Patent Application
20220362300
This disclosure concerns the generation and use of chimeric antigen receptor modified T cells from stem cells or progenitor cells.
Chimeric Antigen Receptor (CAR) T cell therapy is a cancer treatment that genetically alters T cells to redirect and harness their cancer killing potential. Currently FDA approved CAR T cell products are autologous-based, requiring individualized blood apheresis and manufacture. Deriving patient-specific CAR T cell products is expensive, laborious, and time consuming, with numerous logistical and regulatory challenges.
Generating CAR T cells from induced pluripotent stem cells (iPSC) holds encouraging prospect for generating ‘off-the-shelf’ CAR T cell products and overcoming these challenges. iPSCs can proliferate almost infinitely while keeping their pluripotency and lineage differentiation potential. However, the complexity of T cell development and disturbance of T cell differentiation by CAR expression creates a challenge for successful iPSC-derived CAR T cell generation.
Described herein, inter alia, are methods for making and using phenotypically defined, functional, and/or expandable T cells or NK cells expressing a chimeric antigen receptor (CAR) from pluripotent stem cells embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The CAR T cells and CAR NK cells described herein target a specific predetermined antigen expressed on the cell surface of a target cell, possess enhanced functional potential, enhance survival and treatment of cancers and/or targeted diseases, and/or possess cytotoxic potential and anti-tumor activity. The CAR T cells and CAR NK cells described herein may be used as “off-the-shelf” cells for administration to multiple recipients, which crosses immunogenic barriers and alleviates at least symptom of Graft versus Host disease (GVHD).
In some embodiments, naïve and memory T (Tn/mem) cells-derived iPSCs are start material for generating iPSC-derived CAR T cells. In some embodiments, peripheral blood mononuclear cells (PBMCs), naïve T (Tn) cells, memory T (Tmem) cells, naïve and memory T cells (Tn/mem), or a combination thereof-derived iPSCs are start material for generating iPSC-derived CAR T cells. Without being bound by theory, T cells already have the TCR gene rearranged during development, and the T-derived iPSCs maintain the rearranged TCR loci, which is important for T cell development during in vitro differentiation. In some embodiments and without being bound by theory, Tn/mem is a subpopulation of young T cells with premium fitness comparing to terminal differentiated effector T cells. The generated Tn/mem-derived iPSCs may also have unique properties because of less epigenetic footprints.
In some embodiments, described herein in a method for preparing a composition of CAR T cells, the method comprising:
In some embodiments, described herein in a method for preparing a composition of CAR NK cells, the method comprising:
In some embodiments, the PBMCs, Tn cells, Tmem cells, or Tn/mem cells, or combination thereof are human or are isolated from human blood. In some embodiments, the PBMCs, Tn cells, Tmem cells, or Tn/mem cells, or combination thereof are CD14−, CD25−, and CD26L+.
In some embodiments, the PBMCs, Tn cells, Tmem cells, or Tn/mem cells, or combination thereof are reprogrammed to generate iPSCs. In some embodiments, the iPSCs are generated by contacting the PBMCs, Tn cells, Tmem cells, or Tn/mem cells, or combination thereof with one or more of OCT3/4, OCT3, OCT4, SOX2, KLF4, L-MYC, C-MYC, LIN28, or short hairpin RNA targeting TP53 (shRNA-TP53). In some embodiments, transduced cells are cultured in X-Vivo15 medium supplemented with 50 U/mL IL-2, 0.5 ng/ml IL-15 and CD3/CD28 Dynabeads (bead:cell ratio of 1:1). In some embodiments, one, two, or three days after the transfection, equal volume of PSC medium containing bFGF and 10 μM Y27632 is added. In some embodiments, three, four, five, six, or seven days, the medium is then completely changed to PSC medium. In some embodiments, the iPSC cells are cultured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 26, 27, 28, 29, or 30 days. In some embodiments, individual colonies are picked to further cultivation and evaluation.
In some embodiments, CAR iPSCs are generated contacting the iPSCs with a nucleic acid or vector encoding a CAR. In some embodiments, transduced CAR iPSCs are cultured for at least 2 passages before single cell sorting and iPSC colonization. In some embodiments, colonized CAR IPSCs are expanded and banked for differentiation.
In some embodiments, the IPSCs or CAR iPSCs are genetically modified. In some embodiments, one or more genes are knocked out, down regulated, or upregulated.
In some embodiments, the one or more genes comprise one or more of TRAC, TRBC, B2M, CIITA, or combinations thereof. In some embodiments, TRAC, TRBC, B2M, CIITA are knocked out. In some embodiments, TRAC, TRBC, B2M, CIITA are down regulated. In some embodiments, genetic modification is achieved by methods described herein and those known in the art. In some embodiments, genetic modification methods comprise gene editing, homologous recombination, nonhomologous recombination, RNA-mediated genetic modification, DNA-mediated genetic modification, zinc finger nucleases, meganucleases, TALEN, or CRISPR/CAS9.
In some embodiments, CAR iPSCs are differentiated into embryonic mesodermal progenitor (EMP) cells and further differentiated into CAR T cells. In some embodiments, the EMP cells are CD56+ and CD326−.
In some embodiments, the CAR-expressing iPSCs are differentiated into embryonic mesodermal progenitor (EMP) cells and further differentiated into CAR NK cells. In some embodiments, the EMP cells are CD56+ and CD326−.
In some embodiments, the CAR iPSCs are differentiated into CD34+ hematopoietic stem and progenitor cells (HSPCs) and further differentiated into CAR T cells.
In some embodiments, the CAR iPSCs are differentiated into CD34+ HSPCs and further differentiated into CAR NK cells. In some embodiments, the CAR iPSCs are differentiated into CAR T cells using a nanofiber matrix-based culture system.
In some embodiments, the CAR iPSCs are differentiated into CAR NK cells using a nanofiber matrix-based culture system.
In some embodiments, the CAR is specific for a tumor, cell surface marker, and/or toxin. In some embodiments, the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), or combinations thereof.
In some embodiments, the CAR is bispecific.
In some embodiments, the chimeric antigen receptor comprises: at least one targeting domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR is a 1928z.
In some embodiments, described herein is a composition comprising the iPSC-derived CAR T cells or CAR NK cells. In some embodiments, a composition comprising iPSC-derived CAR T cells or CAR NK cells has enhanced therapeutic properties. In some embodiments, the iPSC-derived CAR T cells or CAR NK cells demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g. as activity that reduces the amount of tumor load.
In some embodiments, a composition comprising the CAR T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, naïve T cells, regulatory T cells, natural killer T cells, or combinations thereof. In some embodiments, a composition comprising the CAR T cells comprise CD3+, CD5+, CD7+, and TCRαβ+. In some embodiments, a composition comprising the CAR T cells comprise CD8+ CAR T cells are CD8αβ T cells, which have strong cytotoxicity against tumor cells in an antigen specific manner and can potently secret cytokines such as IFNγ. In some embodiments, CAR T cells have predominant homogenous TCR phenotype. In some embodiments, a composition comprising the CAR T cells comprise CD3+CD5+CD7+TCRαβ+CD8αβ+, CD3+CD5+CD7+TCRαβ+CD4+, CD62L+CD45RA+ stem memory T cells, CD62L-CD45RA-CD45RO+effector memory T cells and CD62L-CD45RA+ effector T cells, and combinations thereof.
