EP4572775A2 - Atténuation de la maladie du greffon contre l'hôte à l'aide de cellules inkt modifiées - Google Patents

Atténuation de la maladie du greffon contre l'hôte à l'aide de cellules inkt modifiées

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Publication number
EP4572775A2
EP4572775A2 EP23855611.2A EP23855611A EP4572775A2 EP 4572775 A2 EP4572775 A2 EP 4572775A2 EP 23855611 A EP23855611 A EP 23855611A EP 4572775 A2 EP4572775 A2 EP 4572775A2
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EP
European Patent Office
Prior art keywords
cells
hsc
subject
inkt
allogeneic
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German (de)
English (en)
Inventor
Lili Yang
Yan-Ruide LI
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University of California San Francisco UCSF
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University of California
University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • 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
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells

Definitions

  • the present invention relates to methods and materials for alleviating graft versus host disease.
  • Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematologic malignancies such as leukemia/lymphoma owing to the graft-versus leukemia/lymphoma (GvL) effect elicited by alloreactive donor T cells (Appelbaum, 2001; Gribben and O’Brien, 2011; Shlomchik, 2007).
  • GvHD graft-versus-host disease
  • alloreactive donor T cells responding to minor or major histocompatibility antigen disparities between donor and recipient remains a major cause of patient morbidity and mortality for patients receiving T-cell replete allo-HSCT (Chakraverty and Sykes, 2007; Ferrara et al., 2009; Hill et al., 2021).
  • T cell depletion of the graft can reduce the incidence and severity of GvHD in patients but is associated with an increased risk of graft rejection, infections, and leukemia relapse (Apperley et al., 1986).
  • NK Yamamoto et al., 2003
  • B Shiabukuro-Vomhagen et al., 2009
  • CD4 + CD25 w FoxP3 + T regulatory (Treg) cells Pabst et al., 2007; Wolf et al., 2007).
  • allo-HSCT is a curative therapy for hematologic malignancies owing to the GvL effects mediated by alloreactive T cells.
  • these same T cells also mediate GvHD, a severe side effect which limits the wide-spread application of allo-HSCT therapies in the clinic.
  • Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity.
  • iNKT invariant natural killer T
  • the 3rd HSC-iNKT cells of the invention closely resemble the CD4‘CD8‘ /+ subsets of endogenous human iNKT cells in phenotype and functionality. We have further discovered that these cells display potent anti-GvHD functions and can, for example, eliminate antigen-presenting myeloid cells in vitro and in xenograft preclinical models of lymphoma and leukemia without negatively impacting tumor eradication by allogeneic T cells. The disclosure presented herein therefore indicates that 3rd HSC- iNKT cells can be used in off-the-shelf cell methods for GvHD prophylaxis and therapy.
  • Embodiments of the invention include, for example, methods of inhibiting or treating a graft versus host disease in a subject in need thereof, for example in a patient diagnosed with hematologic malignancy such as a leukemia or a lymphoma. These methods include administering to the subject a therapeutically effective amount of allogeneic HSC-engineered human iNKT cells (e.g., at least 0.031 X 10 6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells) so that graft versus host disease is inhibited or treated in the subject.
  • a therapeutically effective amount of allogeneic HSC-engineered human iNKT cells e.g., at least 0.031 X 10 6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells
  • embodiments of the invention can be used to treat acute graft-versus-host-disease (aGVHD) and/or chronic graft-versus-host-disease (cGVHD).
  • the engineered iNKT cells typically comprise one or more exogenous nucleic acids transduced therein such as a Va24-Jal8 iNKT cell receptor gene, a suicide gene or the like.
  • the subject/patient is someone undergoing treatment for a hematologic malignancy.
  • the subject is selected to be patient who has undergone or will undergo an allogeneic hematopoietic stem cell transplantation procedure.
  • the subject is administered the allogeneic HSC-engineered human iNKT cells at the time the subject undergoes the allogeneic hematopoietic stem cell transplantation procedure.
