METHOD TO PRODUCE T CELLS AND USES THEREOF

Abstract

The present invention refers to a method to produce a T cell with advantageous properties. The invention also refers to a T cell or an engineered T cell produced by the method and its use in therapy.

Claims

1. A method to produce a T cell, comprising: a) isolating a population of CD45RA.sup.+/CD62L.sup.+/CD95.sup.− T cells and CD45RA.sup.+/CD62L.sup.+/CD95.sup.+ T cells from a biological sample of a subject; b) activating said population of T cells by stimulating CD3 and CD28; and c) contacting said activated population of T cells with IL-7 and IL-15.

2. The method according to claim 1 wherein the method further comprises expanding the activated population of T cells in culture with IL-7 and IL-15, preferably for 5-30 days, more preferably for about 15 days.

3. The method according to claim 1, wherein said T cell has at least one of the following properties: prevent cytokine release syndrome, prevent neurotoxicity, display a high expansion rate, preserved early memory phenotype, a poor exhausted profile and long-term persistence.

4. The method according to claim 1, further comprising introducing in said population of T cells a nucleic acid sequence encoding an exogenous gene, thereby producing an engineered T cell.

5. The method according to claim 4 wherein the exogenous gene encodes a member of the group consisting of an antigen-recognizing receptor, an ortho-receptor, an immunomodulatory cytokine, a chemokine receptor, a dominant negative receptor, and a transcription factor able to prevent exhaustion.

6. The method according to claim 5 wherein said antigen recognizing receptor is a T cell receptor (TCR).

7. The method according to claim 5 wherein said antigen recognizing receptor is a chimeric antigen receptor (CAR).

8. The method according to claim 5, wherein said antigen recognizing receptor is exogenous.

9. The method according to claim 4, wherein said nucleic acid sequence is introduced by a vector.

10. The method according to claim 9 wherein the vector is a lentiviral vector.

11. The method according to claim 4 wherein said nucleic acid sequence is placed at an endogenous gene locus of the T cell.

12. The method according to claim 4 wherein said insertion of the nucleic acid sequence disrupts or abolishes the endogenous expression of a TCR.

13. A T cell or an engineered T cell obtainable by the method of claim 1.

14. A CAR T cell obtainable by the method of claim 7.

15. A TCR-engineered T cell obtainable by claim 6.

16. An isolated engineered cell population derived from a population of CD45RA.sup.+/CD62L.sup.+/CD95.sup.− T cells and CD45RA.sup.+/CD62L.sup.+/CD95.sup.+ T cells and engineered to comprise a nucleic acid sequence encoding an exogenous gene wherein said population reduces at least one symptom of cytokine release syndrome (CRS) or reduces at least one symptom of neurotoxicity in a subject or wherein said population has high expansion rate.

17. The isolated engineered T cell population of claim 16 wherein the exogenous gene encodes a member of the group consisting of an antigen-recognizing receptor, an ortho-receptor, an immunomodulatory cytokine, a chemokine receptor, a dominant negative receptor, and a transcription factor able to prevent exhaustion.

18. The isolated engineered T cell population of claim 17 wherein said antigen recognizing receptor is a T cell receptor (TCR).

19. The isolated engineered T cell population of claim 17 wherein said antigen recognizing receptor is a chimeric antigen receptor (CAR).

20. The isolated engineered T cell population of claim 17 wherein said antigen recognizing receptor is exogenous.

21. The isolated engineered T cell population of claim 17 wherein said nucleic acid sequence is introduced by a vector.

22. The isolated engineered T cell population of claim 17 wherein said nucleic acid sequence is placed at an endogenous gene locus of the T cell.

23. The isolated engineered T cell population of claim 17 wherein said insertion of the nucleic acid sequence disrupts or abolishes the endogenous expression of a TCR.

24. A pharmaceutical composition comprising at least one T cell or the engineered T cell according to claim 13.

25. The T cell or the engineered T cell according to claim 13, for use in a therapy, preferably for use in reducing tumor burden or for use in treating and/or preventing a neoplasm or for use in lengthening survival of a subject having a neoplasm or for use in the treatment of an infection or for use in the treatment of an autoimmune disease, preferably the neoplasm is selected from the group consisting of solid or blood cancer, preferably B cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, acute myeloid leukemia (AML), non-Hodgkin's lymphoma, preferably the neoplasm is B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.

26. The T cell or the engineered T cell according to claim 13 for use in preventing and/or reducing at least one symptom of cytokine release syndrome (CRS) or for use in reducing at least one symptom of neurotoxicity in a subject.

27-36. (canceled)

Description

[0117] The present invention will be described by means of non-limiting examples in reference to the following figures.

[0118] FIG. 1. CAR T.sub.N/SCM display an indolent effector signature in vitro. A) Schematic representation of CAR T-cell manufacturing. Briefly, double positive CD62L.sup.+/CD45RA.sup.+ T cells were FACS-sorted and bulk unselected T cells were employed as control. T.sub.N/SCM and T.sub.BULK were activated with TransAct, transduced with a LV encoding for a CD19.28z CAR and expanded in culture with IL-7 and IL-15. B) T.sub.N/SCM enrichment (n=16), C) CD4/CD8 ratio (n=20) and D) HLA-DR expression (n=18) at the end of CAR T-cell manufacturing. E) Fold expansion at different time points during culture (n=12). F) De-granulation assay performed by co-culturing CAR T.sub.N/SCM, CAR T.sub.BULK and Mock control with CD19+ targets for 24 hours (n=14 donors challenged against NALM-6, BV173 and ALL-CM CD19+ target cell lines). G) Killing activity expressed as Elimination Index (see Methods) and measured by co-culturing CAR T cells with CD19+ tumor cells for 4 days at different Effector to Target (E:T) ratios (n=15 for CAR T.sub.BULK, n=14 for CAR T.sub.N/SCM against NALM-6 cell line; n=9 for CAR T.sub.BULK, n=8 for CAR T.sub.N/SCM against ALL-CM cell line). H) Cytokine production after 24 h co-culture of T cells with CD19+ tumor cells at the 1:10 E:T ratio (n=5 donors challenged against NALM-6, BV173 and ALL-CM cell lines). I) T-cell proliferation after a 4-day co-culture with CD19+ targets, measured by intracellular staining of Ki-67 (n=15 donors challenged against NALM-6, BV173 and ALL-CM cell lines).

[0119] Data are represented as the result of mean±SEM or mean±SEM together with overlapping scattered values. Results of paired t-test (B, D, F, I) or two-way (C, E, G, H) ANOVA are reported when statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0120] FIG. 2. CAR T.sub.N/SCM display superior anti-tumor activity and expansion in HuSGM3 mice.