In some embodiments, described herein is a method of increasing survival of a subject having cancer comprising administering a composition comprising a CAR T cell or CAR NK cell described herein.
In some embodiments, described herein is a method of treating a cancer in a patient comprising administering a composition comprising a CAR T cell or CAR NK cell described herein.
In some embodiments, described herein is a method of reducing or ameliorating a symptom associated with a cancer in a patient comprising administering a composition comprising a CAR T cell or CAR NK cell described herein.
In some embodiments, a composition comprising a CAR T cell or CAR NK cell described herein is administered locally or systemically. In some embodiments, a composition comprising a CAR T cell or CAR NK cell described herein is administered by single or repeat dosing. In some embodiments, a composition comprising a CAR T cell or CAR NK cell described herein is administered to a patient having a cancer, a pathogen infection, an autoimmune disorder, or an allogeneic transplant.
In some embodiments, the cancer is selected from the group consisting of blood cancer, B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, and sarcoma, acute myeloid leukemia (AML).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
DNA Constructs
CD19-targeted CAR (1928zCAR) and IL13Ra2-targeted CAR constructs were the same as currently used in clinical studies targeting B cell leukemia/lymphoma (clinicaltrials.gov #NCT01815749) (Wang, X., et al., Phase 1 studies of central memory-derived CD19 CAR T-cell therapy following autologous HSCT in patients with B-cell NHL. Blood, 2016. 127(24): p. 2980-90) and recurrent/refractory GBM (clinicaltrials.gov #NCT02208362) (Brown, C. E., et al., Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med, 2016. 375(26): p. 2561-9).
The 1928zCAR comprised a CD19 scfv domain, a CD28z costimulatory domain, IgG4 spacer with two point mutations (L235E and N297Q) within the CH2 region, the cytoplasmic a truncated human epidermal growth factor receptor (huEGFRt) as a safety switch (Jonnalagadda, M., et al., Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Mol Ther, 2015. 23(4): p. 757-68; Urak, R., et al., Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy. J Immunother Cancer, 2017. 5: p. 26; Wang, X., et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood, 2011. 118(5): p. 1255-63).
The IL13Rα2 CAR construct comprised of a human GM-CSF receptor a chain leader peptide, a human IL-13 with an E13Y mutation, an IgG4 spacer with 2 point mutations (L235E and N297Q), a CD4 transmembrane domain, a human 4-1BB costimulatory domain, and the cytoplasmic domain of human CD3ζ. In some embodiments, a truncated CD19 was also introduced in the construct to allow for potential enrichment and tracking of transduced cells (Brown, C. E., et al., Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med, 2016. 375(26): p. 2561-9; Brown, C. E., et al., Optimization of IL13Ralpha2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-tumor Efficacy against Glioblastoma. Mol Ther, 2018. 26(1): p. 31-44).
Tn/mem Isolation
Blood products were obtained from healthy donors under protocols approved by the COH IRB, and naïve and memory T (Tn/mem) cells were isolated following the similar procedures described in previous studies (e.g. Wang, X., et al., Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J Immunother, 2012. 35(9): p. 689-701). In brief, PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and then underwent sequential rounds of CliniMACS/AutoMACS (Miltenyi Biotec) depletion to remove CD14- and CD25-expressing cells, followed by a CD62L-positive selection for Tn/mem.
Generatation iPS Cells from PBMC or Tn/mem
Reprogramming PBMC or Tn/mem cells into iPS cells was conducted with procedures similar to published protocol (e.g. Okita, K., et al., An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458-66). In brief, 1-3 million PBMC or Naïve and memory T (Tn/mem) were electroporated with 3 μg plasmids mixture by using Nucleofector 4D electroporation device (Lonza). The plasmid mixture was composed of episomal plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. The transduced cells were cultured in X-Vivo15 medium supplemented with 50 U/mL IL-2, 0.5 ng/ml IL-15 and CD3/CD28 Dynabeads (bead:cell ratio of 1:1). Two days after the transfection, equal volume of PSC medium containing bFGF and 10 μM Y27632 was added. The medium was then completely changed to PSC medium 4 days after transfection. iPSC colonies were shown at day 20-30 and individual colonies were picked to further cultivation and evaluation.
Generation of CAR Positive iPS Cells and Colonized iPS Cell Line
iPS cells were routinely cultured in cGMP grade mTeSR1 medium (StemCell Technologies) in matrigel coated plates. Before lentivirus transduction, the iPS cells were dissociated with accutase treatment and single iPS cells were seeded in density of 105 per well in 12 well plate in mTeSR1 medium with supplement of 1X cloneR and 10 μM Y27632 (StemCell Technologies). After overnight culture, clinical grade lentivirus encoding CAR was added into the culture with 10 μg/mL protamine sulfate to transduce the iPS cells (multiplicity of infection [MOI]=1). The transduced cells were cultured for at least 2 passages before do single cell sorting and iPS colonization. Colonized CAR positive cells were expanded and banked for differentiation use.
iPSC Gene Editing
Certain genes, including TRAC, TRBC, B2M, CIITA, were knocked out by CRISPR-Cas9 gene editing technology using ribonucleoprotein (RNP) complex delivery. In brief, 180 pmol of chemical modified guide RNA with scaffold and gene specific target sequence was mixed with 60 pmol Truecut Cas9 protein (Thermofisher Scientific) in 50 ul P3 primary nucleofector solution (Lonza) and incubated for 10 min at room temperature to form RNP complex. iPS cells were dissociated with Accutase treatment. 1×105 single iPS cells were washed in PBC with 10 μM Y27632 and spun down by centrifuging at 300 g for 3 min. The supernatant was carefully removed and the cells were resuspended in 50 uL P3 primary nucleofector solution and followed by combining with the RNP complex solution. The combined cell suspension was transferred to the cuvette and electroporation was performed with Nucloefector 4D instrument (Lonza). After electroporation, 500 ul mTeSR1+1XcloneR was added into the cuvette and incubated for 15 min in incubator before transferring into Matrigel coated 6 well plate. The cells were cultured in mTeSR1 medium with supplement of 1X clonR and 10 μM Y27632 for 2 days. The medium was changed to mTeSR1+cloneR. After passaging for two times, single cell sorting were performed and colonized iPSC cells were cryopreserved. Genomic DNA were extracted and targeted PCR and sequencing were performed to screen for edited colonies.
