . Deconvolution of clinical variance in CAR-T cell pharmacology and response. Nat Biotechnol 2023;41:1606–17. doi:10.1038/s41587-023-01687-x
      OpenUrl
    42. ↵
      1. Metelo AM,
      2. Jozwik A,
      3. Luong LA, et al
      . Allogeneic anti-BCMA CAR T cells are superior to multiple myeloma-derived CAR T cells in preclinical studies and may be combined with gamma secretase inhibitors. Cancer Res Commun 2022;2:158–71. doi:10.1158/2767-9764.CRC-21-0157
      OpenUrl
    43. ↵
      1. Dickinson MJ,
      2. Barba P,
      3. Jäger U, et al
      . A novel autologous CAR-T therapy, YTB323, with preserved T-cell stemness shows enhanced CAR T-cell efficacy in preclinical and early clinical development. Cancer Discov 2023;13:1982–97. doi:10.1158/2159-8290.CD-22-1276
      OpenUrl
    44. ↵
      1. Park A,
      2. Monroy I,
      3. Cook C, et al
      . 847 CF33-CD19t oncolytic virus (oncarlytics) in combination with off-the-shelf allogeneic cycart-19 T cells targeting de novo CD19+ solid tumors. J Immunother Cancer 2022;10:A885-A. doi:10.1136/jitc-2022-SITC2022.0847
    45. ↵
      1. Arcangeli S,
      2. Bove C,
      3. Mezzanotte C, et al
      . CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. J Clin Invest 2022;132:e150807. doi:10.1172/JCI150807
    46. ↵
      1. Zhang H,
      2. Lv X,
      3. Kong Q, et al
      . IL-6/IFN-gamma double knockdown CAR-T cells reduce the release of multiple cytokines from PBMCs in vitro. Hum Vaccin Immunother 2022;18:1–14. doi:10.1080/21645515.2021.2016005
      OpenUrlPubMed
    47. ↵
      1. Rainone M,
      2. Ngo D,
      3. Baird JH, et al
      . Interferon-gamma blockade in CART-cell therapy-associated macrophage activation syndrome/hemophagocytic lymphohistiocytosis. Blood Adv 2023;7:533–6. doi:10.1182/bloodadvances.2022008256
      OpenUrl
    48. ↵
      1. Jackson Z,
      2. Hong C,
      3. Schauner R, et al
      . Sequential single-cell transcriptional and protein marker profiling reveals TIGIT as a marker of CD19 CAR-T cell dysfunction in patients with non-hodgkin lymphoma. Cancer Discov 2022;12:1886–903. doi:10.1158/2159-8290.CD-21-1586
      OpenUrlCrossRefPubMed
    49. ↵
      1. Caël B,
      2. Galaine J,
      3. Bardey I, et al
      . Umbilical cord blood as a source of less differentiated T cells to produce CD123 CAR-T cells. Cancers (Basel) 2022;14:3168. doi:10.3390/cancers14133168
    50. ↵
      1. Graham CE,
      2. Jozwik A,
      3. Quartey-Papafio R, et al
      . Gene-edited healthy donor CAR T cells show superior anti-tumour activity compared to CAR T cells derived from patients with lymphoma in an in vivo model of high-grade lymphoma. Leukemia 2021;35:3581–4. doi:10.1038/s41375-021-01324-z
      OpenUrl
    51. ↵
      1. Liu L,
      2. Bi E,
      3. Ma X, et al
      . Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9. Nat Commun 2020;11:5902. doi:10.1038/s41467-020-19672-2
      OpenUrlPubMed
    52. ↵
      1. Noh K-E,
      2. Lee J-H,
      3. Choi S-Y, et al
      . TGF-beta/IL-7 chimeric switch receptor-expressing CAR-T cells inhibit recurrence of CD19-positive B cell lymphoma. Int J Mol Sci 2021;22:8706. doi:10.3390/ijms22168706
    53. ↵
      1. Das RK,
      2. Vernau L,
      3. Grupp SA, et al
      . Naive T-cell deficits at diagnosis and after chemotherapy impair cell therapy potential in pediatric cancers. Cancer Discov 2019;9:492–9. doi:10.1158/2159-8290.CD-18-1314
      OpenUrlAbstract/FREE Full Text
    54. ↵
      1. Kawalekar OU,
      2. O’Connor RS,
      3. Fraietta JA, et al
      . Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity 2016;44:380–90. doi:10.1016/j.immuni.2016.01.021
      OpenUrlCrossRefPubMed

    Supplementary materials

    Footnotes

    • Contributors KK, NRB, WvdT, SH, and RH designed research studies. KT, MD, TD, and NRB performed experiments. NRB, MD, TD, and KK analyzed the data and performed statistical analysis. WvdT, KK, and NRB wrote the manuscript. All authors contributed and approved the final version. KK serves as acting guarantor for the manuscript.

    • Funding This work was supported by Celularity Inc.

    • Competing interests All authors are current or former employees of Celularity Inc. and hold stock and/or stock options in the company. KK, KT, SH, and RH are all listed as inventors on the patent application for placenta-derived allogeneic CAR-T cells and uses thereof (PCT/US2020/063473). RH is the CEO of Celularity.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.