[0121] A) Schematic representation of the HSCP-humanized mouse model. SGM3 mice were infused with HSPCs and, after hematopoietic reconstitution, injected with Lucia+/NGFR+/NALM-6 leukemia and treated with low doses of CAR T.sub.N/SCM (n=17), CAR T.sub.BULK (n=17) or Mock control (n=7). B) NALM-6-derived bioluminescence signal measured at different time points after treatment and expressed as Relative Light Units (RLU). C) T-cell expansion in the peripheral blood of NALM-6 bearing mice measured at different time points after treatment. D) IFN-γ serum levels measured at day 4 after treatment and day 5 after NALM-6 re-challenge. E) T-cell memory phenotype of CAR T.sub.BULK and CAR T.sub.N/SCM at day 14 after treatment. Left panel: dot plot of two representative mice. (T.sub.SCM: CD45RA+CD62L+; T.sub.CM: CD45RA-CD62L.sup.+; T.sub.EM: CD45RA-CD62L−; T.sub.EMRA: CD45RA+CD62L−). Right panel: frequency of the central memory T-cell subset (analysis performed for n=7 mice/group). F, G) Evaluation of signs and symptoms typical of CRS development in HuSGM3 leukemia bearing mice after treatment, represented by weight loss (F), and serum levels of IL-6 (G, left) and murine serum amyloid A (SAA, G right).

[0122] Data are represented as the result of mean±SEM or mean±SEM together with overlapping scattered values. Results of two-way ANOVA (B, C, D, F) and unpaired t-test (E, G) are reported when statistically significant (***p<0.001; ****p<0.0001).

[0123] FIG. 3. CAR T.sub.N/SCM retain an enhanced in vivo fitness after leukemia encounter. SGM3 mice were infused with HSPCs and, after hematopoietic reconstitution, injected with Lucia+/NGFR+/NALM-6 leukemia and treated with low doses of CAR T.sub.N/SCM (n=3) or CAR T.sub.BULK (n=5) as described in FIG. 2. A) A median of ˜74000 CD3+ lymphocytes deriving from peripheral blood of both CAR T.sub.N/SCM and CAR T.sub.BULK treated mice at day 14 after leukemia infusion were inquired by BH-SNE and K-means algorithms. Data were plotted according to BH-SNE1 and BH-SNE2 specifically calculated variables and the events were split in two density plots according to the CAR T-cell population they belong to. B) CAR T.sub.N/SCM and CAR T.sub.BULK specifically identified clusters after application of Flow-SOM algorithm to both BH-SNE1 and BH-SNE2 variables. C, D) CAR T.sub.N/SCM and CAR T.sub.BULK specific clusters were described in terms of T-cell memory subset composition, together with expression of inhibitory and activation receptors. E) Heatmap visualization of both inhibitory and activation receptors expressed by CAR T.sub.N/SCM and CAR T.sub.BULK specific meta-clusters, in which mean fluorescence intensity (MFI) levels were normalized on the basis of the maximum expressed value of each analyzed parameter in the whole analyzed sample.

[0124] FIG. 4. CAR T.sub.N/SCM display an intrinsically reduced toxic profile.

[0125] A) SGM3 mice were infused with HSPCs and, after hematopoietic reconstitution (HuSGM3), injected with Lucia+/NGFR+/NALM-6 leukemia. When a high tumor burden was reached, mice were treated with high doses of CAR T.sub.N/SCM (n=9), CAR T.sub.BULK (n=9) or Mock control (n=6). B) NALM-6-derived bioluminescence signal measured at different time points after treatment and expressed as Relative Light Units (RLU). C) T-cell expansion in the peripheral blood of mice, D) weight loss evaluation and E) IL-6 serum levels at different time points after treatment. F) SAA levels 24 hours after T-cell infusion (n=6 for CAR T.sub.BULK, n=6 for CAR T.sub.N/SCM, n=3 for Mock). G) Peak serum cytokine levels and H) heat-map visualization of peak serum cytokine levels at day 4 after treatment. Data are represented as the result of mean±SEM and values are scaled according to a colored-graded range depending on relative minimum and maximum levels. I) Severe CRS (sCRS)-related Kaplan-Meyer survival analysis of mice. J) CRS grading. Left panel: Kaplan-Meyer curves. Right panel: Histograms summarizing CRS grading. K) Hematoxylin and eosin-stained sections of brains belonging to representative Mock control, CAR T.sub.BULK and CAR T.sub.N/SCM treated mice (20× magnification; bar: 50 micron).

[0126] Data are represented as the result of mean±SEM together with overlapping scattered values and box and violin plots. P values (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001) were determined by unpaired t-test (F, G), two-way ANOVA (B-E), Mantel-Cox two-sided log-rank test (I) and Gehan-Breslow-Wilcoxon test (J).

[0127] FIG. 5. CAR T.sub.N/SCM expand more in HuSGM3 mice without causing detrimental side effects. HuSGM3 mice were infused with Lucia+/NGFR+/NALM-6 cells and treated with 3×10.sup.{circumflex over ( )}6 CAR T.sub.N/SCM, CAR T.sub.BULK or Mock control and analyzed for antitumor activity and toxic manifestations. A) Left panel: bioluminescence detection of tumor growth after treatment. Data are represented as single interspersed lines representing individual treated mice. Right panel: Kaplan-Meyer survival analysis of mice. B) T-cell expansion in the peripheral blood of mice. C, D) Evaluation of signs and symptoms typical of CRS development in HuSGM3 leukemia bearing mice, represented by weight loss (C) and serum levels of IL-6 (D, left), murine serum amyloid A (SAA, D middle) and other pro-inflammatory cytokines, namely IL-10, TNF-α, IL-1a, IFN-g, MIP-1a, IP-10, MCP-1, IL-8, IL-2 and again IL-6 (D, right).

[0128] Data are represented as the result of mean±SEM and box and violin plots. Results of unpaired t-test (D, right panel) and two-way ANOVA (A-D) are reported when statistically significant (*p<0.05; **p<0.01; ***p<0.001).

[0129] FIG. 6. CAR T.sub.N/SCM and CAR T.sub.BULK are effective in vivo and equally represented in the meta-cluster analysis. A) Bioluminescence detection of Lucia+/NGFR+/NALM-6 systemic growth in HuSGM3 mice after treatment with 1×10{circumflex over ( )}6 CAR T.sub.N/SCM, CAR T.sub.BULK or Mock control. Data are represented as single interspersed lines representing individual treated mice (n=7 for CAR T.sub.BULK and n=3 for CAR T.sub.N/SCM). B) Distribution of each sample in the relevant clusters after BH-SNE analysis.

[0130] FIG. 7. CAR T.sub.N/SCM better calibrate monocyte-like activation and cytokine production.