Generation and Isolation of Human Embryonic Mesodermal Progenitors (hEMPs)
Mesoderm commitment was induced as previously described (e.g. Montel-Hagen, A., et al., Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell, 2019. 24(3): p. 376-389 e8; Chin, C. J., et al., Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors. Stem Cells, 2016. 34(5): p. 1239-50; Evseenko, D., et al., Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells. Proc Natl Acad Sci USA, 2010. 107(31): p. 13742-7). Briefly, human pluripotent stem cells (hPSC) were harvested as a single cell suspension after Accutase (StemCell Technologies) treatment, washed, and counted. Cells were resuspended directly in X-VIVO 15 medium supplemented with rhActivin A (10 ng/ml) (R&D Systems, Cat. 338-AC-010), rhBMP4 (10 ng/ml) (R&D Systems, Cat. 314-BP-010), rhVEGF (10 ng/ml) (R&D Systems, Cat. 298-VS-005), rhFGF (10 ng/ml) (R&D Systems, Cat. 233-FB-025), and ROCK inhibitor Y-27632 dihydrochloride (10 μM) (Tocris, Cat. 1254). Cells were plated on Matrigel coated 6-well plates at 3×106 cells per well in 3 ml. Medium was then changed daily with X-VIVO 15 supplemented with rhBMP4 (10 ng/ml), rhVEGF (10 ng/ml, and rhFGF (10 ng/ml). At day 3.5, cells were washed 3 times with PBS and incubated with Accutase (1 mL per well, for 10 min. at 37° C.). Cells were harvested using a cell scraper, washed in PBS, and stained with antibodies for flow cytometry. CD326-CD56+ hEMP were isolated by FACS on a FACSARIA cell sorter (BD Biosciences, San Jose, Calif.) or by CD56 enrichment kit (StemCell Technologies).
Differentiation of CAR+/− Tn/mem iPSC into CAR T/T cells by EMO ATO Culture System (Protocol IA)
Tn/mem iPS cells with or without CAR expression were induced to differentiate into EMP (CD56+CD326−) cells and then further to T cells according to published protocol (e.g. Montel-Hagen, A., et al., Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell, 2019. 24(3): p. 376-389 e8). Firstly, embryonic mesodermal organoids (EMO) culture were set up by aggregating EMP cells and MS5-hDLL4 feeder cells. MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza) supplemented with 10 μM ROCK inhibitor Y-27632 (StemCell Technologies) and 10 uM TGF-βRI inhibitor SB-431542 (SB blocker). At day −14, 5×105 MS5-hDLL4 cells were combined with 0.5-1×104 purified hEMP per PSC-ATO in 1.5 mL Eppendorf tubes and centrifuged at 300 g for 5 min at 4° C. in a swinging bucket centrifuge. Multiple (up to 12) EMOs were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing and resuspended in hematopoietic induction medium at a volume of 6 μl per EMO. 6 μl of cells were plated as EMOs on a 0.4 μm Millicell transwell insert (EMD Millipore) and placed in 6-well plates containing 1 mL hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 week, with medium composed of EGM2 with SB-431542 10 μM. This medium was changed every 2-3 days. At day −7, medium was change to EGM2+SB blocker (10 μM) with the hematopoietic cytokines rhTPO 5 ng/ml (Peprotech 300-18), rhFLT3L 5 ng/ml (Peprotech, Cat. 300-19), and rhSCF 50 ng/ml (Peprotech, Cat. 300-07). At day 0, PSC-ATOs were initiated simply by changing the medium to “RB27” supplemented with 10 ng/ml rhSCF, 5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7. Medium was changed completely every 3-4 days. After differentiation culture for 5-7 weeks, PSC-ATO CAR T cells or T cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2 mM EDTA) to each well and briefly dis-aggregating the ATO by pipetting with a 1 mL “P1000” pipet, followed by passage through a 50 μm nylon strainer.
Differentiate CAR+/− Tn/mem iPSC into CAR NK/NK Cells by EMO-ATO Culture System (Protocol 1B)
Tn/mem iPS cells with or without CAR expression were induced to differentiate into EMP (CD56+CD326−) cells and then further to CAR NK or NK cells by a similar protocol with protocol 1A (above) with modification of feeder cells and cytokine combination in the step of ATO culture. Briefly, the feeder cells in 1B would use MS5_DL1 instead of MS5_DL4. The 10 ng/mL IL15 was added along with other cytokines (10 ng/ml rhSCF, 5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7) from day 0. Tn/mem iPSC CAR NK/NK cells were harvested on day 28-50.
Generation and Isolation of CD34+ HSPC from iPSC Cells
iPS cells were differentiated into CD34+ hematopoietic stem and progenitor cells (HSPC) by using STEMdiff Hematopoietic Kit (Stemcell Technologies). Briefly, iPS cells were harvested and seeded as small aggregates in mTeSR1 medium. After one day culture, the medium was changed to differentiation medium A to induce the cells toward a mesoderm-like state. On day 2, half-medium was changed with fresh medium A. On day 3, medium was changed to B and half-medium changed was performed on day 5, 7, and 10 to promote further differentiation into hematopoietic cells. The hematopoietic progenitor cells were harvested from the culture supernatant on day 10-12. CD34 positive enrichment kit (Stemcell technologies) was used to enrich the CD34+ HSPC cells.
Differentiation of CAR+/− Tn/mem iPSC Derived HSPC into CAR T/T Cells by ATO Culture System (Protocol 2A)
Tn/mem iPSC derived HSPC cells were differentiated into CAR T/T cells using published ATO culture system {Montel-Hagen, 2019 #9; Seet, 2017 #19}. In brief, MS5-hDLL4 (or MS5-DLL1, as noted) cells were harvested by trypsin treatment and resuspended in serum free ATO culture medium (‘RB27’), which was composed of RPMI 1640, 4% B27 supplement (thermofisher scientific), 30 uM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 1% penicillin-streptomycin, 1% Glutamax, 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7. MS-hDLL4 were combined with enriched CD34+HSPC in 1.5 ml microcentrifuge tubes and centrifuged at 300 g for 5 min at 4° C. in a swinging-bucket centrifuge. Supernatants were carefully removed and the cell pellet was resuspended in ATO culture medium at a volume of 6 μl per ATO. 6 μl of cells slurry were plated as ATOs on a 0.4 mm Millicell transwell insert (EMD Millipore) and placed in 6-well plates containing 1 mL RB27 per well. Medium was changed completely every 3-4 days. After several weeks' culture, the generated CAR T/T cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2 mM EDTA) to each well and briefly dis-aggregating the ATO by pipetting with a 1 mL pipet, followed by passage through a 50 μm nylon strainer.
Differentiation of CAR+/− Tn/mem iPSC Derived HSPC into CAR NK/NK Cells by ATO Culture System (Protocol 2B)
Tn/mem iPS cells with or without CAR expression were induced to differentiate into CAR NK or NK cells by a similar protocol with protocol 2A (above) with modification. Briefly, the feeder cells in 2B were MS5_DL1 instead of MS5_DL4. 10 ng/ml rhSCF and 10 ng/ml IL15 was supplied in RB27 along with other cytokines (5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7). Tn/mem iPSC CAR NK/NK cells were harvested on day 28-50.