  • the subject is administered allogeneic HSC-engineered human iNKT cells mixed or in combination with allogeneic hematopoietic stem cells.
  • inventions include methods of depleting allogeneic CD14 + myeloid cells from a subject transfused with allogenic leukocytes.
  • these methods comprise administering to said subject amounts of allogeneic HSC- engineered human iNKT cells sufficient to target the allogeneic CD14 + myeloid cells in the subject, thereby depleting the HSC-engineered human iNKT cells in the subject.
  • the HSC -engineered human iNKT cells comprise one or more exogenous nucleic acids that includes a T cell receptor gene (e.g., a iNKT receptor gene such as Va24-Jal8, or a classical a or T cell receptor gene), and/or a suicide gene, and/or a gene encoding a polypeptide that promotes growth or a function of the HSC-engineered human iNKT cells.
  • a T cell receptor gene e.g., a iNKT receptor gene such as Va24-Jal8, or a classical a or T cell receptor gene
  • a suicide gene e.g., a gene that promotes growth or a function of the HSC-engineered human iNKT cells.
  • Embodiments of the invention also include methods of inhibiting or suppressing expansion of Th 1 -type pathogenic donor T cells in a subject (e.g. a patient diagnosed with hematologic malignancy) treated with allogenic T cells in a therapeutic regimen, the methods comprising administering to the subject amounts of allogeneic HSC-engineered human iNKT cells sufficient to inhibit or suppress the expansion of Thl-type pathogenic donor T cells in the subject (e.g., at least 1 X 10 6 allogeneic HSC-engineered human iNKT cells or at least 0.031 x 10 6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells).
  • the subject is administered allogeneic HSC-engineered human iNKT cells at the time that the subject is treated with the allogenic T cells in the therapeutic regimen.
  • the allogeneic HSC-engineered human iNKT cells used in the methods are derived from hematopoetic stem cells transduced with an exogenous nucleic acid comprising a iNKT receptor such as a Va24-Jal8 T cell receptor gene.
  • HSC-engineered human iNKT cells are lacking or have reduced surface expression of at least one HLA-1 or HLA-II molecule.
  • the lack of surface expression of HLA-I and/or HLA-II molecules is achieved by disrupting the genes encoding individual HLA-I/II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin) that is a common component of all HLA-I complex molecules, or by discrupting the genes encoding CIITA (the class II major histocompatibility complex transactivator) that is a critical transcription factor controlling the expression of all HLA-II genes.
  • B2M beta 2 microglobulin
  • CIITA the class II major histocompatibility complex transactivator
  • Li et al. iScience volume 25, issue 9, 104859, September 16, 2022 (hereinafter “Li et al.”) the contents of which are incorporated by reference.
  • Fig. 1 Ex vivo generation and characterization of HSC-engineered iNKT (HSC-iNKT) cells.
  • HSC HSC-engineered iNKT
  • A Experimental design. HSC, hematopoietic stem cell; CB, cord blood; aGC, a-galactosylceramide; Lenti/iNKT-sr39TK, lentiviral vector encoding an iNKT TCR gene and an sr39TK suicide/PET imaging gene; ATO, artificial thymic organoid; CMC, chemistry, manufacturing, and controls; MOA, mechanism of action.
  • B and C FACS monitoring of HSC-iNKT cell development during the 2-stage Ex Vivo HSC-iNKT Cell Culture.
  • iNKT cells were identified as iNKT TCR + TCRaP + cells. iNKT TCR was stained using a 6B11 monoclonal antibody.
  • B Generation of HSC-iNKT cells using an ATO approach.
  • C Generation of HSC- iNKT cells using a Feeder-Free approach.
  • D Table summarizing the production of HSC-iNKT cells.
  • E FACS detection of surface markers, intracellular cytokines, and cytotoxic molecules of HSC-iNKT cells. Healthy donor periphery blood mononuclear cell (PBMC)-derived conventional aP T (PBMC-Tcon) and iNKT (PBMC-iNKT) cells were included for comparison. Representative of over 10 experiments.
