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WO2025045979A1 - Constructions de récepteurs antigéniques chimériques et leurs utilisations - Google Patents

Constructions de récepteurs antigéniques chimériques et leurs utilisations Download PDF

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WO2025045979A1
WO2025045979A1 PCT/EP2024/074131 EP2024074131W WO2025045979A1 WO 2025045979 A1 WO2025045979 A1 WO 2025045979A1 EP 2024074131 W EP2024074131 W EP 2024074131W WO 2025045979 A1 WO2025045979 A1 WO 2025045979A1
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car
cells
sgrp
cell
polynucleotide molecule
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Gregor HUTTER
Tomas DE AQUINO DOS SANTOS MARTINS
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Universitaet Basel
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Universitaet Basel
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/055—Fusion polypeptide containing a localisation/targetting motif containing a signal for localisation to secretory granules (for exocytosis)

Definitions

  • the present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SZRPy)-related protein (SGRP).
  • SZRPy signal regulatory protein gamma
  • GBM Glioblastoma
  • SOC current standard of care
  • chemo- and radiotherapy regimens are not curative, invariably leading to recurrent disease.
  • the survival time for patients with GBM is around 15 months, underscoring the need for a significant breakthrough in effective medical treatment 1 3 .
  • CAR T cell-based immunotherapies have had remarkable outcomes in the clinical treatment of hematological malignancies, yet the development of effective CAR T cell therapies against solid tumors remains challenging 4 ’ 5 .
  • a critical limitation of CAR T cells is the scarcity of known tumor-specific surface antigens and their heterogeneous expression profiles in GBM 6 .
  • One of the most well-studied target antigens in GBM is EGFRvIII, a tumorspecific, mutated form of EGFR expressed in approximately 40% of GBM cases 7,8 .
  • EGFRvIII mutations arise with concomitant EGFR amplification during clonal evolution events in GBM development, resulting in EGFRvIII- mosaic tumors 8,9 .
  • Immune checkpoint inhibitors have recently shown promising responses against solid tumors 13 .
  • the highly immunosuppressive tumor microenvironment (iTME) of GBM severely limits the efficacy of immune checkpoint blockade (ICB) 14 .
  • IRB immune checkpoint blockade
  • GBM glioma-associated macrophages and microglia
  • GBMs glioma-associated macrophages and microglia
  • Microglia are professional phagocytes of the brain. They play an important role in the brain's innate immune surveillance and strongly influence the outcome and response to pathological states through the release of cytokines, chemokines, and growth factors 18,19 .
  • CD47-SIRPa phagocytosis axis whereby SIRPa, expressed on the surface of microglia and macrophages, interacts with the ubiquitously- expressed CD47 transmembrane protein, thereby inhibiting phagocytosis 20,21 . Therefore, CD47 is an innate immune checkpoint co-opted by tumor cells as a ‘don't eat me’ signal, which results in immune evasion by tumor cells through reduced recognition by phagocytic cells 11,22 .
  • Blockade of CD47 has been shown to rescue GAM phagocytic function in GBM-bearing mice leading to a strong antitumoral response in vivo 23 25 .
  • clinical studies of systemic monotherapy with CD47 blockade have only recently begun to assess efficacy against solid tumors, showing promising clinical activity 26,27 .
  • the published data from these trials suggest overall safety and considerable activity but also report low bioavailability within the tumor as well as treatment-associated toxicity 28 .
  • the present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPY)-related protein (SGRP).
  • SIRPY signal regulatory protein gamma
  • the inventors of the present invention have developed a new combinatorial approach of intratumoral (i.t.) CAR T cell therapy and GAM modulation for an additive elimination of GBM with a fourth-generation CAR design, whereby anti-EGFRvIII CAR T cells constitutively release a soluble SIRPy-related protein (SGRP) with high affinity to CD47 29 . It has been found by the inventors of the present invention that the combination of tumortargeting CAR T cells with SGRP dramatically improved the elimination of EGFRvIII-mosaic GBM in vivo in orthotopic GBM and peripheral lymphoma xenograft mouse models.
  • the present invention provides a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPY)-related protein (SGRP).
  • SIRPY signal regulatory protein gamma
  • the present invention provides an amino sequence comprising a chimeric antigen receptor (CAR) or a fragment thereof comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); wherein the nucleotide sequence encoding the chimeric antigen receptor (CAR) is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPY)-related protein (SGRP).
  • SIRPY signal regulatory protein gamma
  • the present invention provides a construct comprising the polynucleotide molecule described herein.
  • Fig- 2 Outline of the SGRP engineering strategy including specific AA substitutions to the endogenous human SIRPy-Vl sequence and addition of an N-terminal IL-2 signal sequence (IL2sig) leading to constitutive SGRP secretion.
  • Full CAR sequences are listed in Table 2.
  • Fig. 3 AlphaFold 30 ’ 31 -generated in silico modeling displaying predicted protein-protein interactions of SGRP, hSIRPa-Vl and mSIRPa-Vl with hCD47. Amino acid sequence information regarding hCD47, IL2sig, hSIRPa-Vl, hSIRPy-Vl, IL2sig-SGRP, mSIRPa-Vl and SGRP are listed in Table 1.
  • TBP TATA-box binding protein
  • Fig. 14 Flow cytometric representation of surface target expression of tumor cell lines U251vIII, U251, U87, BS153, Raji and control neural stem cell line NSC197. Note: all subsequent in vitro and in vivo experiments encompassing EGFRvIIF U251 and U87 were performed using sorted cells on the EGFRvIIF population.
  • Fig. 16 Assessment of CAR T cell on-target killing capacity by co-culture time-lapse of nEGFP + U251vIII with mCherry + target-specific (aEGFRvIII CAR +/- SGRP) or nonspecific (aCD19 CAR +/- SGRP) at a 1 : 1 E:T ratio for 72 h.
  • Fig. 17 72 h timelapse co-culture experiment of endogenously EGFRvIIF BS153 cells with aCD19 or aEGFRvIII CARs, respectively. Tumor confluence (cells/well) is plotted over time. Curves represent the mean of duplicate measurements. Differences between each co-culture compared to the ‘tumor alone’ control condition were analyzed using one-way ANOVA and Dunnett’s multiple comparisons tests.
  • Fig. 18 Tumor confluence (Green nuclei per well) in co-cultures of EGFRvIIF U251 with either aCD19, aCD19-SGRP, aEGFRvIII or aEGFRvIII- SGRP CAR T cells in 3 different effectortarget ratios at 24, 48 and 72 h timepoints. Differences between each co-culture compared to the ‘tumor alone’ control condition were analyzed using one-way ANOVA and Dunnett’s multiple comparisons tests.
  • Fig. 19 Dose-dependent CAR T cell killing capacity in a co-culture with U251vIII at defined time points. Straight horizontal lines represent the mean confluence in control wells with U251vIII only.
  • Fig. 24 Schematic (top panel) of the experimental setup of an SGRP/aCD47 blocking assay on CD47 + target tumor cells where (1) BS153 were treated with aEGFRvIII- S GRP CAR conditioned-medium or aCD47, (2) exposed to bt-SIRPa which competitively bound to available CD47, and (3) CD47-bound bt-SIRPa was assessed by SA-FITC and MFIs calculated; Representative dot-plots (bottom panel) depicting CD47-blocking capacity determined by FC-based detection of SA-FITC coupled to GBM-bound bt-SIRPa. Conditions were performed in triplicates and the experiment was repeated once.