Differentiation of CAR+/− iPSC into CAR T/T Cells by Nanofiber Matrix Based Culture System (Protocol 3A)
In this protocol, when hMEP cells were ready at day −14, MS5-hDLL4 cells were harvested by trypsinization and cell suspension was irradiated for 80 Gy. For each well of 6 well plate, one Millicell transwell insert (EMD Millipore, pore size 0.4 μm˜3 μm) containing a polymeric nanofiber insert (Nanofiber solutions, ECM matrix) was placed and 2 mL medium was added into the well outside the insert, 2.5×105 EMP cells and 5×106 MS5-DLL4 cells were mixed, resuspended in 250 ul medium, and seeded directly on the nanofiber matrix insert. The hematopoietic induction medium comprised EGM2 (Lonza) supplemented with 10 μM ROCK inhibitor Y-27632 (StemCell Technologies) and 10 uM TGF-βRI inhibitor SB-431542 (SB blocker). Medium was changed completely every 2-3 days for 1 week, with medium composed of EGM2 with SB-431542 10 μM. At day −7, medium was change to EGM2+SB blocker (10 μM) with the hematopoietic cytokines rhTPO 5 ng/ml (Peprotech 300-18), rhFLT3L 5 ng/ml (Peprotech, Cat. 300-19), and rhSCF 50 ng/ml (Peprotech, Cat. 300-07). At day 0, the medium was changed to ‘RB27’ supplemented with 10 ng/ml rhSCF, 5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7. Medium was changed completely every 3-4 days. After differentiation culture for 5-7 weeks, the generated CAR T cells or T cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2 mM EDTA) to each well and briefly dis-aggregating the culture by pipetting with a 1 mL pipet, followed by passage through a 50 μm nylon strainer.
To differentiate iPSC into HSPC then further differentiate into CAR T or T cells, 250 uL cell mixture containing 2.5×105 enriched CD34+HSPC cells and 5×106 irradiated MS-hDLL4 cells were directly seeded on a nanofiber matrix insert in a 6 well plate with 2 ml RB27 medium with 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7. Medium was changed completely every 2-3 days for 5-7 weeks.
The nanofiber matrix based co-culture was also set up by directly adding hMEP/HSPC cells and irradiated MS5-DLL4 cell suspension in nanofiber plates (Nanofiber solutions) and centrifuging at 300 g for 3 min.
The nanofiber matrix based co-culture were prepared by mixing EMP/HSPC and MS5-DLL4 cells with micronized nanofibers in RB27 medium or methylcellulose based semi-solid medium, followed by seeding into ultralow attachment plates.
Differentiation CAR+/− iPSC into CAR T/T Cells by Nanofiber Matrix Based Culture System (Protocol 3B)
iPS cells with or without CAR expression were induced to differentiate into CAR T or T cells by a similar protocol with protocol 3A (above) with modification. Briefly, the feeder cells in 3B would use MS5_DLL1 as feeder cells instead of MS5_DLL4.
Differentiation from iPSC directly to CAR NK cells or NK cells, 10 ng/mL IL15 was added along with other cytokines (10 ng/ml rhSCF, 5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7) from day 0.
Differentiation from iPSC to HSPC then to CAR NK cells or NK cells, 10 ng/ml rhSCF and 10 ng/ml IL15 was supplied in RB27 along with other cytokines (5 ng/ml rhFLT3L, and 5 ng/ml rhIL-7).
CAR T Cell Preparation
PBMC or Tn/mem were stimulated with Dynabeads Human T expander CD3/CD28 (Invitrogen) at a ratio of 1:3 (T cell:bead) and transduced with lentivirus to express CAR in X-VIVO 15 (Lonza) medium containing 10% FCS with 20 μg/ml protamine sulfate (APP Pharmaceuticals), 50 U/ml recombinant human IL-2 (rhIL-2), and 0.5 ng/ml rhIL-15. Cultures were then maintained at 37° C., 5% CO2 under the same condition of media and cytokines. Cytokines were supplied every other day. On day 7 after transduction, the CD3/CD28 Dynabeads were removed from cultures using the DynaMag-50 magnet (Invitrogen).
Flow Cytometry
iPSC cells were dissociated with Accutase (Innovative Cell Technologies) and resuspended in mTeSR1 medium with 1X CloneR supplement (Stemcell Technologies). T cells were harvested and stained as described previously {Jonnalagadda, 2015 #3; Jonnalagadda, M., et al., Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Mol Ther, 2015. 23(4): p. 757-68}. iPSC phenotype was examined using fluorochrome-conjugated antibodies against SSEA3, SSEA4, TRA1-60, TRA1-81, CD30. T cell phenotype was examined using fluorochrome-conjugated antibodies against CD3, CD4, CD8a, CD8b, CD5, CD7, CD45, CD45RA, CD45RO, TCRab, TCRgd, CD16, CD56, CD27, CD28, NKP44, NKP46, NKG2A, NKG2D, CD178 (FasL), CD19. Transgene expression was determined by staining for the truncated EGFR or truncated CD19. Memory-associated phenotypes were analyzed with fluorochrome-conjugated antibodies against CD45RO, CD45RA, CD62L. All samples were analyzed via a MacsQuant Analyzer (Miltenyi Biotec) and processed via FlowJo v10.
TCR Vβ Repertoire Expression Analysis
T cell receptor Vβ staining was determined using three-color flow cytometry with the IOTest Beta Mark TCR Repertoire Kit (Beckman Coulter) which consists of monoclonal antibodies (mAbs) designed to identify 24 distinct TCR Vβ families. Each set consisted of three distinct anti-Vβ family-specific mAb labelled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or doubly labelled with FITC and PE. T cell population was also co-stained with APC-anti-CD3 antibody and CD3+ population was gated for analysis.
In Vitro T Cell Assays
To test for cytotoxicity and activity, target tumor cells were planted in 96 round bottom well plates at indicated density. T cells were then washed and resuspended in the same media and added to the target cells. To test for degranulation, CAR T or control T cells were incubated with target cells for 5 hours in the presence of CD107a antibody and Golgistop protein transport inhibitor (BD Biosciences). After the co-culture, cells were harvested, fixed, permeabilized, and stained for intracellular cytokines. Degranulation (CD107a staining) and intracellular cytokine staining were examined by flow cytometry. For cytotoxicity tests, co-culture would last 4 hour for short term assay and 48 hour for long term assay as indicated. After co-culture, all cells were harvested and stained with indicated antibodies, followed by quantification by flow cytometry.
In Vitro Cytokine Production Assay
CAR T or T cells were co-incubated for 24 hours with different target cells at an effector-to-target (E:T) ratio of 1:1. Supernatant was collected and the cytokines were examined by cytokine 10-plex human panel kit (Invitrogen) with Bio-Plex reader (Bio-Rad).
In Vivo Xenograft Studies
All mouse experiments were approved by the COH IACUC. Tumor xenograft models were generated using 6- to 8 week-old NOD/SCID/IL2R−/− (NSG) mice as previously described (e.g. Urak, R., et al., Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy. J Immunother Cancer, 2017. 5: p. 26). Briefly, on day 0, ffLuc+NALM6 cells (1×106) were intraperitoneal injected (i.p.) into the NSG mice. After 4 days, mice were then treated intraperitoneally with CAR T cells or T cells as indicated for each experiment. Tumor growth was determined by in vivo biophotonic imaging using a Xenogen IVIS 100. Mice were also monitored for survival, with euthanasia applied according to the American Veterinary Medical Association Guidelines.