[0131] A) Activation kinetic of CAR T cells at different time points after stimulation with NALM-6 cells measured as upregulation of CD25/CD69 and HLA-DR activation markers (n=11). B) Number of T cells co-expressing activation markers (CD25/CD69/HLA-DR) 24 hours after co-culture. C) Schematic representation of tripartite co-cultures comprising NALM-6 leukemia, CAR T cells and THP-1 monocyte-like cells, together with Mock control. D) IL-6 production (left panel) and heat-map visualization of cytokine release (right panel) 24 hours after plating (n=4). E) Activation receptors (ARs) upregulation on T cells (CD54/CD86/HLA-DR, left) and THP-1 cells (CD54/CD86/CD163/HLA-DR, middle) expressed as MFI 24 hours after plating, together with correlation analysis between T-cell and THP-1 activation statuses (considering CD54/CD86/HLA-DR, right; n=4). Data are represented as the result of mean±SEM together with overlapping scattered values and box and violin plots. Results of paired t-test (B, D, E) and two-way ANOVA (A) are reported when statistically significant (*p<0.05, **p<0.01).

[0132] FIG. 8. CAR T.sub.N/SCM fine-tune monocyte activation and cytokine production and trigger less sCRS independently of CAR co-stimulation.

[0133] Experiments were conducted as described in FIG. 4A but with CAR T cells carrying the 4-1BB costimulatory domain. A) NALM-6-derived bioluminescence signal measured at different time points after treatment and expressed as Relative Light Units (RLU) (n=13 for CAR T.sub.BULK, n=12 for CAR T.sub.N/SCM, n=6 for Mock). B) T-cell expansion in the peripheral blood of mice. C) Weight loss evaluation at different time points after treatment. D, E) IL-6 and other cytokine serum levels, with their heat-map visualization, at day 4 after treatment (n=18 for CAR T.sub.BULK n=19 for CAR T.sub.N/SCM; n=6 for Mock). F) Severe CRS (sCRS)-related Kaplan-Meyer survival analysis of mice. G) CRS grading. Left panel: Kaplan-Meyer curves. Right panel: Histograms summarizing CRS grading. H) Monocyte absolute number immediately before T-cell infusion (n=13 for CAR T.sub.BULK, n=12 for CAR T.sub.N/SCM, n=6 for Mock). I) Percentage of activated monocytes co-expressing CD80/CD86/CD54/HLA-DR activation markers (ARs) 1 day after treatment (n=7 for CAR T.sub.BULK and CAR T.sub.N/SCM, n=3 for Mock). J) Evaluation of AR upregulation on CAR T cells (CD54/CD86) and monocytes (CD54/CD86/CD163) expressed as MFI at day 1 after treatment (n=11 for CD54 and n=7 for CD86 evaluated on CAR T.sub.N/SCM; n=9 for CD54 and n=6 for CD86 evaluated on CAR T.sub.BULK; n=6 for CD163 in the CAR T.sub.BULK cohort; n=7 for CD163 in the CAR T.sub.N/SCM cohort). K) Correlation between CAR T-cell and monocyte activation statuses at day 1 after treatment.

[0134] Data are represented as the result of box and violin plots, mean±SEM together with overlapping scattered values, or scaled according to a colored-graded range depending on relative minimum and maximum levels, when referring to the heat-map. P values (*p<0.05, **p<0.01) were determined by unpaired t-test (D, E, H, I, J), two-way ANOVA (A-C), Mantel—Cox two-sided log-rank test (F) and Gehan-Breslow-Wilcoxon test (G).

[0135] FIG. 9. CAR T.sub.N/SCM lessen monocyte reactions, due to an overall reduced activation signature.

[0136] A) Differently from CAR T.sub.BULK, CAR T.sub.N/SCM expand more while displaying a lower activation profile that results in reduced monocyte activation and cytokine release.

[0137] FIG. 10. CAR T.sub.N/SCM are less differentiated and display an overall reduced exhausted-like status compared to CAR T.sub.BULK in vitro. A) Frequency of central memory, effector memory and terminally differentiated T-cell subsets at the end of culture (n=16). B) Left plots and panel: Tsne representation and percentage of T cells co-expressing TIM-3, LAG-3 and PD-1 inhibitory receptor (IRs) after co-culture with CD19+ targets (NALM-6 and ALL-CM cell lines; n=19 for CAR T.sub.BULK, n=15 for CAR T.sub.N/SCM). Right panel: IRs mean fluorescence intensity (MFI) values (n=11 donors).

[0138] Data are represented as the result of mean±SEM together with overlapping scattered values and box and violin plots. Results of two-way ANOVA (A) and paired t-test (B) are reported when statistically significant (*p<0.05).

[0139] FIG. 11. Monocyte levels before CAR T-cell infusion were comparable in all the experimental groups. A) Monocyte absolute number immediately before CAR T-cell infusion in HuSGM3 leukemia bearing mice (n=9 for CAR T.sub.BULK, n=9 for CAR T.sub.N/SCM, n=6 for Mock).

[0140] FIG. 12. CAR T.sub.N/SCM display an overall reduced activation profile after encounter of CD19+ target cells. A) Number of T cells co-expressing CD25/CD69/HLA-DR activation markers 48 hours after co-culture with tumor cells (n=11).

[0141] Data are represented as the result of mean±SEM together with overlapping scattered values and results of paired t-test is reported when statistically significant (*p<0.05).

[0142] FIG. 13. CAR T.sub.N/SCM BBz are less differentiated and display lower activation and expansion in vitro. A) Memory phenotype at the end of T-cell manufacturing (n=7 donors), together with B) CD4/CD8 ratio (n=9 donors) and C) HLA-DR expression at the end of culture (n=9 donors). D) Fold expansion at different time points during culture (n=7).

[0143] Data are represented as the result of mean±SEM together with overlapping scattered values. Results of paired t-tests (C) and two-way ANOVA (A, B, D) are reported when statistically significant (*p<0.05; ***p<0.001).

[0144] FIG. 14: hierarchical model of human T cell differentiation.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

Transduction and Culture Conditions

[0145] Buffy coats from healthy donors were obtained after written informed consent and IRB approval. CD45RA+/CD62L+ Naive/Stem Cell Memory T cells (T.sub.N/SCM) were FACS-sorted. Unselected T cells (T.sub.BULK) and T.sub.N/SCM were stimulated through MACS-GMP T Cell TransAct (Miltenyi), transduced with a bidirectional lentiviral vector encoding for a CD19.CAR.28z or a CD19.CAR.BBz (Amendola M, Nat Biotech 2005) and the LNGFR marker gene. Cells were kept in culture in TexMacs medium (Miltenyi), supplemented with low-doses IL-7/IL-15 (Miltenyi) for 15 days. CAR+ cells were enriched by sorting through magnetic labelling of the LNGFR marker gene. Phenotypic and functional analysis of each CAR T-cell product were performed at the end of manufacturing.