  • Fig. 25 FC representation of surface CD47 expression of aEGFRvIII CAR and aEGFRvIII- SGRP CAR T cells from 3 HDs. Unstained T cells served as a negative control.
  • Fig. 26 FC assessment of phagocytosis and macrophage polarization/effector function in cocultures of EGFRvIII-mosaic tumor cells with donor-matched macrophages and CAR T cells from 4 HDs. Heatmap showing the MFI of markers and the fractions of phagocytosed U87 and U251vIII cells. Gated on CD1 lb + cells. All differences between conventional CARs and SGRP-secreting CARs were not statistically significant using one-way ANOVA and Tukey’s multiple comparisons tests.
  • Fig. 27 Experimental setup of the EGFRvIII xenograft GBM tumor model and subsequent monotherapeutic CAR and antibody treatment schemes. 7 and 14 d after orthotopic tumor implantation with U251vIII-NLuc tumor cells, animals were treated with either intratumoral (i.t.) CAR T cells or antibodies followed by BLi and scoring until the humane endpoint was reached.
  • Fig. 28 Overview of experimental groups/therapeutic conditions and treatment dosages
  • Fig. 29 Kaplan-Meier plot of overall survival (in days). Log-rank tests were used to compare selected treatment/control groups.
  • Fig. 30 Tumor progression in EGFRvIII xenografts was monitored for each individual mouse using BLi time course.
  • Fig. 31 Experimental treatment and monitoring schedule for EGFRvIII mosaic model. Animals were treated twice - at 7 and 14 days - after intracranial tumor implantation using the same stereotactic coordinates, and routinely monitored for clinical signs, weekly dual bioluminescence imaging (BLi) and morbidity/ survival assessment. Plasma for cytokine analysis was collected on day 15 - 24 h after the second intratumoral (i.t.) treatment. Anti- CD47 therapy was prolonged for 4 additional intraperitoneal (i.p.) injections on days 19, 22, 26 and 29. Animals were euthanized upon reaching the humane endpoint. Upon reaching 90 d tumor-free survival, 5 aEGFRvIII-SGRP CAR-treated animals were tumor-rechallenged in the contralateral hemisphere using the same stereotactic coordinates.
  • Fig. 32 Experimental setup of orthotopic xenograft experiments in NSG mice encompassing co-implanted EGFRvIII U251vIII and EGFRvIII' U87 GBM cell lines mimicking tumor heterogeneity, and therapeutic/control cohorts including local CAR T cell or antibody monotherapies or combinations with local SGRP or local and systemic CD47 blockade.
  • Fig. 33 Kaplan-Meier plot of overall survival (in days). Log-rank tests were used to compare selected treatment/control groups. Survival and bioluminescence data were pooled from 3 independent experiments.
  • Fig. 34 Kaplan-Meier plot of tumor-free survival (in weeks), combining survival assessment with BLi monitoring scores. Log-rank tests were used to compare selected treatment/control groups. Survival and bioluminescence data were pooled from 3 independent experiments.
  • Fig. 35 Tumor progression in EGFRvIII-mosaic xenografts was monitored for each individual mouse using differential BLi time course imaging with either FFz or D-luciferin substrates (in weeks); U251vIII NLuc-reporter BLi curves are depicted here.
  • Fig. 36 Tumor progression in EGFRvIII-mosaic xenografts was monitored for each individual mouse using differential BLi time course imaging with either FFz or D-luciferin substrates (in weeks); U87 Luc2-reporter BLi curves are depicted here.
  • Fig. 37 Cumulative differential monitoring in weeks by BLI for both grafted mosaic EGFRvIII and EGFRvIII' tumors per experimental condition as outlined in Fig. 32 Growth of U251vIII + tumors was measured by luminescence elicited by FFz, whereas growth of U87vlir tumors was detected by D-luciferin luminescence. Curves end whenever the humane endpoint was reached.
  • Fig. 38 Representative overlay images of dual BLi studies at week 7 of aEGFRvIII CAR, aEGFRvIII CAR + aCD47 and aEGFRvIII- S GRP CAR treated animals highlighting the suppression of EGFRvIIF tumors in aEGFRvIII- S GRP CAR treated animals.
  • Fig. 39 Quantification of BLi signal intensities (mean photon counts) as a surrogate for tumor burden for both EGFRvIII and EGFRvIIF tumors from Fig.38 at 7 weeks after tumor implantation. Each dot represents an individual mouse. Statistical comparisons were performed with one-way ANOVA with multiple comparisons corrections.
  • Fig. 40 Outline of in vivo experiment comparing the effect of aCD19-SGRP CAR and aEGFRvIII- S GRP CAR on the GBM iTME in the context of EGFRvIIF or EGFRvIII-mosaic intracerebral tumors.
  • Fig. 41 Heatmap of scaled median cluster-defining cell lineage marker expression on 10 immune cell populations indicated on the left y-axis. Cluster frequency is indicated on the right y-axis.
  • Fig. 47 Clustered heatmap of targeted post-therapy plasma proteomics showing the relative expression of proteins between all treatment groups; the analysis included samples from complementary datasets which were bridged and normalized (from lighter to darker grey: dataset 1, dataset 2, bridge samples). Individual protein expressions across the datasets are depicted on the y-axis, each cell represents the Z-score of all the measurements in this row. Data are clustered according to Euclidean distance. The barplot on the right shows the proportion of measurements under the limit of detection for each protein in each dataset.
  • Fig. 48 Non-metric multidimensional scaling (NMDS) plot of 92 examined human soluble proteins in mouse plasma determined by proximity extension assay.
  • NMDS Non-metric multidimensional scaling
  • Fig. 49 Volcano plot comparing aEGFRvIII- S GRP CAR and aEGFRvIII CAR treatment groups, showing significant enrichment of immune markers CCL3, IL13, and reduction of CD27. Significant differences in protein expression are represented by symbols: adj. P ⁇ 0.001, star; adj. P ⁇ 0.05, pentagon; P ⁇ 0.05, triangle; P > 0.05 or fold change (FC) ⁇ 0.5, dot.
  • Fig. 50 Box plots showing the normalized protein expression of significant innate immune surrogate markers in plasma (CD27, CCL3, IL13, ARG1, ILIA and IFNy). Each data point represents one mouse. Statistics were calculated using two-sided Mann-Whitney-U tests for the comparisons of interest (aEGFRvIII CAR vs aEGFRvIII CAR + aCD47, aEGFRvIII CAR vs aEGFRvIII- S GRP CAR, aEGFRvIII CAR vs aCD19 CAR, aCD19-SGRP CAR vs aEGFRvIII- S GRP CAR, aCD19 CAR vs aCD19-SGRP CAR, aEGFRvIII CAR + aCD47 vs aEGFRvIII- S GRP CAR, aCD47 vs aEGFRvIII CAR + aCD47) with Benjamini-Hochberg correction.
  • Fig. 51 Experimental treatment and monitoring schedule of the CCL3 blockade in vivo cohort. Animals were treated i.t. twice - at 7 and 14 days - after i.c. implantation of EGFRvIII U251vIII and EGFRvIII' U87 GBM cell lines. Tumor and CAR T cells were injected using the same stereotactic coordinates. Systemic aCCL3 therapy was administered 3x per week for 5 weeks, starting on day 8. Animals were euthanized upon reaching the humane endpoint or at day 90. CAR T cell dose: 5 x 10 5 cells delivered i.t.; Antibody dose: 50 ng delivered i.p.
  • Fig. 52 Kaplan-Meier plot of overall survival (in days). Log-rank tests were used to compare the indicated treatment/control groups.