Certain reagents and resources used in Example 4 are described in a table at the end of this section.
Mice
All animal experiments were conducted under a protocol approved by City of Hope Animal Research Committee. This study used 6-8 week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice from Jackson Laboratory.
DNA Constructs
The CD19-targeted chimeric antigen receptor (CD19-CAR) construct is the same as currently used in our clinical studies targeting B cell leukemia/lymphoma (clinicaltrials.gov #NCT02146924) [1, 2]. The CD19-CAR contains an anti-CD19 scFv domain derived from the FMC63 mAb [3], an IgG4 Fc spacer with two point mutations (L235E and N297Q) within the CH2 region, a CD28 transmembrane domain, a CD28ζ costimulatory domain, and a CD3 signaling domain. A T2A ribosome skip sequence [4] then separates this CAR sequence from a truncated human epidermal growth factor receptor sequence (huEGFRt) which can be used as a selection marker and safety switch[5-7]. The episomal plasmids encoding OCT3/4/shp53, SOX2/KLF4, L-MYC/LIN28, and EBNA were gifts from Shinya Yamanaka[8].
Tn/mem Isolation
Blood products were obtained from healthy donors under protocols approved by the COH IRB, and naïve and memory T (Tn/mem) cells were isolated following similar procedures described in previous studies[9]. In brief, human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and then underwent sequential rounds of CliniMACS/AutoMACS (Miltenyi Biotec) depletion to remove CD14- and CD25-expressing cells, followed by a CD62L-positive selection for Tn/mem cells.
Generation of iPSCs from Tn/mem
Tn/mem cells were reprogrammed into pluripotent stem cells (iPSCs) by an integration-free method modified from a published protocol N. In brief, one million Tn/mem cells were electroporated with 3 μg of plasmid mixture using the Human T Cell Nucleofector Kit and the Nucleofector 4D electroporation device (Lonza). The plasmid mixture was composed of episomal plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53 N. The transfected cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 10% FBS (HyClone), 50 U/mL rhIL-2 (Novartis Oncology), 0.5 ng/mL rhIL-15 (CellGenix) and Dynabeads Human T-Expander CD3/CD28 (ThermoFisher Scientific) (bead to cell ratio of 1:1). Two days after the transfection, an equal volume of pluripotent stem cell (PSC) medium containing rhFGF-basic and 10 μM Y27632 was added [8]. The medium was then completely changed to PSC medium 4 days after transfection. iPSC colonies were visible at day 20-30 and individual colonies were picked under a microscope for further culture/expansion in cGMP-grade mTeSR1 medium (StemCell Technologies) in Matrigel-coated (Corning) plates.
Generation of CAR-Positive, Clonal iPSC Lines
Before lentivirus transduction, iPSC cultures were dissociated with Accutase (ThermoFisher Scientific) treatment and the cells were seeded at a density of 105 per well in 12-well plates in mTeSR1 medium supplemented with 1X CloneR and 10 μM ROCK inhibitor Y-27632 dihydrochloride (StemCell Technologies). After overnight culture, cGMP lentivirus encoding CD19-CAR was added to the culture with 10 μg/mL protamine sulfate (APP Pharmaceuticals) to transduce the iPSCs (multiplicity of infection [MOI]=1). The transduced cells were cultured for at least two passages before single cell sorting by flow cytometry and iPSC colonization. Clonal CAR-positive cells were again expanded in mTeSR1 medium on Matrigel-coated plates, and banked for subsequent differentiation.
Integration Detection by PCR
EBNA1 is a common component of all episomal vectors [10]. To detect genomic integration of episomal plasmids used for iPSC reprogramming from T cells, PCR was performed to amplify integrated EBNA components from genomic DNA using primers as follows: EBNA1_For: ATCAGGGCCAAGACATAGAGATG, EBNA1_Rev: GCCAATGCAACTTGGACGTT. Plasmid integration free iPSC clones did not show EBNA1 signal. FBX15, which was expressed on pluripotent stem cells, was used as house-keeping gene here and was amplified by the following primers: FBX15_For: GCCAGGAGGTCTTCGCTGTA; FBX15 Rev: AATGCACGGCTAGGGTCAAA.
Teratoma Formation Assay
Two million dissociated iPSCs were suspended in 200 uL medium (100 uL PBS (Irvine Scientific) and 100 uL Matrigel) and injected subcutaneously into NSG mice. After 5-8 weeks, teratomas were harvested in PBS, fixed overnight in 4% paraformaldehyde (Boston BioProducts) at room temperature, and maintained thereafter in 70% ethanol for processing. Samples were submitted to the City of Hope Histology Core Facility for sectioning and hematoxylin and eosin staining. Sections were examined, interpreted, and photographed microscopically.
Differentiation of Tn/mem-Derived, CAR+ iPSC into CAR+ T Cells by PSC-ATO Culture
The schema of the sequential differentiation protocol is outlined in
iPSC mesodermal organoids (iMOs) were generated by aggregating iMP cells and MS5-hDLL4 feeder cells. On day −14, MS5-hDLL4 cells were harvested with trypsin and washed into hematopoietic induction medium composed of EGM-2 (Lonza) with 10 μM Y-27632 and 10 μM TGF-βRI inhibitor SB-431542 (StemCell Technologies). After using a 40 μm nylon mesh strainer to remove aggregates, 5×105 MS5-hDLL4 cells were combined with 0.5-1×104 purified iMP cells in 1.5 mL microfuge tubes and centrifuged at 300×g for 5 min at 4° C. in a swinging bucket centrifuge. Up to 12 iMOs were prepared in each tube. After carefully removing the supernatant, the MS5-hDLL4/iMP cell pellet was resuspended by brief pulse vortexing in hematopoietic induction medium (i.e., EGM-2 with 10 μM SB-431542) at 6 μl per iMO. Two 6 μL aliquots of cells were plated as iMOs on one Millicell transwell insert (Millipore Sigma) per well in 6-well plates containing 1.5 mL hematopoietic induction medium. Medium was changed completely every 2-3 days for one week. On day −7, medium was changed to EGM-2 with 10 μM SB-431542 plus 5 ng/mL rhTPO (Peprotech), 5 ng/mL rhFLT3L (Peprotech), and 50 ng/mL rhSCF (Peprotech).