  • PBMC periphery blood mononuclear cell
  • PBMC-Tcon derived conventional aP T
  • PBMC-iNKT iNKT
  • Fig. 2 Third-party HSC-iNKT ( 3rd HSC-iNKT) cells ameliorate graft- versus-host disease (GvHD) in NSG mice engrafted with donor-mismatched human PBMCs.
  • A Experimental design.
  • B Clinical GvHD score (p was calculated using data on day 40).
  • a clinical GvHD score was calculated as the sum of individual scores of 6 categories (body weight, activity, posture, skin thickening, diarrhea, and dishevelment; score 0-2 for each category).
  • C Body weight (p was calculated using data on day 40).
  • D Kaplan-Meier survival curves.
  • E FACS detection of human T cells in peripheral blood.
  • F Representative image of experimental mice on day 40.
  • G H&E-stained tissue sections. Scale bar: 100 pm.
  • H Quantification of (G).
  • Fig. 3rd HSC-iNKT cells ameliorate GvHD through rapid depletion of donor CD14 + myeloid cells that exacerbate GvHD.
  • A-C Sublethally irradiated NSG mice received intravenous injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells and were sacrificed 3 days later.
  • A Experimental design.
  • B FACS detection of CD14 + myeloid cells in the lymphohematopoietic system (i.e., blood, spleen and lymph nodes) and GvHD target organs (i.e., liver and lung).
  • C Quantification of (B).
  • N 4.
  • Fig. 4 3rd HSC-iNKT cells ameliorate GvHD through eliminating donor CD14 + myeloid cells through CD Id recognition.
  • MLR mixed lymphocyte reaction
  • PBMCs healthy donor PBMCs
  • irradiated donor-mismatched allogeneic PBMCs stimulateators
  • purified anti-human CDld antibody or its IgG isotype control was also added.
  • HLA-A2 + responders and HLA-A2" stimulators were used in the study.
  • Fig. 5 3rd HSC-iNKT cells ameliorate GvHD while preserving GvL in a human B cell lymphoma xenograft NSG mouse model.
  • Sublethally irradiated NSG mice were inoculated with 1 x 10 5 Raji-FG cells, followed by intravenous injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3ld HSC-iNKT cells. Mice were monitored for tumor burden and GvHD development.
  • Raji-FG human B cell lymphoma Raji cell line engineered to overexpress firefly luciferase and green fluorescence protein (FG) dual reporters.
  • BLI bioluminescence imaging.
  • (G) Human T cells in peripheral blood of experimental mice over time. N 10. Representative of 2 experiments. All data are presented as the mean ⁇ SEM. **** > ⁇ 0.0001 by Student’s / test (D, E, G), one-way ANOVA (C), or by log rank (Mantel-Cox) test adjusted for multiple comparisons (F).
  • Fig. 6 3rd HSC-iNKT cells ameliorate GvHD while preserving GvL in a human acute myeloid leukemia (AML) xenograft NSG mouse model.
  • AML acute myeloid leukemia
  • Sublethally irradiated NSG mice were inoculated with 2 x 10 5 HL60-FG human AML cells, followed by intravenous injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells. Mice were monitored for tumor burden and GvHD development.
  • iNKT cells Invariant nature killer T (iNKT) cells have been studied extensively for their roles in modulating GvHD and GvL.
  • iNKT cells are a small subset of aP T cells that express both a semi-invariant T cell receptor (Va24-Jotl8 in humans and Val4-Jal8 in mice paired with a limited selection of VP chains) and natural killer cell markers (e.g., CD161 in humans and NK1.1 in mice) (Bendelac et al., 2007; Brennan et al., 2013; Brigl and Brenner, 2004; Kronenberg, 2005; Kumar et al., 2017; Lantz and Bendelac, 1994; Taniguchi et al., 2003).
  • embodiments of the invention can be used to treat acute graft-versus-host-disease (aGVHD) and/or chronic graft-versus-host-disease (cGVHD).
  • the engineered iNKT cells comprise one or more exogenous nucleic acids that has been transduced therein.