  • Fig. 53 Overview of experimental groups/therapeutic conditions and treatment dosages for brain multiplex IF.
  • Fig. 54 H&E-stained sections of representative tumor-burdened brains at day 21 post tumor implantation and 7 days after the second treatment dose.
  • Fig. 55 DAPI nuclear-stained stitched assemblies of brain sections of experimental groups used for subsequent immunofluorescence multiplexing; Scale bars: 1000 pm.
  • Fig. 57 Brain collection intermediate post-therapeutic time points for conventional immunohistochemistry per experimental condition. Representative IHC images at d 13 for aEGFRvIII- S GRP CAR treatment and d 21 for all other groups showing CD3 + human T cells within the tumor core and adjacent brain (human CD3 IHC). Left column', overview of tumor- burdened brain sections, scale bar: 1 mm; right column', close-up according to the inserts at the tumor-brain interface. Scale bar: 100 pm. Bar graphs: Quantification of CD3 + T cells in the tumor rim and tumor core.
  • Fig. 58 Pie charts displaying relative comparisons of the percentage of marker-positive cells/all cells within the whole tumor or tumor cores in experimental conditions. Two slides per condition were assessed creating a ratio between overall positive/negative cells. No tumors were detected in any of the aEGFRvIII- S GRP CAR-treated brains analyzed.
  • Fig. 59 Brain collection intermediate post-therapeutic time points for conventional immunohistochemistry per experimental condition. Representative IHC images at d 13 for aEGFRvIII-SGRP CAR treatment and d 21 for all other groups showing CD68 stained brain sections per therapeutic condition. Left column', overview of tumor-burdened brain sections, scale bar: 1 mm; right column', close-up according to the inserts at the tumor-brain interface. Scale bar: 100 pm. Percentage of CD68 + cells within the tumor rim (defined as 100 pm expansion around the tumor). Percentage of CD68 + cells within the tumor core (delineated based on nuclear stain (or DAPI) density).
  • Fig. 60 Assessment of spleen to bodyweight ratio on day 21 post tumor implantation, after two treatments on days 7 and 14. Comparisons of all treatment groups to vehicle were nonsignificant using a one-way ANOVA with Dunnett’s multiple comparisons tests.
  • Fig. 61 Weekly weight monitoring of animals in the EGFRvIII-mosaic GBM survival experiment, showing aEGFRvIII CAR, aEGFRvIII CAR + aCD47, and aEGFRvIII-SGRP CAR groups. Dashed vertical lines mark the treatments in weeks 1 and 2 post tumor implantation.
  • NPX normalized protein expression
  • Fig. 64 Longitudinal monitoring of hematological parameters: erythrocyte count (RBC), platelet count (PLT) and neutrophil count (NEUT) for up to 20 days after a single treatment.
  • the scale was normalized to Z-score to allow direct comparisons between time points.
  • a mixed effects model was applied to each variable using the lme4 package in R. Post hoc tests for significant effects were conducted using estimated marginal means (emmeans package) to compare conditions within each time point. Significant differences between conditions were found only in neutrophil counts at day 3, 13, and 20.
  • Fig. 64 Longitudinal monitoring of hematological parameters: erythrocyte count (RBC), platelet count (PLT) and neutrophil count (NEUT) for up to 20 days after a single treatment.
  • the scale was normalized to Z-score to allow direct comparisons between time points.
  • a mixed effects model was applied to each variable using the lme4 package in R. Post hoc tests for significant effects were conducted using estimated marginal means (emmeans package)
  • Fig. 69 Left panel: Representative pseudocolor plots visualizing mCherry + CAR T cells and mTagBFP2 + tumor cells per experimental conditions. Right panel: quantification of CAR T cells and tumor cells per condition. Statistics: One-way ANOVA with Tukey’s multiple comparison test. * P ⁇ 0.05.
  • Fig. 70 Pseudocolor plots displaying the 4 most prominent CD45 + myeloid subsets in the TME of Vehicle or CAR-treated animals, differentiated by expression of P2RY12 and F4/80.
  • Bottom plot Backgating of the 4 populations termed ‘activated microglia’ (dark green, P2RY12 int F4/80 hi ), ‘microglia’ (light green, P2RY12 hi F4/80 int ), ‘moMacs’ (orange, P2RY12 lo F4/80 hi ), and ‘granulocytes’ (red, P2RY12'F4/80').
  • Fig. 71 Gating strategy to yield CD45 + or CD45' live singlets. Further myeloid subdivision on CD45+ cells was performed by identifying 4 populations with differential expression of P2RY12 and F4/80, respectively, named monocyte-derived cells (MdCs), activated microglia, resting microglia, and neutrophils. Human CAR T cells (moCD45‘) and BFP2 + tumor cells were assessed in the CD45' gate.
  • Fig. 72 Representative heatmap overlay of expression values of CD11c (top), CD86 (center) and MHCII (bottom) per experimental condition on the 4 different subsets from Fig.70 . The color bar insert represents MFI values.
  • Fig. 73 Quantification of mTagBFP2 signal intensities within different phagocytic subsets.
  • Top panel Staggered histograms of the median fluorescent intensities of mTagBFP2 for all Vehicle (light blue), aEGFRvIII CAR (dark blue) and aEGFRvIII-SGRP CAR (red) -treated animals.
  • Fig. 74 MFI of TNF and MHCII on the activated microglia subpopulation.
  • Statistics Oneway ANOVA with Tukey’s multiple comparison test. Adj. P values listed.
  • Fig. 75 TSNE (top) and UMAP (bottom) plots depicting the initial 15 clusters within the CD45 + pre-gated population.
  • Fig. 76 Clustered heatmap of scaled median marker expression per cluster. The number of cells per cluster is indicated on the right y-axis.
  • Fig. 77 Histograms of marker expression per cluster.
  • Fig. 79 TSNE plot after merging and annotation of initial clustering. 8 clusters (microglia, activated microglia, MoMac, MoDC, monocytes, pDC, neutrophils, and unknowns) were identified.
  • Fig. 80 UMAP (plots after merging and annotation of initial clustering. 8 clusters (microglia, activated microglia, MoMac, MoDC, monocytes, pDC, neutrophils, and unknowns) were identified.
  • Fig. 82 Stacked bar plots representing cluster frequencies per condition.
  • Fig. 83 Representative heatmap overlay of expression values of CD11c (top), CD86 (center) and MHCII (bottom) per experimental condition on the 4 different subsets identified by conventional gating and flow cytometric analysis.
  • the color bar insert represents MFI values.
  • Fig. 84 Overlay of P2RY12/F4/80 (upper panel) and P2RY12/CD11c on the TSNE plot from Fig. 79 displaying differential contribution of these markers to the microglia subclusters.
  • Fig. 85 TSNE plot of microglia-specific sub clustering, resulting in 3 microglia populations: resting microglia, MHCII-high microglia and activated microglia.
  • Right upper panel merged TSNE plot for aEGFRvIII CAR-treated animals; right lower panel: TSNE plot for aEGFRvIII-SGRP CAR treated animals.
  • Fig. 86 Histograms displaying individual marker expression per microglia subcluster.
  • Fig. 87 Stacked bar plots displaying the frequency distribution of microglia subpopulations per individual experimental animal.
  • Fig. 88 Median expression heatmap displaying XCR1 expression levels in microglia subpopulation per experimental condition/individual animal.
  • Fig. 89 Overview of median expression heatmaps per marker assessed per microglia subpopulations.
  • Fig. 90 EGFRvIII-expression analysis by quantitative real-time PCR (qPCR) in U87, U251 and U251vIII cell lines.