In Vitro Functional Assays

[0146] CAR T.sub.BULK or CAR T.sub.N/SCM cells were co-cultured with CD19+ leukemic cell lines (Lucia+/NGFR+/NALM-6; ALL-CM; BV-173) at different E:T ratios. Untransduced T cells were used as control (Mock). After 24 h hours, supernatants were collected and analyzed with the LEGENDplex bead-based cytokine immunoassay (Biolegend). After 4 days, residual cells in culture were analyzed by FACS using Flow-Count Fluorospheres (BeckmanCoulter). The elimination index was calculated as follows: 1−(number of residual target cells in presence of target antigen-specific CAR T cells/number of residual target cells in presence of CTRL CAR T cells). For de-granulation assays, CAR T.sub.BULK or CAR T.sub.N/SCM cells were labeled with FITC-anti-CD107a immediately after co-culture with different CD19+ cell lines at the 1:3 E:T ratio. After 24 hours, cells were collected and analyzed by FACS. For proliferation assays, CAR T.sub.BULK or CAR T.sub.N/SCM cells were co-cultured with CD19+ targets at the E:T ratio of 1:1. After 4 days, cells were stained for intracellular Ki-67 and analyzed by FACS. Concerning assays for CAR T-cell activation kinetics, T cells and NALM-6 cells were co-cultured at the 1:10 E:T ratio and CD69/CD25 upregulation, together with HLA-DR expression were evaluated at several time points. Finally, a tripartite co-culture comprising NALM-6 leukemia, T cells and wild type THP-1 monocyte-like cells was conceived for 24 hours at a 1:1 E:T ratio. At the end of the experiment, supernatants were collected and analyzed as previously mentioned for cytokine detection, while the expression of CD163, CD86, HLA-DR and CD54 activation markers was evaluated on T cells and monocyte-like cells.

In Vivo Experiments

[0147] All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of San Raffaele University Hospital and Scientific Institute and by the Italian Governmental Institute of Health (Rome, Italy).

[0148] Six to 8-week-old NOD.Cg-Prkdcscid IL-2rgtm1Wjl/SzJ (NSG) mice were obtained from Jackson Laboratory. In the indolent tumor model, NSG mice were injected i.v. with 8×10.sup.6 ALL-CM cells and, upon tumor engraftment, treated i.v. with 2×10.sup.6 CAR T.sub.BULK, CAR T.sub.N/SCM or Mock T cells. In the aggressive tumor setting, NSG mice were injected i.v. with 0.5×10.sup.6 Lucia+/NGFR+/NALM-6 cells and after 5 days treated i.v. with 1×10.sup.6 or 3×10.sup.6 CAR T.sub.BULK, CAR T.sub.N/SCM or Mock T cells. Lucia+/NGFR+/NALM-6 cells were monitored by bioluminescence detection, using the QUANTI-Luc detection reagent (InvivoGen), while ALL-CM cells and CAR T cells were monitored by FACS using Flow-Count Fluorospheres (BeckmanCoulter)..sup.30

[0149] Six to 8-week-old NSGTgCMV-IL3, CSF2, KITLG1Eav/MloySzJ (SGM3) mice were sub-lethally irradiated and infused i.v. with 1×10.sup.5 human cord blood CD34+ cells (Lonza). Upon reconstitution, HuSGM3 mice were infused i.v. with 0.5×10.sup.6 Lucia+/NGFR+/NALM-6 cells and 5 or 7 days later, in the low and high tumor burden setting respectively, treated i.v. with 1×10.sup.6 or 1×10.sup.7 CD19.CAR T.sub.BULK, CD19.CAR T.sub.N/SCM or control Mock T cells. Mice were sacrificed when Relative Bioluminescent Units exceeded the threshold of 1.5×10.sup.6 or when manifesting clinical signs of suffering. For evaluating CRS development, weight loss was daily monitored and the concentration of serum human cytokines (LegendPLEX, Biolegend) and mouse SAA (ELISA kit abcam) were weekly assessed according to the manufacturer instructions. CRS incidence and grading were calculated by taking into account several sCRS related parameters, ie., weight loss, mice death, together with IL-6, MCP-1 and IP-10 myelo-derived cytokines, assigning a CRS grade to each treated mouse. These parameters were specifically scored and pondered within an algorithm that was designed taking into consideration the statistical differences occurring between sCRS-related deaths and recovering animals.

BH-SNE Analysis

[0150] BH-SNE (Barnes-Hut Stochastic Neighborhood Embedding) was applied on concatenate down sampled CD3+ events (7400 events/sample) collected from the peripheral blood of HuSGM3-NALM-6 bearing mice treated with CAR T cells, 14 days after infusion. BH-SNE algorithm analysis settings were perplexity=30000 and theta=0.5. Flow-SOM algorithm was then calculated for the cytometry variables of interest and clustered data in 50 different groups. Clusters were first studied in their composition by means of raw percentages and, when attributed to one experimental group, the mean fluorescence for the variables of interest was calculated and normalized according to the mean fluorescence of the total experimental dataset.

Histopathological Analysis

[0151] Brains from HuSGM3 mice were collected at necropsy, fixed in buffered 4% formalin, embedded in paraffin, cut and stained in Good Laboratory Practice (GLP) SR-TIGET Pathology laboratory following Good Laboratory Practices principles. Haematoxylin and eosin stained 3-μm paraffin sections were blindly and independently examined for histopathological analysis by two pathologists. Selected slides were stained with rabbit monoclonal anti-CD3 (2GV6), employing automated BenchMark Ultra Ventana in the Pathology Unit (accredited ISO 9001:2008, certification n. IT-25960). Photomicrographs were taken using the AxioCam HRc (Zeiss) with the AxioVision System SE64 (Zeiss).

Statistical Analysis

[0152] Statistical analyses were performed with Prism Software 9.1 (GraphPad). Data are shown as Mean±SEM with at least n=3 replicates. Datasets were analyzed with paired or unpaired Student's t-test, two-way ANOVA, or Gehan-Breslow-Wilcoxon and Mantel-Cox two-sided log-rank tests depending on the experimental design. Differences with a P value<0.05 were considered as statistically significant.