  • the one or more exogenous nucleic acids comprise a Va24-Jal8 iNKT cell receptor gene; and/or the one or more exogenous nucleic acids comprise a classical a or p T cell receptor gene.
  • Embodiments of the invention also include methods of depleting allogeneic CD14 + myeloid cells from a subject transfused with allogenic leukocytes (including the allogeneic CD14 + myeloid cells). See, for example, the data presented in Figure 3 and Figures S4A-S4E in Li et al.
  • these methods comprise administering to said subject amounts of allogeneic HSC-engineered human iNKT cells sufficient to target the allogeneic CD14 + myeloid cells in the subject, thereby depleting the HSC- engineered human iNKT cells in the subject.
  • the patient is administered the third-party HSC-engineered human iNKT ( 3rd HSC-iNKT) cells at the time the subject undergoes the allogeneic hematopoietic stem cell transplantation procedure.
  • the patient is administered third-party HSC-engineered human iNKT ( 3rd HSC-iNKT) cells mixed with allogeneic hematopoietic stem cells.
  • the patient is administered at least 1 X 10 6 third-party HSC-engineered human iNKT ( 3rd HSC-iNKT) cells.
  • the patient is administered at least 0.031 x io 6 cells/kg of body weight of the third-party HSC-engineered human iNKT ( 3rd HSC-iNKT) cells.
  • the engineered iNKT cells comprise one or more exogenous nucleic acids transduced therein.
  • the one or more exogenous nucleic acids comprise a T cell receptor alpha chain gene and/or a T cell receptor alpha chain gene; and the engineered iNKT cells comprise clonal populations of cells comprising the T cell receptor alpha chain gene and/or the T cell receptor beta chain gene.
  • a patient/subject can be someone diagnosed with a disease or disorder.
  • the disease or disorder may be at least one of a hemoglobinopathy, a congenital hemoglobinopathy, P-Thalessemia major (TM), sickle cell disease (SCD), severe aplastic anemia, Fanconi's anemia, dyskeratosis congenita, Blackfan-Diamond anemia, Thalassemia, congenital amegakaryocytic thrombocytopenia, severe combined immunodeficiency, T cell immunodeficiency, T cell immunodeficiency-SCID variants, Wiskott-Aldrich syndrome, a hemophagoc tic disorder, a lymphoproliferative disorder, severe congenital neutropenia, chronic granulomatous disease, a phagocytic cell disorder, TPEX syndrome, juvenile rheumatoid arthritis, systemic sclerosis, an autoimmune disorder, an immune dysregulation disorder, mucopolysaccharoidoses,
  • the subject can be diagnosed with a cancer.
  • the cancer may be a hematological cancer.
  • the cancer can be at least one of Acute myeloid leukemia, myelodysplastic syndrome, follicular lymphoma, diffuse large B cell lymphoma, acute lymphoblastic leukemia, multiple myeloma, Hodgkin lymphoma, chronic myeloid leukemia, T cell non-Hodgkin lymphoma, lymphoblastic B cell non-Hodgkin lymphoma (non-Burkitt), Burkitt's lymphoma, anaplastic large cell lymphoma, germ cell tumor, Ewing's sarcoma, soft tissue sarcoma, neuroblastoma, Wilms' tumor, osteosarcoma, medulloblastoma, acute promyelocytic leukemia, mantle cell lymphoma, T cell lymphoma, lymphoplasmacytic lymphoma, cutaneous T cell lymphom
  • the subject can be diagnosed with a cancer and be designated to undergo an allogeneic transplant.
  • the subject can be diagnosed with a cancer and has previously undergone an allogeneic transplant.
  • a subject can be diagnosed with a cancer and be designated to undergo a transplant in order to treat the cancer.
  • a subject can be diagnosed with a cancer and has previously undergone a transplant in order to treat the cancer.
  • a subject can be diagnosed with a cancer and be designated to undergo an allogeneic transplant in order to treat the cancer. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a cancer and has previously undergone an allogeneic transplant in order to treat the cancer.