  • RFU relative fluorescent units.
  • Fig. 91 Top panel'. Experimental setup of pharmacoscopy experiment. Batched, frozen single-cell suspensions from GBM patients (tumor center) were thawed and plated at equal numbers into 384 well plates. Beforehand, EGFRvIII status was determined on RNA extracts of matching samples. Single-cell suspensions were co-cultured with aEGFRvIII, aEGFRvIII- SGRP, aCD19 or aCD19-SGRP CAR T cells for 48 h, fixed, stained and imaged via confocal microscopy. Center panel'. Exemplary immunofluorescence readouts of co-cultures of CAR T cells with single-cell suspensions derived from 5 GBM patients; Scale: 100 pm. Bottom panel'.
  • Fig. 92 Results of EGFRvIII screening by qPCR of patient-derived GBM EGFRvIII positive samples.
  • Fig. 93 Results of EGFRvIII screening by qPCR of patient-derived GBM EGFRvIII negative samples.
  • Fig. 94 Boxplots summarizing expression of EGFRvIII (upper panel) and EGFR wt (lower panel) in the 2 cohorts.
  • Fig. 95 Experimental setup and timeline of interventions of peripheral CD19 + lymphoma model treated with systemic CAR T cell infusions. Three days after tumor implantation in the right flank, mice were treated i.v. with CAR T cells, followed by 3 times weekly tumor volume assessment and clinical scoring. Mice were sacrificed upon reaching the humane endpoint.
  • Fig. 96 Overview of experimental groups/therapeutic conditions and treatment dosages.
  • Fig. 97 Kaplan-Meier plot of overall survival (in days). Log-rank tests were used to compare indicated treatment/control groups.
  • Fig. 98 Tumor volume measurements in mm 3 of individual animals over time (in days post tumor implantation).
  • the present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SZRPy)-related protein (SGRP).
  • SZRPy signal regulatory protein gamma
  • the present invention relates further to a chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule and methods of treating cancer using the polynucleotide molecule and/or the chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule.
  • CAR chimeric antigen receptor
  • the term "about” refers to a range of values ⁇ 10% of a specified value.
  • the phrase “about 200” includes ⁇ 10% of 200, or from 180 to 220.
  • fragment thereof or “fragment” in relation to a chimeric antigen receptor (CAR) refer to functionally active fragments of the CAR, preferably to functionally active fragments of the CAR which are capable of exercising the same physiological function as the CAR.
  • CAR chimeric antigen receptor
  • the present invention provides a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SZRPy)-related protein (SGRP).
  • SZRPy signal regulatory protein gamma
  • polynucleotide molecule further comprises a nucleotide sequence encoding a self-cleaving peptide.
  • the polynucleotide molecule comprises in the following order from the 5' to the 3' end: a) a promoter; b) a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof i) comprising an extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen binding region which binds to CD 19; wherein the nucleotide sequence encoding the chimeric antigen receptor (CAR) or a fragment thereof is operably linked to the promoter of a); c) a nucleotide sequence encoding a self-cleaving peptide; and d) a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPy)-related protein (SGRP).
  • SIRPy signal regulatory protein gamma
  • the heterologous signal peptide fused to a signal regulatory protein gamma (SIRPy)-related protein (SGRP) is a signal peptide selected from the group consisting of interleukin 2 (IL-2) signal peptide, interleukin 4 (IL-4) signal peptide, interleukin 9 (IL-9) signal peptide and interferon gamma (IFNy) signal peptide, preferably selected from the group consisting of human IL-2 signal peptide, human IL-4 signal peptide, human IL-9 signal peptide and human IFNy signal peptide.
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • IFNy interferon gamma
  • heterologous signal peptide fused to a signal regulatory protein gamma (SIRPy)-related protein (SGRP) is a human interleukin 2 (IL-2) signal peptide.
  • heterologous signal peptide is fused to the N-terminal region of the signal regulatory protein gamma (SIRPy)-related protein (SGRP).
  • SIRPy signal regulatory protein gamma
  • the promoter which is operably linked to the nucleotide sequence encoding the chimeric antigen receptor (CAR) or a fragment thereof is the elongation factor 1 alpha (EFl A) promoter or the elongation factor 1 alpha short (EFS) promoter, preferably the EFl A promoter.
  • the chimeric antigen receptor (CAR) or a fragment thereof comprises i) an extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) an extracellular domain comprising an antigen binding region which binds to CD 19; wherein the the CAR or a fragment thereof further comprises a CD8a leader, CD8a hinge and transmembrane domains, a 4-1BB costimulatory domain and a CD3( ⁇ signaling domain.
  • EGFRvIII epidermal growth factor receptor variant III
  • the extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III is a single-chain variable fragment (scFv); or ii) the extracellular domain comprising an antigen binding region which binds to CD 19 is a single-chain variable fragment (scFv).
  • the self-cleaving peptide is a T2A peptide or a P2A peptide, preferably a T2A peptide.
  • polynucleotide molecule comprises the sequence as shown in SEQ ID NO: 1:
  • polynucleotide molecule further comprises a peptide tag, preferably a tag selected from the group consisting of CD34, FLAG and MYC.
  • the present invention provides an amino sequence comprising a chimeric antigen receptor (CAR) or a fragment thereof comprising an extracellular domain comprising an antigen-binding region which binds to epidermal growth factor receptor variant III (EGFRvIII); wherein the nucleotide sequence encoding the chimeric antigen receptor (CAR) is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPy)-related protein (SGRP), wheren the amino acid comprises preferably the sequence as shown in SEQ ID NO: 3:
  • the present invention provides an amino sequence comprising a chimeric antigen receptor (CAR) or a fragment thereof comprising an extracellular domain comprising an antigen-binding region which binds to CD 19; wherein the nucleotide sequence encoding the CAR is operably linked to a promoter and wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPy)-related protein (SGRP), wheren the amino acid comprises preferably the sequence as shown in SEQ ID NO: 4:
  • SIRPy signal regulatory protein gamma
  • the present invention provides a construct comprising the polynucleotide molecule as described herein.
  • the construct is comprised by a viral expression vector, preferably by a lentiviral, retroviral or adenoviral expression vector or by a non-viral mammalian expression system, preferably by a PiggyBac, Sleeping Beauty or Tol2 expression system.
  • the present invention provides a chimeric antigen receptor (CAR)-T cell comprising T cells expressing the polynucleotide molecule as described herein and/or the construct as described herein.
  • CAR chimeric antigen receptor
  • the T cells are present in a therapeutically effective amount for the prevention and/or treatment of a cancer entity expressing epidermal growth factor receptor variant III (EGFRvIII) and/or CD 19.
  • EGFRvIII epidermal growth factor receptor variant III
  • the T cells are activated and produce one or more cytokines.
  • the one or more cytokines are selected from the group consisting of interferon gamma (IFNy), TNF, CCL3, IL- 13 and IL-1A.
  • At least a portion of the T cells express one or more surface markers selected from the group consisting of CD25, CD69 and CD 107a.
  • the chimeric antigen receptor (CAR)-T cell further comprises a pharmaceutically acceptable excipient, carrier, and/or diluent which supports maintenance of the T cells.