Cell Lines

[0153] Leukemic cell lines NALM-6 and BV173 were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 (BioWhittaker), supplemented with 10% FBS (Lonza), 100 IU/ml penicillin/streptomycin and glutamine. ALL-CM cell line was kindly provided by Fred Falkenburg, Leiden University Medical Center and kept in culture in X-VIVO (Lonza) with 3% human serum (Euroclone) and 100 IU/ml penicillin/streptomycin. For in vivo experiments NALM-6 cell line was transduced with a lentiviral vector encoding for the secreted luciferase Lucia (Lucia+/NGFR+/NALM-6), as previously reported..sup.30

Multi-Parametric Flow Cytometry

[0154] HuSGM3 peripheral blood samples were obtained at day 14 after CAR T-cell infusion and stained with monoclonal antibodies specific for human CD3 BV605 (clone SK7), CD8 BV650 (clone SK1), CD4 (L3T4) BUV496 (clone SK3), CD57 BB515 (clone NK-1), CD223 (LAG-3) APC-R700 (clone T47-530), CD45RA APC-H7 (clone HI100), TIGIT BV421 (clone 741182), CD279 (PD-1) BV480 (clone EH12.1), CD27 BV750 (clone L128), CD25 (IL-2 Receptor a chain) BUV563 (clone 2A3), CD62L (L-selectin) BUV805 (clone DREG-56), CD95 (Fas/APO-1) PE-Cy™7 (clone DX2), CD28 PE-Cy™5 (clone CD28.2), CD45 APC (clone HI30), CD272 (BTLA) BB700 (clone J168-540), CD197 (CCR7) PE (clone 150503), CD271 (NGF Receptor) BUV395 (clone C40-1457), CD98 BUV661 (clone UM7F8), CD154 BUV737 (clone TRAP1) (BD Biosciences). Samples were stained in brilliant staining buffer (BD). In addition, CAR T-cell and mouse samples were stained with one or more of the following conjugated monoclonal antibodies: CD3 PB (Biolegend, cloneHIT3a), CD45 BV510 (Biolegend, clone HI30), CD271 PE-Cy7 (Biolegend, clone CD40-1457), CD271 PE (BD, clone C40-1457), CD4 FITC (Biolegend, clone SK3), anti-mouse CD45 PerCP (Biolegend, clone 30411), CD14 APC (Biolegend, clone M5E2), CD19 APC/Cy7 (Biolegend, clone HIB19), HLA-DR APC/Cy7 (Biolegend, clone L243), CD45RA FITC (Biolegend, clone HI100), CD62L APC (Biolegend, clone DREG-56), CD8 PerCP (BD, clone SK1), CD107a (FITC), Ki-67 (Pacific Blue), CD69 APC (Biolegend, clone FN50), CD25 APC/Cy7 (Biolegend, clone BC96), CD163 FITC (Biolegend, clone GHI/61), CD54 PE (Biolegend, clone HA58), CD80 PE-Cy7 (Biolegend, clone 2D10), CD86 APC (Biolegend, clone IT2.2). Flow-cytometry data were acquired using BD Symphony and BD Canto II cell analyzers and visualized with FlowJo_V10 software.

Results

[0155] CAR T.sub.BULK Display a More Pronounced Effector Signature Compared to CAR T.sub.N/SCM In Vitro

[0156] With the aim of uncovering if pre-selection of early memory subsets as starting sources for manufacturing could enhance the therapeutic potential of CAR T cells, the inventors FACS-sorted CD62L+/CD45RA+T.sub.N/SCM cells with a purity of ˜99.1% and employed bulk unselected T cells for comparison. Both T.sub.N/SCM and T.sub.BULK were activated with the TransAct nanomatrix, transduced to express a CD28 co-stimulated CD19 CAR and expanded with IL-7 and IL-15 (FIG. 1A).

[0157] Quite surprisingly, phenotypical characterization at the end of culture pointed out a higher proportion of T.sub.SCM cells in CAR T.sub.N/SCM compared to CAR T.sub.BULK (FIG. 1B), together with a reduction in effector memory T cells (T.sub.EM) (FIG. 10A), even though a similar CD4/CD8 ratio was maintained (FIG. 1C). In addition, a lower activation profile in terms of HLA-DR expression and a reduced expansion were characteristic of CAR T.sub.N/SCM compared to CAR T.sub.BULK (FIG. 1D, 1E).

[0158] To evaluate if the two CAR T-cell products exhibited different functional capabilities, the inventors challenged them against multiple CD19+ leukemia cell lines. CAR T.sub.N/SCM displayed a reduced de-granulation capability (FIG. 1F), a lower cytotoxic potential (FIG. 1G) and a decreased production of pro-inflammatory cytokines, as compared to CAR T.sub.BULK (FIG. 1H). In contrast, a similar proliferation response was detected between CAR T.sub.N/SCM and CAR T.sub.BULK (FIG. 1I). Interestingly, even though co-expression of PD-1, LAG-3 and TIM-3 inhibitory receptors (IRs) was similar after stimulation with CD19+ targets, the overall exhausted-like status of CAR T.sub.N/SCM was reduced compared to CAR T.sub.BULK, as displayed by the lower IRs mean fluorescence intensity (MFI) values (FIG. 10B).

[0159] These data indicate that the two CAR T-cell products are phenotypically and functionally different, with CAR T.sub.BULK showing a more pronounced effector signature compared to CAR T.sub.N/SCM.

CAR T.SUB.N/SCM .are Uniquely Able to Elicit Recall Anti-Tumor Responses in HSPC-Humanized Mice

[0160] The inventors reasoned that reduced in vivo efficacy by CAR T.sub.N/SCM in NSG mice could be dependent on either tumor aggressiveness or intrinsic CAR T.sub.N/SCM dependence on supportive human cells and cytokines, which are absent in classical xenograft mouse models. To address this issue, the inventors sought to employ the Hematopoietic Stem/Precursor Cell (HSPC)-humanized mouse model in triple transgenic SGM3 mice, which better support human healthy and tumor hematopoiesis compared to standard NSG..sup.29,31 In this model, the inventors previously reported that the presence of human myeloid cells is crucial to trigger CRS and neurotoxicity..sup.29 The inventors here hypothesized that this complex human network, which includes human hematopoietic cells and cytokines, could also be instrumental to appreciate the full antitumor potential and safety profiles of CAR T.sub.N/SCM.

[0161] The inventors therefore reconstituted SGM3 mice with human cord blood CD34+ cells and infused humanized mice (HuSGM3) with NALM-6 leukemia. Leukemia-bearing mice were then treated with high doses of CAR T.sub.N/SCM or CAR T.sub.BULK and monitored for T-cell expansion, tumor progression and overt toxicities. Leukemia control was equally achieved by both CAR T.sub.N/SCM and CAR T.sub.BULK in HuSGM3 mice, even though CAR T-cell expansion was higher when looking at CAR T.sub.N/SCM treated mice (FIGS. 5A and 5B). These results support the hypothesis that CAR T.sub.N/SCM are more dependent on supportive homeostatic cytokines for exerting their full potential.