On day 0, the artificial thymic organoid (ATO) T cell differentiation phase was initiated with a switch to serum-free ATO culture medium containing 10 ng/mL rhSCF, 5 ng/mL rhFLT3L, and 5 ng/mL rhIL-7 in RB27 medium that consisted of RPMI 1640 (Lonza), with 4% B27 Supplement (ThermoFisher Scientific), 30 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 1% GlutaMAX (ThermoFisher Scientific), 1% Penicillin-Streptomycin (Lonza), 55 uM 2-mercaptoethanol (ThermoFisher Scientific), and 1% MEM Non-essential Amino Acids (ThermoFisher Scientific). Medium was changed completely every 2-3 days. After 5-7 weeks of differentiation, iMO-ATO-derived CAR+ T cells were harvested by pipetting 1-2 mL of X-VIVO 15 with 10% FBS onto the surface of each transwell insert and disaggregating the iMO-ATO by repeated aspiration with a P1000 pipettor. Single cells were isolated by passing the disaggregated cell suspension through a 40 μm nylon mesh strainer. An aliquot of the recovered cells was stained with the indicated antibodies for phenotyping by flow cytometry and the remaining cells were cultured in a previously described rapid expansion method (REM) [14, 15], with minor modifications. Briefly, 1×106 iMO-ATO-derived T cells were combined with 50×106 γ-irradiated (35 Gy) PBMCs and 10×106 γ-irradiated LCL cells (80 Gy) in 50 mL X-VIVO 15 medium containing 10% FBS, 20 ng/mL anti-CD3 (Miltenyi Biotec), 50 U/mL rhIL-2 and 10 ng/mL rhIL-7. REM cultures were maintained for 14 days, with half-volume medium changes every 48 hours.
Immunohistochemistry
The PSC-ATO organoids were fixed and permeabilized with the Fixation/Permeabilization Solution Kit (BD Biosciences), stained with PE-anti-CD3 and DAPI in permeabilization buffer for 15 minutes and then rinsed with wash buffer three times. In situ images were taken with a BZ-X810 fluorescence microscope (Keyence).
Generation of Conventional CD19-CAR T Cells
PBMC (or Tn/mem as indicated for
Flow Cytometry
iPSCs were dissociated with Accutase (ThermoFisher Scientific) and resuspended in mTeSR1 medium with 1X CloneR supplement (StemCell Technologies). iPSC phenotype was examined using fluorochrome-conjugated antibodies against EGFR (to detect transgene), SSEA3, SSEA4, TRA1-60, TRA1-81, and CD30. T cells were harvested and stained as described previously[5]. T cell phenotype was examined using fluorochrome-conjugated antibodies against CD3, CD4, CD8α, CD8β, CD5, CD7, TCRαβ, TCRγδ, CD16, CD56, CD27, CD28, NKP44, NKP46, NKG2A, NKG2D, CD178 (FasL), and CD19. CAR expression was determined by staining for the truncated EGFR. Memory-associated phenotypes were evaluated with fluorochrome-conjugated antibodies against CD45RO, CD45RA, and CD62L.
T cell receptor Vβ staining was performed with the IOTest Beta Mark TCR Repertoire Kit (Beckman Coulter) which consists of monoclonal antibodies (mAbs) designed to identify 24 distinct TCR Vβ families. Each set consisted of three distinct anti-Vβ family-specific mAbs labelled with fluorescein isothiocyanate (FITC), phycoerythrin (PE) or doubly labelled with FITC and PE. The T cell population was also co-stained with APC-anti-CD3 antibody and the CD3-positive population was gated on for analysis. Data were acquired on MacsQuant Analyzer 10 (Miltenyi Biotec) or Fortessa (Becton Dickinson) flow cytometers and analyzed with FlowJo (v10.6.1).
PCR Based TCR β Clonality Assay
Genomic DNA was extracted by DNeasy kit (Qiagen) and used as PCR template. The PCR assay was set up according to the protocol of IdentiClone TCRB+TCRG T-Cell Clonality Assay Kit (Invivoscribe) [16, 17], TCRB tube A and B primer master mix target framework regions within the variable region and joining region of the TCR beta chain locus. TCRB Tube C targets the diversity and joining regions of the TCR beta chain locus. The specimen control size ladder master mix targets multiple genes and generates a series of amplicons to serve as quality control of input DNA. The primers are fluorescence labelled and fragment analysis was performed to detect the fragment size of PCR products concomitantly with regular DNA agarose gel examination.
In Vitro T Cell Functional Assays
Effector cells (iPSC CD19-CAR T, iPSC Mock T, conventional CD19-CAR T or conventional Mock T cells) were washed, resuspended in fresh medium containing 50 U/mL rhIL-2 and 0.5 ng/mL rhIL-15 and co-cultured in 96-well U-bottom plates with the indicated tumor cells at the indicated effector-to-target (E:T) ratios for 4 hours or 48 hours. Cytotoxic activity was then routinely evaluated by flow cytometry by enumerating viable (i.e., DAPI-negative) GFP-expressing tumor cells; for primary ALL cells, DAPI-/CD19+ cells were enumerated. Alternatively, for luciferase based cytotoxicity assays, at each timepoint, D-luciferin potassium salt (PerkinElmer) was added to each well at a final concentration of 0.14 mg/mL and plates were incubated at 37° C. for 10 minutes.
Following the incubation with luciferin, the contents of each culture plate were mixed carefully and transferred to an opaque 96-well U-bottom plate with a multichannel pipettor. Bioluminescent flux was read with a Cytation 3 plate reader (Biotek). For each tumor line, replicate wells of tumor cells alone were used to generate internal MIN (0% viability) and MAX (100% viability) references for the calculation of percent lysis; the MIN was obtained by the addition of SDS to a final concentration of 1% ten minutes before the addition of luciferin [18].
To evaluate T cell activation, iPSC-derived or conventional CAR T or Mock T cells were incubated with the indicated tumor cells at an E:T ratio of 1:1 for five hours in the presence of CD107a antibody and GolgiStop protein transport inhibitor (BD Biosciences). Cells were then harvested, fixed, permeabilized, and stained for intracellular cytokines. Degranulation (CD107a staining) and intracellular cytokine staining (e.g. IFNγ) on CD3-gated cells was then examined by flow cytometry. Similar co-cultures without GolgiStop were harvested for staining of surface activation markers CD25 and CD137/4-1BB on CD3-gated cells was evaluated by flow cytometry.
To further characterize cytokine production, iPSC-derived or conventional CAR T or Mock T cells were co-incubated for 24 hours with the indicated NALM6 tumor cells at an E:T ratio of 1:1 in medium without added cytokines. Supernatants were collected and cytokine levels were quantified with the Cytokine 10-Plex Human Panel Kit (ThermoFisher Scientific) by a Bio-Plex reader (Bio-Rad). Similar co-cultures were harvested for flow cytometric analysis of surface activation markers CD25 and CD137/4-1BB on CD3-gated cells.
For In vitro repetitive challenge assay, 105 CAR T cells were co-cultured with 4×105 CD19+ NALM6 cells at E:T ratio of 1:4, and re-challenged every other day with 4×105 NALM6 cells for 3 times. The cells were then stained with surface exhaustion markers PD-1, TIM-3 and LAG-3 together with T cells markers. Each of exhaustion marks was evaluated on CD3-gated cells by flow cytometry[19].
RNA and Protein Analysis
RNA was extracted with the Quick-RNA Microprep kit (Zymo Research) and treated with DNase I. RNA deep sequencing was performed by the City of Hope Integrative Genomics Core Facility. Briefly, stranded RNA-seq libraries were prepared using the KAPA mRNA HyperPrep kit (Roche), according to the manufacturer's recommended protocol. Libraries were quantified using Qubit quantification kit (Thermofisher Scientific) and loaded onto the HiSeq 2500 sequencing platform (Illumina) for single-end 51-bp sequencing. Base calling was done using Illumina Real Time Analysis (RTA) v1.18.64.