  • the present invention provides a method of treating cancer in a subject suffering from an epidermal growth factor receptor (EGFR)-associated cancer or suffering from a CD19-associated cancer, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • EGFR epidermal growth factor receptor
  • CD19 CD19-associated cancer
  • a chimeric antigen receptor (CAR)-T cell as described herein, for use in a method for the treatment of cancer in a subject suffering from an epidermal growth factor receptor (EGFR)-associated cancer or suffering from a CD19-associated cancer, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • EGFR epidermal growth factor receptor
  • a chimeric antigen receptor (CAR)-T cell as described herein for the manufacture of a medicament for the treatment of cancer in a subject suffering from an epidermal growth factor receptor (EGFR)-associated cancer or suffering from a CD 19- associated cancer.
  • EGFR epidermal growth factor receptor
  • a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein for the treatment of cancer in a subject suffering from an epidermal growth factor receptor (EGFR)-associated cancer or suffering from a CD 19- associated cancer.
  • the present invention provides a method of treating a solid cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • a chimeric antigen receptor (CAR)-T cell as described herein, for use in a method for the treatment of a solid cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • a chimeric antigen receptor (CAR)-T cell as described herein for the manufacture of a medicament for the treatment of a solid cancer in a subject.
  • a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein for the treatment of a solid cancer in a subject comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • the present invention provides a method of treating cancer in a subject, wherein the cancer is located in the central nervous system and wherein the method comprises administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof comprising an extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III (EGFRvIII).
  • CAR chimeric antigen receptor
  • a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof comprising an extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III (EGFRvIII), for use in a method for the treatment of cancer in a subject, wherein the cancer is located in the central nervous system, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein.
  • EGFRvIII epidermal growth factor receptor variant III
  • CAR chimeric antigen receptor
  • the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof comprising an extracellular domain comprising an antigen binding region which binds to epidermal growth factor receptor variant III (EGFRvIII), for the manufacture of a medicament for the treatment of cancer in a subject, wherein the cancer is located in the central nervous system,.
  • EGFRvIII epidermal growth factor receptor variant III
  • CAR chimeric antigen receptor
  • the cancer or solid cancer is selected from the group consisting of glioma, glioblastoma (GBM), medulloblastoma, ependymoma or diffuse intrinsic pontine glioma (DIPG), and is preferably GBM.
  • GBM glioblastoma
  • DIPG diffuse intrinsic pontine glioma
  • the present invention provides a method of treating cancer in a subject, wherein the cancer is selected from the group consisting of a breast cancer, lung cancer, melanoma, lymphoma, acute lymphocytic leukemia (ALL), and non-Hodgkin’s lymphoma (NHL), preferably lymphoma, and wherein the method comprises administering to the subject a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof comprising an extracellular domain comprising an antigen binding region which binds to CD 19.
  • CAR chimeric antigen receptor
  • a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof comprising an extracellular domain comprising an antigen binding region which binds to CD 19, for use in a method for the treatment of cancer in a subject, wherein the cancer is selected from the group consisting of a breast cancer, lung cancer, melanoma, lymphoma, acute lymphocytic leukemia (ALL), and non-Hodgkin’s lymphoma (NHL), preferably lymphoma.
  • CAR chimeric antigen receptor
  • a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof, comprising an extracellular domain comprising an antigen binding region which binds to CD 19, for the manufacture of a medicament for the treatment of cancer in a subject, wherein the cancer is selected from the group consisting of a breast cancer, lung cancer, melanoma, lymphoma, acute lymphocytic leukemia (ALL), and non-Hodgkin’s lymphoma (NHL), preferably lymphoma.
  • CAR chimeric antigen receptor
  • a therapeutically effective amount of a chimeric antigen receptor (CAR)-T cell as described herein, wherein the CAR or a fragment thereof of the CAR-T cell comprises a CAR or a fragment thereof, comprising an extracellular domain comprising an antigen binding region which binds to CD 19, for the treatment of cancer in a subject, wherein the cancer is selected from the group consisting of a breast cancer, lung cancer, melanoma, lymphoma, acute lymphocytic leukemia (ALL), and non-Hodgkin’s lymphoma (NHL), preferably lymphoma.
  • CAR chimeric antigen receptor
  • the cancer is a target antigen-mosaic tumor or a target antigen- homogeneous tumor, preferably a target antigen-mosaic tumor.
  • the present invention provides a method of preparing a population of activated T cells comprising a polynucleotide molecule as described herein or a construct as described herein, the method comprising: (i) contacting in vitro one or more T cells that have been modified to comprise a polynucleotide molecule as described herein or a construct as described herein with a stimulus that induces expansion of the T cells to provide an expanded T cell population; and (ii) activating in vitro the T cells to produce an activated T cell population.
  • the T cells are the chimeric antigen receptor (CAR)-T cell as described herein.
  • the present invention provides a nucleotide sequence encoding a signal regulatory protein gamma (SZRPy)-related protein (SGRP) comprising the sequence as shown in SEQ ID NO: 5:
  • the present invention provides a amino sequence comprising a signal regulatory protein gamma (SIRPY)-related protein (SGRP) comprising the sequence as shown in SEQ ID NO: 6:
  • SIRPY signal regulatory protein gamma
  • SGRP signal regulatory protein gamma
  • the present invention provides a nucleotide sequence encoding a signal regulatory protein gamma (SIRPY)-related protein (SGRP) comprising the sequence as shown in SEQ ID NO: 18:
  • SIRPY signal regulatory protein gamma
  • SGRP signal regulatory protein gamma
  • SGRP 9 amino acid substitutions were made to the endogenous human SIRPy binding domain (hSIRPy-Vl) AA sequence, as previously described by Ring and colleagues 29 .
  • a human IL-2 signal peptide (IL2sig) sequence was added to the N-terminus of SGRP to allow its secretion by T cells to the extracellular space.
  • the SGRP AA sequence was optimized for production by human T cells using GenSmart Codon Optimization (GenScript Biotech, USA) and reverse-translated into a nucleotide (nt) sequence. All AA and nt sequences are listed in Table 1 below.
  • Protein structures of SGRP, human SIRPa binding domain (hSIRPa-Vl), murine SIRPa binding domain (mSIRPa-Vl) and human CD47 (hCD47) were predicted by AlphaFold 30 ’ 31 , using the source code, trained weights and inference script available under an open-source license: https://github.com/deepmind/alphafold.
  • AA sequences of SGRP, hSIRPa-Vl, mSIRPa-Vl and hCD47 were entered and folded using the multimer model. Sequences were superimposed against a genetic database to generate multiple sequence alignment statistics.
  • Predictions ran on 'relax mode without GPU' and generated 3D interaction models for SGRP and hCD47, hSIRPa-Vl and hCD47, or mSIRPa-Vl and hCD47.
  • Predicted local distance difference tests were calculated to evaluate local distance differences of all atoms in each model and validate stereochemical plausibility (data not shown). All AA and nt sequences are listed in Table 1 below:
  • replication-defective lentiviruses were produced using a second- generation lentiviral system with transfer plasmids encoding a 3C10.BBz 32 ’ 33 (anti-EGFRvIII) or FMC63.BBz 34,35 (anti-CD19) CAR, an mCherry fluorescence reporter protein and, in some iterations, SGRP 29,36 .
  • the CAR structure consisted of a CD8a leader, a single-chain variable fragment (scFv), CD8a hinge and transmembrane domains, a 4- IBB costimulatory domain and a CD3( ⁇ signaling domain.
  • Transgene expression was driven by the EF1A promoter and polyprotein sequences were cleaved by T2A or P2A peptides. All sequences were assembled with Vector Design Studio (VectorBuilder, USA). The vectors included an ampicillin resistance gene for positive selection of transformed E. coli. The lentiviral plasmids were purchased as bacterial glycerol stocks from VectorBuilder (VectorBuilder, USA). CAR vector maps are illustrated in Fig. 5 and CAR vector sequences are listed in Table 2 below: Table !