[0162] Notably, in this experimental setting, characterized by a low tumor burden, mice did not experience severe CRS (sCRS), as indicated by only moderate and reversible weight loss and modest elevation of serum levels of IL-6 and Amyloid A (SAA), a murine homolog to the human CRS biomarker C-reactive protein.sup.29 (FIGS. 5C and 5D).

[0163] To further challenge the therapeutic potential of the two CAR T-cell populations, the inventors performed a similar experiment in HuSGM3 mice, where the inventors injected a lower T-cell dose and provided a second tumor re-challenge (FIG. 2A). In this setting, CAR T.sub.N/SCM showed comparable activity to CAR T.sub.BULK during the first antitumor response but were uniquely able to elicit recall responses upon leukemia re-challenge (FIG. 2B). This improved therapeutic potential was associated with a higher CAR T-cell expansion, which was even more evident during the second response (FIG. 2C). Accordingly, a trend towards higher IFN-γ production by CAR T.sub.N/SCM was observed during the first and second antitumor responses (FIG. 2D). Notably, 14 days after infusion, CAR T.sub.N/SCM comprised an increased percentage of T.sub.CM compared to CAR T.sub.BULK (FIG. 2E), possibly accounting for their superior and long-lasting therapeutic activity. Also in this setting, no signs of sCRS were detected, independently of the CAR T-cell population employed, as indicated by absence of weight loss and only moderate elevation of serum IL-6 and SAA (FIG. 2F, G).

[0164] Collectively, these results indicate that HuSGM3 mice offer the appropriate human environment to support the activity of CAR T.sub.N/SCM, which strongly outperformed CAR T.sub.BULK in terms of long-term therapeutic potential, due to their higher expansion rates and early memory preservation after leukemia encounter.

Barnes-Hut Stochastic Neighborhood Embedding (BH-SNE) Algorithm Identifies a Best Performing Phenotype Typical of CAR T.SUB.N/SCM

[0165] The selective enrichment of T.sub.CM in mice treated with CAR T.sub.N/SCM as compared to CAR T.sub.BULK prompted the inventors to investigate whether the functional differences between the two populations could be reflected in a different phenotype once in vivo. To answer this question, the inventors performed the same experiment as described in FIG. 2A, but with the aim of deepening the phenotypic characterization of CAR T cells after the first leukemia encounter, i.e. at day 14 after CAR T-cell infusion. To this aim, the inventors sought to employ an unsupervised approach based on the BH-SNE dimensionality reduction algorithm for data analysis..sup.32,33

[0166] As formerly observed, no difference in the capability of controlling leukemia growth was observed between CAR T.sub.N/SCM and CAR T.sub.BULK (FIG. 6A). However, unsupervised and stochastic data downscaling, in which ˜74000 CD3+ lymphocytes were chosen for each file, together with the multi-dimensionality reduction operated by BH-SNE analysis, revealed the enrichment of clusters in totally distinct areas between CAR T.sub.N/SCM and CAR T.sub.BULK (FIG. 3A, B). Examination of these clusters, in which a similar distribution of each sample was found (FIG. 6B), highlighted intrinsic differences in the phenotypic composition of CAR T.sub.N/SCM when compared to CAR T.sub.BULK. Of notice, clusters of CAR T.sub.N/SCM were extremely enriched in T.sub.SCM and T.sub.CM, whereas those concerning CAR T.sub.BULK preferentially exhibited an effector memory and effector memory RA+ phenotype (FIG. 3C). Moreover, CAR T.sub.N/SCM displayed an activated phenotype, characterized by co-expression of activation markers and limited enrichment of inhibitory receptors, while CAR T.sub.BULK were typified by an exhausted phenotype, co-expressing multiple inhibitory receptors in the absence of activation markers (FIG. 3D). Indeed, the opposed spatial orientation of CAR T.sub.N/SCM and CAR T.sub.BULK was directed towards the enrichment of either activation or inhibitory receptors, respectively, as evidenced by the heat-map visualization (FIG. 3E).

[0167] In conclusion, this unsupervised approach revealed that CAR T.sub.N/SCM are endowed with enhanced in vivo fitness, which relies on an improved preservation of early memory cells, higher activation and lower exhaustion.

CAR T.sub.N/SCM Display a Negligible Intrinsic Potential to Cause sCRS and Neurotoxicity

[0168] Concerned about the higher expansion rate displayed by CAR T.sub.N/SCM, which may theoretically increase their toxic potential, the inventors modified the previous experimental setting in HuSGM3 mice to exacerbate their intrinsic potential to elicit sCRS and neurotoxicity. Since such adverse events are known to be associated with both tumor burden and the level of CAR T-cell expansion upon infusion.sup.11,34,35 the inventors increased leukemia load and CAR T-cell dose of about one Log (FIG. 4A). In these conditions, CAR T.sub.N/SCM and CAR T.sub.BULK were mutually able to control leukemia growth, even though CAR T.sub.N/SCM showed a slightly slower kinetic of tumor clearance (FIG. 4B). Despite similar antitumor activity, CAR T.sub.N/SCM proliferated more than CAR T.sub.BULK, confirming that these cells are endowed with a superior expansion potential in vivo (FIG. 4C). Strikingly, however, while the majority of mice treated with CAR T.sub.BULK experienced severe, irreversible weight loss, most animals treated with CAR T.sub.N/SCM eventually recovered from toxicity (FIG. 4D). According to what observed in patients and in previous preclinical studies,.sup.11,16,29,36 the development of sCRS in mice treated with CAR T cells was associated with higher serum levels of IL-6 and SAA, both increased in the CAR T.sub.BULK compared to CAR T.sub.N/SCM treated group (FIG. 4E,F). Besides IL-6, a wide plethora of pro-inflammatory cytokines, released by immune components in concert with activated CAR T cells, was measured in all treated mice but, once again, overall cytokine levels were lower in mice receiving CAR T.sub.N/SCM than in those injected with CAR T.sub.BULK (FIG. 4G). Heat-map visualization of cytokine levels and composition confirmed this picture and revealed greater amounts of myeloid-derived cytokines, including IP-10, IL-8 and MCP-1, in the CAR T.sub.BULK treated mice compared to CAR T.sub.N/SCM (FIG. 4H).

[0169] Accordingly, a higher proportion of mice that received CAR T.sub.BULK succumbed to sCRS as compared to mice treated with CAR T.sub.N/SCM (FIG. 4I). In order to more precisely stratify CRS development, we then considered multiple parameters, i.e., weight loss, death event and myelo-derived cytokine levels, to generate an algorithm that assigns to each mouse a CRS score and allows to recapitulate the grading system employed in patients. By applying this strategy, we observed that none of the mice treated with CAR T.sub.N/SCM developed grade 4 CRS, which conversely was observed in the 33% of mice treated with CAR T.sub.BULK (FIG. 4J). Moreover, while absence of CRS was observed only in the 11% of CAR T.sub.BULK-treated mice, this proportion raised to 44% in the cohort infused with CAR T.sub.N/SCM, suggesting that this cell product has a lower potential to cause CRS.