  • a subject can be diagnosed with a disease or disorder and be designated to undergo a transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and has previously undergone a transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and be designated to undergo an allogeneic transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and has previously undergone an allogeneic transplant in order to treat the disease or disorder.
  • a subject can have been previously administered a transplant. Accordingly, 3rd HSC-iNKT cells can be administered to the subject after the latter has been administered a transplant. In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered an allogeneic transplant. Accordingly, in some embodiments an at least one therapeutically effective amount of 3rd HSC-iNKT cells can be administered to the subject after the subject has been administered an allogeneic transplant. In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered a conditioning therapy in connection with a transplant. In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered a conditioning therapy in connection with an allogeneic transplant. In some embodiments a conditioning therapy can comprise the administration of radiation therapy, chemotherapy, radiomimetic therapy or any combination thereof. In some embodiments a radiation therapy can comprise total body irradiation.
  • a conditioning therapy may be administered in connection with the allogenic transplant.
  • a conditioning therapy can comprise, such as consist of, the administration of radiation therapy, chemotherapy, radiomimetic therapy or any combination thereof.
  • the radiation therapy can comprise total body irradiation.
  • Li et al. iScience volume 25, issue 9, 104859, September 16, 2022 (hereinafter “Li et al.”) the contents of which are incorporated by reference.
  • Non-myeloablative conditioning with TLI/anti -Thymocyte Globulin (ATG) prior to allo-HSCT coincided with a higher iNKT/T cell ratio, decreased incidences of GvHD, and retained GvL effect (Kohrt et al., 2009; Lowsky et al., 2005).
  • Patients with GvHD early after transplantation were found to have reduced numbers of total circulating iNKT cells (Haraguchi et al., 2004), whereas enhanced iNKT cell reconstitution following allo-HSCT positively correlated with a reduction in GvHD without loss of GvL effect (Rubio et al., 2012).
  • iNKT cell numbers in donor allograft was associated with clinically significant reduction in GvHD in patients receiving allo- HSCT (Chaidos et al., 2012).
  • increasing the numbers of iNKT cells, particularly the CD4" iNKT cells, in the allograft may provide an attractive strategy for suppressing GvHD while preserving GvL effect.
  • HSC- iNKT human HSC-engineered iNKT
  • CB Cord blood (CB)-derived human CD34 + hematopoietic stem and progenitor cells (denoted as HSCs) were collected and then transduced with a Lenti/iNKT-sr39TK lentiviral vector that encodes three transgenes: a pair of iNKT TCR a and chain genes as well as an sr39TK suicide/imaging report gene ( Figure SI A in Li et al.) (see also Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
  • HSC-iNKT Ex Vivo HSC-Denved iNKT
  • ATO Artificial Thymic Organoid
  • Figure 1A Feeder-Free
  • ATO culture utilizes a MS5 mouse stromal cell line overexpressed delta-like canonical Notch ligand 1 (DLL1)- or 4 (DLL4) and supports robust ex vivo differentiation and maturation of human T cells from HSCs (Li et al., 2021b; Montel- Hagen et al., 2019; Seet et al., 2017); Feeder-Free culture adopts a system of platebound DLL4 and vascular cell adhesion protein 1 (VCAM-1) to induce T cell commitment from HSCs (Huijskens et al., 2014; Iriguchi et al., 2021; Y. R.
  • DLL1 delta-like canonical Notch ligand 1
  • VCAM-1 vascular cell adhesion protein 1
  • the gene-engineered HSCs efficiently differentiated into iNKT cells in the ATO or Feeder-Free cultures system (Stage 1) over 8 weeks or 4 weeks, respectively, with over 100-fold expansion in cell numbers ( Figures 1A-1C).
  • These engineered HSC-iNKT cells were further expanded with irradiated PBMCs loaded with aGC, a synthetic agonist glycolipid ligand that specifically activate iNKT cells, for another 2-3 weeks (Stage 2) ( Figures 1A-1C), resulting in another 100-1000-fold expansion of HSC-iNKT cells with > 98% purity ( Figures 1A and ID).