  • Plasmid DNA was extracted with a QIAprep Spin Miniprep Kit (#27104, QIAGEN, Netherlands) from bacterial cultures grown overnight in the presence of 100 pg/mL ampicillin (#A5354, Sigma- Aldrich, USA). Lentiviral particles were generated by co-delivery of a transfer plasmid, a plasmid encoding a VSV-G envelope (pMD2.G; #12259, Addgene, USA) and an empty backbone plasmid (psPAX2; #12260, Addgene, USA) into HEK293T cells.
  • DNA plasmids were complexed with polyethyleneimine (PEI; #408727, Sigma- Aldrich, USA) for 15 min and added dropwise to the cells, followed by a 48 h incubation.
  • PEI polyethyleneimine
  • the viral supernatants were collected and cleared from cells and debris by centrifugation at 500 x g for 10 min, followed by filtration through a 0.45 pm polyethersulfone filter (#SLHPM33RS, MilliporeSigma, USA).
  • Virus particles were precipitated with Lenti-X Concentrator (#631232, Takara Bio, Japan), suspended in phosphate buffer saline (PBS; #D8537, Sigma- Aldrich, USA), quantified with Lenti-X GoStix Plus (#631280, Takara Bio, Japan), aliquoted and stored at - 80°C.
  • Lenti-X Concentrator #631232, Takara Bio, Japan
  • PBS phosphate buffer saline
  • Lenti-X GoStix Plus #631280, Takara Bio, Japan
  • Peripheral blood leukocytes from healthy donors were obtained from the Blood Donation Center of the University Hospital Basel, Switzerland, after informed consent was obtained from all participants before blood collection.
  • Peripheral blood mononuclear cells PBMCs were isolated by Ficoll Paque-PLUS (# GE17-1440-02, Cytiva, Germany) and density centrifugation. After up to 2 rounds of ACK-lysis (#A10492-01, Gibco, USA) for removal of erythrocytes, PBMC were washed with PBS.
  • CD3 + T cells were magnetically separated by negative selection with a Human Pan T cell isolation Kit (#130-096-535, Miltenyi Biotec, Germany) and stored long-term in Bambanker serum- free cell freezing medium (#BB01, GC Lymphotec, Japan) in liquid nitrogen (LN2).
  • CD14 + cells used in phagocytosis assays were magnetically separated by positive selection using Human CD14 MicroBeads (#130-050-201, Miltenyi Biotec, Germany) and stored in Bambanker serum-free cell freezing medium in LN2.
  • HD T cells were thawed, washed with PBS and rested in X-VIVO 15 (#BE02-060F, Lonza, Switzerland) at a density of 1 x 10 6 cells per mL at 37°C, in a 5% CO2 atmosphere.
  • the T cells were activated in X-VIVO 15 containing 150 U/mL of human IL-2 (#Ro 23-6019, Roche, Switzerland), 10 ng/mL of recombinant IL-7 (#200-07, PeproTech, USA), 10 ng/mL of recombinant IL-15 (#200-15, PeproTech, USA), 20 ng/mL of recombinant IL-21 (#200-21, PeproTech, USA) and Dynabeads human T-activator CD3/CD28 (aCD3/CD28; #1113 ID, Gibco, USA) in a 1 : 1 cell-bead ratio.
  • human IL-2 #Ro 23-6019, Roche, Switzerland
  • 10 ng/mL of recombinant IL-7 #200-07, PeproTech, USA
  • 10 ng/mL of recombinant IL-15 #200-15, PeproTech, USA
  • 20 ng/mL of recombinant IL-21 #200-21, PeproTech
  • aCD3/CD28 beads were magnetically removed and T cells were resuspended in X-VIVO 15 with 5 pg/mL polybrene (#TR-1003-G, Sigma- Aldrich, USA) at a density of 3 * 10 6 cells per mL.
  • Lentiviral suspensions were added to the T cells at different multiplicity of infection (MOI) ratios and spinfected at 2500 rpm, at 30°C for 90 min. After spinfection, the T cells were washed and maintained at a density of 1 x 10 6 cells per mL in X-VIVO 15 containing 500 U/mL IL-2 for 5-7 days.
  • T cells were sorted for mCherry expression using a BD FACSMelody Cell Sorter (BD Biosciences, USA). After sorting, the CAR T cell cultures were expanded in X-VIVO 15 containing 500 U/mL IL-2 and kept at a density of 1 x 10 6 cells per mL by adjusting the cell density every 2-3 days based on automated cell counting with a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems, South Korea) for 5-10 days until used in downstream assays.
  • a LUNA-FL Dual Fluorescence Cell Counter Logos Biosystems, South Korea
  • CAR T cell viability and count were assessed by Trypan blue exclusion.
  • Cells were washed with PBS and seeded into a 96-well plate at a density of 2 x io 5 live cells per well. Cells were immediately stained with a Zombie NIR Viability kit (#423106, BioLegend, USA) diluted 1 :5000 in PBS for 20 min in the dark at RT.
  • Zombie NIR Viability kit #423106, BioLegend, USA
  • the cells were washed with autoMACS Running Buffer (#130-091-221, Miltenyi Biotec, Germany) and then resuspended in 100 pL per well of 10 pg/mL dilutions of biotinylated CD 19, EGFR or EGFRvIII proteins (#CD9-H82E9, #EGR-H82E3 and #EGR-H82E0, ACROBiosystems, USA) for 1 h in the dark at 4°C.
  • autoMACS Running Buffer #130-091-221, Miltenyi Biotec, Germany
  • the cells were washed with autoMACS Running Buffer and stained with 100 pL per well of FITC Streptavidin (#405202, BioLegend, USA) diluted 1 :50 in autoMACS Running Buffer, for 1 h in the dark at 4°C. Afterward, the cells were washed 3 times with autoMACS Running Buffer and resuspended in 100 pL of autoMACS Running Buffer. Samples were acquired with a CytoFLEX Flow Cytometer (Beckman Coulter, USA) and data were analyzed using FlowJo vlO Software (BD, USA).
  • Expanded aEGFRvIII CAR and aEGFRvIII-SGRP CAR cultures were rested in X VIVO medium without additional supplements for 24 h. The following day, cell viability and count were assessed by Trypan blue exclusion, after which cells were washed in PBS, resuspended in RPMI at a density of 1 x 10 6 live cells per mL, and incubated for another 24 h at 37°C. Afterward, cultures were centrifuged at 300 x g for 5 min, and supernatants were collected, passed through 0.22 pm filters, and stored at -20°C.
  • the mass spectrometer was operated in DDA mode with a total cycle time of approximately 1 sec.
  • MSI 3e6 ions were accumulated in the Orbitrap over a maximum time of 100 ms and scanned at a resolution of 70000 FWHM at 200 m/z.
  • HCD high- collision-dissociation
  • MS2 scans were acquired at a target setting of le5 ions, a maximum accumulation time of 100 ms, and a resolution of 35000 FWHM at 200 m/z. Singly charged ions, ions with charge state > 6 and ions with unassigned charge state were excluded from triggering MS2 events.
  • the normalized collision energy was set to 27%; the mass isolation window was set to 1.4 m/z, and one microscope was acquired for each spectrum.