[0170] Finally, with the aim of evaluating sings of possible neurotoxic events concomitant to sCRS development, mouse brains were collected at sacrifice and subjected to histopathological evaluation. Impressively, 3 out of 5 CAR T.sub.BULK treated mice showed multifocal hemorrhages,.sup.37 whereas, in the group treated with CAR T.sub.N/SCM only one mouse presented a small hemorrhagic focus (FIG. 4K; Table 4).

TABLE-US-00004 TABLE 4 CAR T.sub.N/SCM treated mice display negligible neurotoxic events. Incidence table of microscopic findings recorded in collected brains belonging to CAR T.sub.N/SCM, CAR T.sub.BULK and Mock control HuSGM3 leukemia bearing mice. EMH: Extramedullary hematopoiesis. Microsocopic findings Mock CAR T.sub.BULK CAR T.sub.N/SCM N. of brain 2 5 5 analyzed Hemorrhages 0 3 multifocal 1 focal Infarct 0 0 1 EMH 0 0 1

[0171] Taken together, these results indicate that, despite a greater expansion potential, CAR T.sub.N/SCM are intrinsically less prone than CAR T.sub.BULK to trigger detrimental CAR T cell-related toxicities, displaying a better balance between efficacy and safety profiles. Since before treatment the absolute counts of circulating monocytes, which are crucial for both CRS and ICANS pathogenesis.sup.29,36, were superimposable in the two groups (FIG. 11A), the reasons for differential toxicity must be sought in the intrinsic biology of the two CAR T-cell populations.

CAR T.SUB.N/SCM .Fine Tune Monocytes Activation and Pro-Inflammatory Cytokine Production

[0172] Intrigued by the enhanced safety profile of CAR T.sub.N/SCM despite higher expansion rates, we decided to better decipher the mechanisms underlying this behavior. We hypothesized that a different activation status and/or kinetic existing between the two CAR T-cell populations could account for their diversity in driving sCRS manifestations. In line with this, we first evaluated CAR T-cell activation response and kinetic in vitro after stimulation with NALM-6 leukemia cells. Interestingly, CAR T.sub.N/SCM cells activated less intensely than CAR T.sub.BULK, both in terms of CD69/CD25 and HLA-DR upregulation, even though the kinetic was superimposable between the two populations (FIG. 7A). Moreover, when looking at triple-positive CD69/CD25/HLA-DR marker expression during time, we found that the amount of activated CAR T.sub.N/SCM was significantly lower both at 24 (FIG. 7B) and 48 hours post-stimulation (FIG. 12A).

[0173] In order to assess whether reduced activation could play a role in downscaling monocyte activation and cytokine production, we set up a tripartite co-culture comprising NALM-6 leukemia, CAR T cells and THP-1 monocyte-like cells (FIG. 7C). After 24-hour stimulation with NALM-6 cells, we observed that IL-6 production was significantly reduced with CAR T.sub.N/SCM compared to CAR T.sub.BULK and the same was true also for other myelo-derived cytokines (FIG. 7D). Moreover, when looking at activation markers, we noticed a reduced activation profile of both T cells and monocyte-like cells in the condition including CAR T.sub.N/SCM as compared to CAR T.sub.BULK. Interestingly, a positive correlation was observed between CAR T cells and THP-1 cell activation levels, suggesting that by modulating CAR T-cell activation it is possible to modify the triggering of myeloid cells responsible for high cytokine release and systemic toxicity development (FIG. 7E).

[0174] Collectively, these data reveal a close relationship between the activation status of CAR T cells and myeloid cells and show that CAR T.sub.N/SCM regulates monocyte responses more safely than CAR T.sub.BULK.

CAR T.sub.N/SCM are Intrinsically Less Able to Trigger sCRS Independently of CAR Co-Stimulation, by Lowering Monocyte Activation and Cytokine Production

[0175] The data showed until now refer to CAR T cells incorporating a CD28 costimulatory domain. Aiming to assess if the reduced toxic profile is an intrinsic property of CAR T-cell products generated from T.sub.N/SCM, we transduced either T.sub.N/SCM or T.sub.BULK with a 41BB-costimulated CAR. Even in this case, we observed a higher enrichment of T.sub.SCM in CAR T.sub.N/SCM compared to CAR T.sub.BULK, while the CD4/CD8 ratio was similar (FIGS. 13A and 13B). In addition, CAR T.sub.N/SCM were characterized by a lower activation profile (FIG. 13C) and reduced expansion in culture (FIG. 13D).

[0176] Inventors next evaluated the safety profile of 41BB-costimulated CAR T cells in the same model employed in FIG. 4A, including high leukemia burdens and CAR T-cell doses. Both CAR T-cell populations were equally able to control leukemia growth (FIG. 8A), but CAR T.sub.N/SCM featured increased CAR T-cell expansion rates compared to CAR T.sub.BULK (FIG. 8B). Similar to their CD28z counterpart, also CAR T.sub.N/SCM treated mice experienced less severe weight loss compared to mice that received CAR T.sub.BULK (FIG. 8C), together with reduced serum levels of IL-6 (FIG. 8D) and other inflammatory cytokines (FIG. 8E). Along with this, sCRS-related survival rates in mice infused with CAR T.sub.N/SCM were significantly improved compared to CAR T.sub.BULK (FIG. 8F). Accordingly, the incidence of grade 3 and 4 CRS was significantly higher in the CAR T.sub.BULK population then in the CAR T.sub.N/SCM cohort (FIG. 8G), where grade 1 CRS was rather prevalent.

[0177] Strikingly, being provided with similar monocyte counts before treatment (FIG. 8H) and consistent with in vitro data obtained with CD28-costimulated CAR T cells, we observed a lower fraction of monocytes co-expressing activation markers, such as CD80, CD86, HLA-DR and CD54 in mice treated with CAR T.sub.N/SCM compared to mice that received CAR T.sub.BULK (FIG. 8I). Accordingly, the cumulative expression levels of activation markers in CAR T cells and monocytes were reduced in the CAR T.sub.N/SCM cohort compared to CAR T.sub.BULK (FIG. 8J). Finally, a positive correlation between CAR T-cell and monocyte activation levels was confirmed in vivo (FIG. 8K).