  • HSC-iNKT cells followed a typical human iNKT cell development path defined by CD4/CD8 co- receptor expression (Godfrey and Berzins, 2007): HSC-iNKT cells transitioned from CD4 CD8" to CD4 + CD8 + , then to CD4‘CD8 +/ " ( Figures IB and 1C). At the end of cultures, over 98% of the HSC-iNKT cells displayed a CD4 CD8 +/ " phenotype ( Figures IB and 1C).
  • the dosage (about 10 7 HSC-iNKT cells per dose) was estimated based on an earlier clinical study, wherein 0.031 x io 6 CD4" iNKT cells/kg of body weight was associated with amelioration of GvHD (Chaidos et al., 2012).
  • HSC-iNKT cell product To increase the safety profile of HSC-iNKT cell product, we included an sr39TK PET imaging/suicide gene in the lentiviral vector, which allows for the in vivo monitoring of these cells using PET imaging and the elimination of these cells through ganciclovir (GCV)-induced depletion in case of an adverse event ( Figures S1A and IB in Li et al.). In cell culture, GCV treatment induced effective killing of HSC-iNKT cells ( Figures SIB and 1C in Li et al.).
  • GCV ganciclovir
  • HSC-iNKT HSC-iNKT cells
  • PBMC-iNKT healthy donor periphery blood mononuclear
  • PBMC-Tcon a.p T
  • HSC-iNKT cells displayed a phenotype closely resembling PBMC-iNKT cells and distinct from PBMC-Tcon cells: they expressed high levels of memory T cell markers (i.e., CD45RO) and NK cell markers (i.e., CD161, NKG2D, and DNAM-1) and expressed exceedingly high levels of Thl cytokines (i.e., IFN-y, TNF-a, and IL-2) as well as high levels of cytotoxic molecules (i.e.. Perforin and Granzyme B) ( Figure IE).
  • CD45RO memory T cell markers
  • NK cell markers i.e., CD161, NKG2D, and DNAM-1
  • Thl cytokines i.e., IFN-y, TNF-a, and IL-2
  • cytotoxic molecules i.e.. Perforin and Granzyme B
  • HSC-iNKT cells produced high levels of Thl cytokines (i.e, IFN-y, TNF-a, and IL-2) while low levels of Th2 cytokines (i.e., IL-4), suggesting a function like that of the endogenous CD8 + and DN human iNKT subsets, agreeing with the CD4 CD8 +/ " phenotype of these HSC-iNKT cells ( Figures IB, 1C, and IE) (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
  • Thl cytokines i.e, IFN-y, TNF-a, and IL-4
  • Th2 cytokines i.e., IL-4
  • HSC-iNKT 3rd HSC-iNKT cells ameliorate Xeno-GvHD in NSG mice engrafted with human PBMC
  • the engineered HSC-iNKT cells were predominantly CD4" ( Figures IB, 1C, and IE); this subset of human iNKT cells were reported to be associated with reduced GvHD in patients (Chaidos et al., 2012).
  • a xeno-GvHD model wherein NSG mice were engrafted with human PBMCs (Shultz et al., 2007). NSG mice were preconditioned with non-lethal total body irradiation (TBI, 100 cGy), and were injected intravenously (i.v.) with healthy donor PBMCs with or without the addition of 3rd HSC-iNKT cells.
  • the acute and chronic GvHD overlapping target organs i.e., lung, liver and skin
  • chronic GvHD prototypical target organs i.e., salivary glands
  • the salivary gland also showed infiltration and damage of gland follicles ( Figures 2G-2J).
  • the mice receiving additional 3rd HSC-iNKT cells showed marked reduction in T cell infiltration in the liver, lung and salivary gland as well as tissue damage scores ( Figures 2G-2J).
  • Addition of 3rd HSC-iNKT cells also markedly reduced hair loss and epidermis enlargement, although T cell infiltration in the skin tissues was mild and no significant difference was observed between recipient mice with or without the addition of 3rd HSC-iNKT cells ( Figures 2G-2J).