  • the generated mgf file was searched using MASCOT against a human database (consisting of 41094 forward and reverse protein sequences downloaded from Uniprot in April 2020), a manually entered recombinant SGRP AA sequence as well as 392 commonly observed contaminants using the following search criteria: full tryptic specificity was required (cleavage after lysine or arginine residues, unless followed by proline); 3 missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M) and acetyl (Protein N-term) were applied as variable modifications; mass tolerance of 10 ppm (precursor) and 0.6 Da (fragments).
  • the database search results were filtered using the ion score to set the false discovery rate (FDR) to 1% on the peptide and protein level, respectively, based on the number of reverse protein sequence hits in the dataset.
  • Quantitative analysis results from label- free quantification were processed using the SafeQuant R package v.2.3.2 (https://github.com/eahrne/SafeQuant/) (Ahrne, E., Molzahn, L., Glatter, T., & Schmidt, A. (2013). Critical assessment of proteome-wide label-free absolute abundance estimation strategies. Proteomics. Journal of Proteome Research https://www.ncbi.nlm.nih.gov/pubmed/23794183) to obtain peptide relative abundances.
  • This analysis included global data normalization by equalizing the total peak/reporter areas across all LC-MS runs, data imputation using the knn algorithm, summation of peak areas per protein and LC-MS/MS run, followed by calculation of peptide abundance ratios. Only isoform- specific peptide ion signals were considered for quantification. To meet additional assumptions (normality and homoscedasticity) underlying the use of linear regression models and t-tests, MS-intensity signals were transformed from the linear to the log scale. The summarized peptide expression values were used to test for differentially abundant peptides between conditions.
  • BS153, U87, U251 and U251vIII are human glioma cell lines.
  • BS153 cells were maintained in DMEM (#10938025, Gibco, USA) supplemented with 10% inactivated FBS, 1% pen strep, 2 mM GlutaMAX-I and 1 mM sodium pyruvate (#S8636, Sigma- Aldrich, USA).
  • U87, U251 and U251vIII cells were maintained in MEM (#M4655, Sigma- Aldrich, USA) supplemented with 10% inactivated FBS, 1% pen strep, IX MEM NEAA (#11140-035, Gibco, USA), 2 mM GlutaMAX-I and 1 mM sodium pyruvate.
  • Raji is a lymphoma cell line cultured in DMEM supplemented with 10% inactivated FBS, IX MEM NEAA and 1% pen strep.
  • GBM cells were cultured as adherent monolayers whereas Raji cells were cultured in suspension.
  • GBM cell line lentiviral transduction Parental EGFRvIILnegative U87 and U251 cell lines were transduced with a pmp71 lentiviral vector encoding a full-length EGFRvIII, to generate stable EGFRvIII-expressing U87vIII and U251vIII cell lines, respectively.
  • BS153, U251 and U251vIII cells were transduced with an Incucyte NucLight Green lentivirus (#4624, Sartorius, Germany) to express a nuclear-restricted EGFP (nEGFP) fluorescence viability reporter protein.
  • tumor cells were seeded at 1 x 10 5 cells per well of a 24-well plate and rested for 24 h. Growth media were replaced with antibiotic-free growth media containing 8 pg/mL polybrene. Lentiviral suspensions were added to the cells at different MOIs and incubated for 6 h at 37°C. Afterward, transduction media were replaced with fresh growth media and the cells were expanded for 1-2 weeks. Cells expressing the relevant surface receptor or fluorescence protein were sorted using a BD FACSMelody or a BD FACSAria SORP Cell Sorter (BD Biosciences, USA).
  • Killing assays were performed using an Incucyte S3 Live-Cell Analysis System (Sartorius, Germany). nEGFP-labeled target cells were seeded in flat-bottom, clear, 96-well plates at a density of 1 x 10 4 cells per well and incubated for 24 h to allow the formation of a cell monolayer. mCherry-labeled CAR T cells were added at various effector-target (E:T) ratios and co-cultures were followed for 72 h. Brightfield and fluorescence images were recorded every 4 h with a 10X objective.
  • E:T effector-target
  • BS153 viability and count were assessed by Trypan blue exclusion, after which cells were seeded in flat-bottom 96-well plates at a density of 3 * 10 5 cells per well. Cells were then treated for 30 min at 4°C with 50 pL per well of 10 pg/mL of InVivoMAb anti-human CD47 (clone B6.H12, #BE0019-l, Bio X Cell, USA) or InVivoMAb mouse IgGl isotype control (clone MOPC-21, #BE0083, Bio X Cell, USA) or conditioned-media from 24 h-rested, antigen-naive aEGFRvIII or aEGFRvIII-SGRP CAR T cells seeded at a density of 1 x 10 6 cells per mL of unsupplemented RPMI.
  • InVivoMAb anti-human CD47 clone B6.H12, #BE0019-l, Bio X Cell, USA
  • Macrophages were detached from culture dishes using cell scrapers after a 15-minute incubation at 37°C in TrypLE Express (#12604-021, Gibco, USA). Cells were washed in PBS, counted, seeded at 5 x 10 4 cells per well in a 96-well flat-bottom plate (#353072, Coming, USA), and incubated for 48 h to allow attachment. CAR T cells were counted and diluted to 1 x 10 6 cells per mL in X VIVO 15. Tumor cells were washed in PBS, dissociated, and counted. All cell counts were performed by Trypan Blue exclusion. U251vIII cells were diluted to 1 x 10 6 cells per mL in IMDM.
  • U87 cells were stained with 62.5 x 10 nM CellTracker Green (#C2925, Thermo Scientific, USA) diluted in IMDM (#12440-053, Gibco, USA) for 30 min at 37°C at a density of 1 x 10 6 cells per mL. After two washes with PBS, stained cells were counted and adjusted to 1 x 10 6 cells per mL in IMDM. U87 and U251vIII cells were then mixed in a 1 :1 ratio. The tumor cell mixture (1 x 10 5 cells) and CAR T cells (5 x 10 4 cells) were added to each well containing macrophages. The contents were resuspended in IMDM and incubated at 37 °C for 3 h.
  • NOD.Cg-Prkdc scid I12rg/SzJ (NSG) mice with identifier RRID:IMSR_JAX:005557 were obtained from in-house breedings or externally (Janvier Labs, France) under protocols approved by the SFVO and CVO of Basel-Stadt. Co-housed animals were assigned to treatment or control groups using a randomized approach and euthanized upon reaching the humane endpoint, including significant reduction of locomotion, significant weight loss and mild-to-severe neurologic symptoms. Tumor cell implantation was set as day 0 and the survival time was set as the day of euthanasia.
  • mice were injected intracranially (i.c.) with 5 x 10 4 U251vIII-NLuc cells.
  • mice were injected i.c. with a total of 5 x io 4 GBM cells consisting of 2.5 x 10 4 U87-Luc2 and 2.5 x 10 4 U251-NLuc, resulting in EGFRvIII- mosaic tumors.
  • the scalp was briefly swabbed with povidone-iodide solution, and a midline incision was made.
  • the scalp was briefly swabbed with povidone-iodide solution and a midline incision was made.
  • a burr hole was manually drilled 2 mm lateral from the cranial midline and 1 mm posterior of the bregma suture using a microdrill.
  • a digitally-controlled injection was performed with the Stereodrive Software (Neurostar, Germany) using a 10 pL Microliter Syringe (#80300, Hamilton, USA). The syringe was lowered into the burr hole to a depth of 3 mm below the surface of the dura and retracted by 0.5 mm to form a small reservoir in the cortex.
  • a volume of 4 pL of single-cell suspension was injected at 1 pL per min.
  • the needle was left in place for at least 1 min and carefully retracted by 0.5 mm every 30 sec. After injection, the incision was sutured (#D7585, Ethicon, USA).