[0178] Overall, we can conclude that CAR T.sub.N/SCM, while displaying a higher expansion capability, are characterized by a lower potential to cause detrimental toxicities, thanks to their milder activation signature that translates in reduced monocyte activation and cytokine release (FIG. 9). Importantly, this feature is intrinsic to CAR T-cell products generated from T.sub.N/SCM and independent of the costimulatory domain included in the CAR construct, offering a general way for developing CAR T-cell therapies with ameliorated therapeutic indexes.

DISCUSSION

[0179] CAR T-cell fitness and antitumor activity can be enhanced through the enrichment of early memory subsets in the final cell product, by exploiting optimized manufacturing protocols..sup.12,17,23 However, whether pre-selecting specific T-cell populations before manipulation would be really beneficial is still an open issue, due to the paucity of comprehensive in vivo data and lack of toxicity profiling. Moreover, so far, the majority of studies have compared memory T-cell subsets with each other and not with total T lymphocytes, which are the principal cell source employed in clinical trials. Even when bulk T cells were considered as reference, stimulation with suboptimal manufacturing protocols was employed..sup.17,27,38 In this work, the inventors adapted the HSPC-humanized mouse model the inventors recently developed.sup.29 to investigate the efficacy and safety profiles of CAR T cells generated from pre-selected T.sub.N/SCM or total T lymphocytes employing a gold-standard procedure, based on stimulation with αCD3/CD28 nanomatrix and culture with IL-7/IL-15. Compared to the standard NSG mice, the HSPC-humanized model is characterized by the presence of innate immune cells and cytokines, offering thus a unique human network to uncover the full antitumor potential and safety profile of different CAR T-cell populations.

[0180] Accordingly, while being less potent in vitro, CAR T cells generated from naïve and stem cell memory T cells (CAR T.sub.N/SCM) mediated strong and durable antitumor responses in HSPC-humanized mice compared to CAR T-cell products generated from unselected T cells (CAR T.sub.BULK). Improved activity was by higher expansion rates, which allowed unbalancing the Effector:Target ratio in favor of T cells. Of notice, highly proliferating CAR T.sub.N/SCM maintained a relevant pool of early memory T cells after the first response and were less exhausted and more activated.

[0181] Accordingly, CAR T.sub.N/SCM were uniquely able to counteract tumor re-challenge, envisaging an increased ability to protect patients from tumor relapse.

[0182] High CAR T-cell expansion has been associated with increased incidence and severity of CRS and ICANS in patients..sup.10,14-16 Unexpectedly however, CAR T.sub.N/SCM showed a limited capability to induce severe toxicity, with negligible occurrence of grade 4 CRS and the majority of mice developing grade 1 or even no CRS (˜66%). On the contrary, CAR T.sub.BULK induced grade 4 CRS in a significant proportion of mice (˜30%) and only few had grade 1 CRS or remained CRS-free (˜20%). A clinical correlate to this finding is the observation that the employment of unselected CD8.sup.+ T cells compared to sorted T.sub.CM CD8+ cells for CAR T-cell manufacturing was associated with an increased risk of developing sCRS..sup.14,18 In keeping with this, it has been recently shown that heterogeneity of CAR T-cell products further associates with variation not only in efficacy but also as regards toxicity, especially in the case of CRS and ICANS development.sup.50.

[0183] Importantly, the inventors also observed that mice receiving CAR T.sub.BULK and experiencing sCRS showed multifocal brain hemorrhages, which were absent in mice treated with CAR T.sub.N/SCM. Being similar to the events described in patients suffering from severe neurotoxicity in clinical trials, the inventors interpreted these manifestations as clear signs of ICANS, resulting from endothelial damage..sup.15,16 Interestingly, while CRS and neurotoxicity induction by CAR T.sub.BULK was dependent on the tumor burden and T-cell dose, CAR T.sub.N/SCM proved to be intrinsically safer, independently of CAR co-stimulation, offering a unique option to limit patients' risk of developing fatal toxicities while increasing efficacy.

[0184] It is known that endo-costimulation dramatically influences CAR T-cell fitness, with CD28 imprinting a prominent effector signature and 4-1BB inducing enhanced persistence and reduced differentiation..sup.4,39,40 Therefore, the choice of the most suitable costimulatory domain may presumably change depending on the context. For example, coupling the self-renewal potential of T.sub.N/SCM with the typical effector capabilities of CD28 and its lower sensitivity to antigen density compared to 4-1BB.sup.51, could provide the right balance to increase long-term persistence, without threatening efficient and rapid tumor de-bulking when dealing with solid malignancies or tumors expressing low antigen levels.

[0185] Toxic manifestations and antitumor activity are the result of complex pleiotropic and contact-dependent interactions taking place between activated CAR T cells and innate immune cells, with monocytes being primarily involved in the pathogenesis of both CRS and ICANS..sup.29,36

[0186] We thus hypothesized that CAR T.sub.N/SCM inferior yet progressive activation was capable of stimulating innate immune cells at sufficient levels for mediating supportive antitumor activity, without triggering detrimental side effects. Indeed, even though CAR T.sub.N/SCM and CAR T.sub.BULK activation kinetic was similar, the former activated to a lesser extent, thus better tuning monocyte activation status and consequent cytokine production.

[0187] Recent data suggest that diminishing signal strength in CAR T cells can result in lower toxicity and enhanced antitumor activity.sup.42-44 Based on their indolent functionality, we hypothesized that CAR T.sub.N/SCM are capable of differently processing the signal strength delivered by the CAR molecule per se, thus resulting in improved efficacy and safety profiles. Indeed, we found that a positive correlation exists between CAR T-cell and monocyte activation, with CAR T.sub.N/SCM featuring a reduced activation profile with both the CD28 and 4-1BB costimulatory domains. In this way, selectively manipulating sorted T.sub.N/SCM should result in a final CAR T-cell product endowed with superior expansion potential but lower activation aptitude, capable to better calibrate the dynamic cellular and molecular mediators responsible for sCRS and ICANS development.

[0188] It has been reported that the frequency of T.sub.N/SCM in heavily-pretreated cancer patients can be extremely variable..sup.12,45-48 However, the pre-selection step could be highly beneficial to get rid of dysfunctional T cells, increasing CAR T-cell quality and lowering the dose required to achieve antitumor efficacy..sup.28 Moreover, the superiority of CAR T.sub.N/SCM could be successfully exploited in the allogeneic setting, thus overcoming patient-intrinsic T-cell defects and ensuring a widespread accessibility to therapy..sup.49 In both scenarios, pre-selection of T.sub.N/SCM could allow reducing patient-to-patient variability and better comparing the results among different clinical trials.

[0189] Taken together, our results clearly indicate that pre-selection of T.sub.N/SCM can lead to a better balance between T-cell efficacy and safety profiles, significantly improving the therapeutic index of current T-cell therapies in particular CAR T-cell therapies.

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