For protein analysis by western blot, the harvested cells were lysed in RIPA buffer (ThermoFisher Scientific) and protein extraction was quantified with a BCA protein assay kit (ThermoFisher Scientific). The Bolt Mini Gel System (ThermoFisher Scientific) was used for gel electrophoresis and protein transfer. Anti-p44/42 MAPK (Erk1/2) and anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); anti-PLCγ1, anti-phospho-PLCγ1 (Tyr783) and anti-phospho-PLCγ1 (Ser1248); anti-CD3, and anti-phospho-CD3ζ(Y142); and anti-phospho-ZAP70 were used to interrogate CAR T and T cell signaling pathways (see Resource Table for antibody details).
Bioinformatics Analysis of RNA Seq Data
To analyze the RNA seq data, the 2-D visualization of PCA was implemented using R package “DESeq2” (v.3.10) based on the PCA algorithm. Heatmaps of z-scores were generated by Cluster (v.3.0) and JavaTreeView (v.1.1.6r4) using a hierarchical clustering approach. Differentially expressed gene (DEG) analysis was performed with R package “edgeR” (v.3.28.0)[20]. The pipelines of deriving DEG involved the quantile-adjusted conditional maximum likelihood (qCML), and the quasi-likelihood (QL) F-test. Bubble plots were acquired with R package “ggplot2” (v.3.2.1). The Gene Set Enrichment Analysis (GSEA) algorithm was run on GSEA (v.4.0.3) [2, 22]. The resources of bioinformatic software packages are listed in the table of ‘Key resources’.
Bisulfite Conversion, PCR, and Sequencing
Genomic DNA was prepared by DNeasy kit (Qiagen). 500 ng genomic DNA was treated with sodium bisulfite to convert unmethylated cytosines using the EZ DNA Methylation-Lightning Kit (Zymo Research). Reactions were carried out per manufacture's protocol. Methylation-specific PCR was performed, and 245 bp PCR fragments of EF1a promoter from bisulfite converted gDNA of iPSC CD19-CAR T cells and conventional CD19-CAR T cells were amplified. The PCR fragments were subcloned into a pCR4-TOPO vector (Thermo Fisher Scientific) and six clones of each group were sequenced by Sanger Sequencing. The sequencing results were aligned to original and putative methylated sequences to determine the methylation status of CG sites.
Animal Studies
All mouse experiments were conducted with protocols approved by City of Hope Institutional Animal Care and Use Committee. Tumor xenograft models were generated using 6 to 8 week-old NOD/SCID/IL2Rγ−/− (NSG) mice as previously described (Jackson Laboratory)[6]. Briefly, on day 0, ffLuc+ NALM6 cells (2.5×105) were injected either intraperitoneally (i.p.) or intravenously (i.v.) into the NSG mice. After 4 days, mice were then treated with iPSC-derived or conventional CAR T or Mock T cells as described for each experiment. Mice in the indicated groups were injected i.p. three times per week with 20×106 irradiated (80 Gy) human hIL-15-secreting nurse cells (IL15-NS0) [14]. Reference schematics of
In this example, iPSC lines from healthy donor T cells subpopulation—naïve and memory T cells (Tn/mem) were generated by an integration-free method using iPSC reprograming episomal vectors (as described above and in, for example, Okita, K., et al., An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458-66). The resultant iPS cells were transduced with clinical grade lentivirus to express CD19-specific CARs (CD19CAR) or other CAR. Single cell was sorted, colonized and screened to generate a homogeneous CAR+ iPSC cell bank.
By using EMO-ATO culture system, as described above as Protocol 1A, Tn/mem iPSC-derived CAR T cells were successfully generated. The produced iPSC CD19CAR T cells have a conventional T cell surface marker phenotype with CD3+CD5+CD7+TCRαβ+CD8+ and CD3+CD5+CD7+TCRαβ+CD4+ (
A flow cytometry based TCR Vβ repertoire expression assay demonstrated that the Tn/mem iPSC 1928zCAR T cells showed one type of TCR repertoire exclusively (
The Tn/mem iPSC 1928zCAR T cells expanded robustly within two weeks (˜100 fold), and showed potent antigen-specific cytotoxicity against CD19+ target cells such as CD19+ 3T3 cells, parental tumor cells NALM6, and Raji as compared to their CD19 knockout control cells (
The iPSC-derived CD19CAR T cells also demonstrated efficient degranulation and activation phenotype (
The anti-tumor activity of Tn/mem iPSC 1928zCAR T cells in vivo was examined in a NSG mouse model engrafted with NALM6 cells. Tn/mem iPSC 1928zCAR T cells significantly eliminated the engrafted tumor cells and improved the mice survival. The combination of Tn/mem iPSC 1928zCAR T cells and IL15 secretory nurse cells IL15_NS0 further improved the therapeutic effects (
Tn/mem iPSC HSPC-derived CAR NK cells were generated as described above, for example, as in protocol 2B. The generated CAR NK cells demonstrated typical NK marker profile of CD3-CD56+ NKP46+ (
Tn/mem iPSC HSPC-derived 1928zCAR NK cells were functional and demonstrated potent cytotoxicity against a panel of tumor cell lines in antigen-dependent and antigen-independent manners (
The iPSC 1928zCAR T cells were also generated by nanofiber matrix based culture as described above, for example, as in Protocol 3A. The generated 1928zCAR T cells also demonstrated conventional T cells phenotype with CD3+CD8αβ+ or CD3+CD4+ (
As seen in
This example also shows use of iPSC differentiation for generation of CAR T cells with canonical T cell phenotype and CAR T function. The publications referenced in this example are listed at the end of the example.
Summary
This example shows that unlimited generation of chimeric antigen receptor (CAR) T cells from induced pluripotent stem cells (iPSCs) can be used for the development of ‘off-the-shelf’ CAR T cell immunotherapy. Approaches that enable efficient directional differentiation of iPSCs into canonical αβ T cell lineages, along with maintenance of CAR expression and functionality, however, are challenging. Described below is q continuous 3D-organoid system facilitates the generation of T cells from CAR-engineered iPSCs and confer products with conventional CAR T cell characteristics. The iPSCs were reprogrammed from an enriched CD62L+ naïve and memory subsets (Tn/mem) followed by CAR transduction, single cell sorting, and colonization. Induction of T cell directional differentiation via 3D-organoid culture was evident in that the resulting CD19-CAR T cells (iPSC CD19-CAR T cells) were predominantly CD3/CD5/CD7/TCRαβ/CD8αβ-positive and TCRγδ-negative. While iPSC CD19CAR T cells exhibited lower CAR expression levels due to hyper-methylation of the EF1α promoter as compared to conventionally derived CAR T cells, they exhibited better antigen specificity in cytokine release and more robust TCR/CAR signaling. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity and cytokine secretion compared to conventional CD19-CAR T cells generated from donor matched PBMC, and they maintained homogenous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also exhibited antitumor activity in vivo, prolonging survival of CD19+ human tumor xenografted mice. In summary, these methodologies generate highly functional conventional CAR T cells from iPSCs to support the development of ‘off-the-shelf’ manufacturing strategies.