  • Buprenorphine analgesia (Bupaq-P, Streuli Tier NU, Switzerland) was given intraperitoneally (i.p.) at 0.05 mg per kg immediately post-op.
  • the following treatments and controls were administered intratumorally (i.t.) in a volume of 4 pL on days 7 and 14: Vehicle (PBS), antibody Isotype (InVivoMAb mouse IgGl isotype control, clone MOPC-21; 5 pg), aCD47 (InVivoMAb anti-human CD47, clone B6.H12; 5 pg), aCD19 CAR (5 x 10 5 cells), aCD19-SGRP CAR (5 x 10 5 cells), aEGFRvIII CAR (5 x 10 5 cells), aEGRFvIII CAR + aCD47 (5 x 10 5 cells and 5 pg, respectively) or aEGFRvIII- S GRP CAR (5 x 10 5 cells).
  • Vehicle PBS
  • antibody Isotype InVivoMAb mouse IgGl isotype control, clone MOPC-21
  • aCD47 InVivoMAb anti-human CD47, clone B6.H12;
  • aCD47 and aEGFRvIII CAR + aCD47 treatment groups received additional doses of anti-CD47 (100 pg) administered i.p. in a volume of 100 pL on days 19, 22, 26 and 29.
  • CCL3 blockade survival experiment CAR T cells were applied as described above and 50 ng of antibody per mouse (aCCL3 or isotype) were administered i.p. in a volume of 100 pL on days 8, 10, 13, 15, 17, 20, 22, 24, 27, 29, 31, 34, 36, 38, and 41. Mice were monitored for clinical signs until day 90, upon which all remaining survivors were either euthanized or assigned to a tumor rechallenge experiment.
  • GBM engraftment and growth were monitored by bioluminescence imaging (BLi).
  • Mice implanted i.c. with EGFRvIII-mosaic tumors were subjected to dual BLi with specific substrates to separately monitor the growth of the EGFRvIII and EGFRvIIF tumor fractions.
  • the animals clinical scores and luminescence images were taken weekly, after i.p. injection of 150 mg kg' 1 of D-luciferin or intravenous (i.v.) injection of 0.325 pmol of fluorofurimazine per mouse.
  • Peripheral blood samples were collected approximately 24 h after each treatment dose (days 8 and 15 after tumor implantation). Unanestethized mice were briefly put under a heat lamp and then placed in a cylindrical restrainer. A small puncture was made on the tail to allow the dripping of approximately 100 pL of blood into lithium heparin-coated Microvette 100 capillary blood collection tubes (#20.1282.100, Sarstedt, Germany) and kept at RT. Blood samples were centrifuged at 4500 rpm for 15 min at RT and the top layer of plasma was transferred into sterile 0.5 pL Eppendorf tubes. Samples were immediately frozen at -80°C until analysis.
  • CAR- HEMATOTOX a model for CAR T-cell-related hematologic toxicity in relapsed/refractory large B-cell lymphoma.
  • influxing myeloid cells consisted of P2YR12 neg F4/80 int monocyte-derived macrophages and P2YR12 neg F4/80 neg myeloid cells, most of which were determined to be Ly6g + neutrophils (Fig. 70, 71).
  • eBFP intracellular signal intensity of eBFP as a surrogate for tumor cell phagocytosis.
  • aEGFRvIII- SGRP treated tumor-associated activated microglia, microglia and macrophages displayed a significantly increased eBFP-signal intensity compared to aEGFRvIII CAR T cell suggesting SGRP-mediated phagocytosis of BFP + tumor cells in the early treatment phase (Fig. 73).
  • aEGFRvIII- SGRP CAR-treated activated microglia displayed increased intracellular TNFa measurements by trend compared to EGFRvIII-CARs alone (Fig. 74).
  • microglia activated microglia, monocyte-derived macrophages (MoMacs), monocyte-derived dendritic cells (MoDCs), monocytes, plasmacytoid dendritic cells (pDCs), neutrophils, and unknowns (TSNE plots and UMAP in Fig. 79, 80), which exhibited similar population frequencies among conditions (Fig. 81, 82).
  • MoMacs monocyte-derived macrophages
  • MoDCs monocyte-derived dendritic cells
  • pDCs plasmacytoid dendritic cells
  • neutrophils neutrophils
  • XCR1 is described to be a specific classical dendritic cell marker involved in antigen presentation 48 , however, in our dataset, the cluster ‘activated microglia’ is characterized by P2RY12 int , SiglecH hlgh , MHCII hlgh , F480 hlgh , which most probably classifies them as of microglial origin.
  • aEGFRvIII CARs outperform aCD19 CARs in co-cultures with patient- derived single cell suspensions
  • Anti-CD19-SGRP CAR T cells have superior efficacy over conventional aCD19 CAR T cells in a peripheral lymphoma xenograft model
  • the therapeutic setup consisted of targeted CAR T cells (aCD19 CAR or aCD19-SGRP CAR) or non-targeted CAR T cells (aEGFRvIII CAR or aEFGRvIII-SGRP CAR) serving as controls (Fig. 96).
  • all aCD19 CAR-treated animals had a significant survival benefit compared to either Vehicle or non-targeted CAR controls (Fig. 97).
  • the contribution of SGRP-mediated innate immune modulation might be of relevance and clinical applicability in other, noncerebral cancer entities, depending on efficient CAR T cell homing to the respective tumor site.
  • SIRPa Signal Regulatory Protein Alpha
  • Microglia are effector cells of CD47-SIRPa antiphagocytic axis disruption against glioblastoma. Proc. Natl. Acad. Sci. U. S. A. 116, 997-1006 (2019).
  • Schindelin, J. et al. Fiji an open-source platform for biological-image analysis. Nat. Methods 9, 676-682 (2012).
  • Du, L. etal. IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities. Front. Mol. Biosci. 8, 675179 (2021).

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Abstract

La présente invention concerne une molécule polynucléotidique comprenant une séquence nucléotidique codant pour un récepteur antigénique chimérique (CAR) ou un fragment de celui-ci i) comprenant un domaine extracellulaire comprenant une région de liaison à l'antigène qui se lie au variant III du récepteur du facteur de croissance épidermique (EGFRvIII) ; ou ii) comprenant un domaine extracellulaire comprenant une région de liaison à l'antigène qui se lie à CD19 ; la séquence nucléotidique codant pour le CAR étant liée de manière fonctionnelle à un promoteur et la molécule polynucléotidique comprenant en outre une séquence nucléotidique codant pour un peptide signal hétérologue fusionné à une protéine régulatrice du signal gamma (SIRPγ)-protéine apparentée (SGRP). La présente invention concerne en outre un récepteur antigénique chimérique (CAR)-lymphocyte T exprimant la molécule polynucléotidique et des méthodes de traitement du cancer à l'aide de la molécule polynucléotidique et/ou du récepteur antigénique chimérique (CAR)-lymphocyte T exprimant la molécule polynucléotidique.
PCT/EP2024/074131 2023-08-31 2024-08-29 Constructions de récepteurs antigéniques chimériques et leurs utilisations Pending WO2025045979A1 (fr)

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US20160340397A1 (en) 2015-05-18 2016-11-24 Ab Initio Biotherapeutics, Inc. Sirp polypeptide compositions and methods of use
WO2021233411A1 (fr) * 2020-05-22 2021-11-25 重庆精准生物技术有限公司 Protéine de fusion permettant d'inverser un microenvironnement tumoral et son utilisation

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