Results
Generation of iPSC-Derived CAR T Cells with a Conventional T Cell Phenotype
The iPSC clones were derived from primary CD62L+ naïve and memory T cells (Tn/mem), a T cell population that has been proposed to have superior persistence and improve clinical outcomes in CAR T cell therapy (McLellan and Ali Hosseini Rad, 2019; Morgan and Schambach, 2018; Popplewell et al., 2018; Samer K. Khaled, 2018; Zah et al., 2020). The Tn/mem cells enriched from the peripheral blood of healthy human donors were transduced and reprogrammed by episomal plasmids encoding KLF4, SoX2, OCT-4, C-MYC and LIN28, along with P53 shRNA (Okita et al., 2013), and multiple integration-free iPSC clones were screened and characterized (
Transcriptional Profile of iPSC-Derived CAR T Cells
Bulk RNA deep sequencing analysis was used to explore differences between iPSC CD19-CAR T cells and conventional, PBMC-derived CD19-CAR T cells from the same donor. NK cells from the same donor were also used for comparison. Principle components analysis (PCA) showed that iPSC Mock T and iPSC CD19-CAR T cells displayed similar transcription profiles as conventional mock-transduced T cells, CD19-CAR T cells or NK cells derived from the same donor (PC score —7% variance), but were dramatically distinguished from iPSCs (PC score˜84% variance) (
As shown by flow cytometry that the iPSC CD19-CAR T cells expressed much lower levels of CAR transgene than conventional CAR T cells (
Functional Analysis of iPSC-Derived CAR T Cells
We next evaluated the effector function of REM expanded iPSC CD19-CAR T cells to lyse CD19 expressing targets in vitro. iPSC CAR T cells mediated potent CAR-directed cytolytic activity against CD19+ 3T3 cells (
Next, we explored CAR T cell signaling upon co-culture with either parental CD19+ or CD19 KO NALM6 cells. iPSC CD19-CAR T cells demonstrated ERK1/2 Thr202/Thr204, and PLCγ Ser1248 phosphorylation in an antigen specific manner that was comparable to that of conventional CD19-CAR T cells (
The disclosed iPSC CD19-CAR T cells yield products with comparable or superior in vitro effector activity as compared to conventional CAR T cell expanded using clinically relevant methodologies.
Anti-Tumor Efficacy of iPSC-Derived CAR T Cells
While the reduced CAR expression resulted in less activation in the absence of antigen (
Discussion
Generation of T cells and CAR T cells using extrathymic culture systems, whether they are single-layer or 3D-organoid co-cultures, is challenge (Maeda et al., 2016; Montel-Hagen et al., 2019; Vizcardo et al., 2018; Vizcardo et al., 2013; Zhao et al., 2007). The first reported iPSC CAR T cells generated by a mono-layer co-culture system displayed an innate-like phenotype (i.e., CD8αα+), as well as less-efficient antigen-specific cytotoxicity and cytokine secretion compared to conventional CAR T cells (Themeli et al., 2013). We modified and optimized a 3D-organoid culture system that facilitated generation of mature and functional CD3+CD8αβ+ and CD3+CD4+ conventional T cells and TCR-transgenic T cells (Montel-Hagen et al., 2019), and we demonstrated for the first time the successful generation of iPSC CAR T cells with a conventional T cell phenotype and CAR T cell function. Specifically, by using Tn/mem-derived iPSCs that were gene modified to express the CAR, and an PSC-ATO culture system to drive differentiation, iPSC CAR T cells were generated expressing conventional CD5+CD7+TCRαβ+TCRγδ-CD8αβ+ T cell phenotypes, exhibited potent cytotoxic killing, and Th1 cytokine secretion activity that was comparable to conventional CAR T cells derived from the same donor. Such improvements validate the potential utility of iPSCs for generating therapeutic CAR T cell products.
It is also advantageous that our Tn/mem-derived iPSC CAR T cells displayed a more homogenous, monoclonal TCR repertoire, which was different from the polyclonal phenotype in ESC-derived T cells (Montel-Hagen et al., 2019; Nishimura et al., 2013). Even the use of terminal differentiated effector T cells to generate the iPSCs resulted in regenerated CD8αβ T cells that lost their antigen specificity by additional TCR rearrangement, with TCR stability only being induced upon TCR transduction of the iPSCs (Minagawa et al., 2018). This shows that starting with a less-differentiated Tn/mem population may have unique effects on TCR rearrangement during re-differentiation, which may or may not relate to the allelic exclusion effect of pre-existing TCR loci (Brady et al., 2010). Regardless, selection of Tn/mem-derived iPSC clones of a known and/or innocuous TCR to minimize potential graft-versus-host toxicities are relevant to the manufacture of an ‘off-the-shelf’ iPSC CAR T cell products. All disclosed iPSC CAR T cells and iPSC CAR NK cell described herein can be used as such.
The lower expression levels of MHC and dominance of CD8 on our iPSC CAR T cells may also relate to the unique effects of starting with Tn/mem-derived iPSC clones, or it may be related to the lack of thymic epithelial cells in the culture system (Vizcardo et al., 2018). While low MHC expression may be desirable for reducing T cell mediated rejection and facilitating iPSC CAR T cell persistence after adoptive transfer, it might be important to improve the balance between the CD4+ and CD8+ populations, since CD4+ CAR T cells have recently been shown to play important role in adoptive immune cell therapy (Wang et al., 2018). A more balanced CD4/CD8 lineage differentiation may be obtained by manipulating either the culture conditions during differentiation or the lineage selection pathways by gene editing (Singer et al., 2008).
We demonstrated for the first time that decreased CAR expression in the iPSC CAR T cells was related to the hyper-methylation of the EF1a promoter. While promoter methylation has been known to regulate gene expression (Hofmann et al., 2006), the differentiation-induced hyper-methylation of the CAR transgene promoter represents a new mechanism by which one might regulate CAR expression. Lowering CAR expression might be more desirable than previously appreciated, as it has been reported that optimal basal low expression of CAR can reduce tonic signaling and sustain CAR T cell functions (Eyquem et al., 2017). In our study, the basal pERK phosphorylation level and the basal GM-CSF/IFNγ secretion levels of iPSC CD19-CAR T cells without antigen stimulation was much lower than that seen in conventional CD19-CAR T cells. In contrast, upon antigen encounter, cell signaling by iPSC CD19-CAR T cells, as measured by pERK, PLCγ(Y782) and ZAP70 phosphorylation, as well as Th1 cytokine secretion was comparable to or higher than that of conventional CD19-CAR T cells, in spite of the lower CAR expression. Interestingly, while phosphorylation of both the endogenous and CAR-associated CD3ζ sequences was suppressed by NALM6 tumor cells (
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It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/931,125, filed on Nov. 5, 2019. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/059216 | 11/5/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62931125 | Nov 2019 | US |