  • Flow cytometry analysis also revealed significantly less numbers of donor T cells in the blood and spleen, as well as less T cell infiltration in GvHD target organs (i.e., lung, liver and bone marrow; Figures S2A and S2B in Li et al.).
  • X-VIVO 15 Serum-Free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Coming Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including Penicillin- Streptomycine-Glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and Sodium Pyruvate, were purchased from GIBCO. Beta-Mercaptoethanol (0-ME) was purchased from Sigma. Normocin was purchased from InvivoGen.
  • Complete lymphocyte culture medium (denoted as CIO medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 rnM), Sodium Pyruvate (1 rnM), 0- ME (50 mM), and Normocin (100 mg/ml).
  • Medium for culturing human Raji and HL60 tumor cell lines (denoted as RIO medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
  • Medium for culturing HEK 293T cell line (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1 % vol/vol).
  • Fluorochrome-conjugated antibodies specific for human CD45 (Clone Hl 30), TCRaP (Clone 126), CD4 (Clone OKT4), CD8 (Clone SKI), CD45RO (Clone UCHL1), CD161 (Clone HP-3G10), CD69 (Clone FN50), CD56 (Clone HCD56), CD62L (Clone DREG-56), CD14 (Clone HCD14), CDld (Clone 51.1), NKG2D (Clone 1D11), DNAM-1 (Clone 11A8), IFN-y (Clone B27), Granzyme B (Clone QA16A02), Perforin (Clone dG9), TNF-a (Clone Mabl l), IL-2 (Clone MQ1-17H12), HLA-A2 (Clone BB7.2) were purchased from BioLegend; Fluorochrome-conjugated antibodies specific for human CD34 (
  • the ELISAs for detecting human cytokines were performed following a standard protocol from BD Biosciences. Supernatants from co-culture assays were collected and assayed to quantify IFN-y. Capture and biotinylated pairs for detecting cytokines were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. Tetramethylbenzidine (TMB) substrate was purchased from KPL. The samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan).
  • HSCs cord blood-derived human CD34 + hematopoietic stem and progenitor cells
  • HSC-culture medium comprised ofX-VIVO 15 Serum-Free Hematopoietic Cell Medium supplemented with human recombinant SCF (50 ng/ml), FLT3-L (50 ng/ml), TPO (50 ng/ml), and IL-3 (10 ng/ml) for 24 hours.
  • Cells were then transduced with Lenti/iNKT-sr39TK viruses for another 24 hours (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
  • the transduced HSCs were then collected and put into an Artificial Thymic Organoid (ATO) culture or a Feeder-Free culture.
  • ATO Artificial Thymic Organoid
  • transduced HSCs were mixed with MS5-DLL4 feeder cells to form ATOs and cultured over ⁇ 8 weeks following a previously established protocol (Li et al., 2021b; Montel-Hagen et al., 2019).
  • Feeder-Free culture transduced HSCs were cultured using a StemSpanTM T Cell Generation Kit (StemCell Technologies) over ⁇ 5 weeks following the manufacturer’s instructions (Y. R. Li et al., 2022).
  • the resulting HSC-iNKT cells isolated from ATOs or Feeder-Free culture were expanded with aGC-loaded PBMCs (aGC-PBMCs).
  • HSC-iNKT cells were mixed with irradiated aGC-PBMCs at ratio 1:1, followed by culturing for 2 weeks in CIO medium supplemented with human IL-7 (10 ng/ml) and IL-15 (10 ng/ml); cell cultures were split, and fresh media/cytokines were added if needed.
  • the resulting HSC-iNKT cell products were then collected and cryopreserved for future use.
  • PBMC-Tcon PBMC-Derived Conventional T
  • PBMC- iNKT PBMC- iNKT
  • Healthy donor PBMCs were obtained from the UCLA/CFAR Virology Core Laboratory and were used to generate the PBMC-Tc and PBMC-iNKT cells.
  • PBMCs were stimulated with CD3/CD28 T- activator beads (ThermoFisher Scientific) and cultured in CIO medium supplemented with human IL-2 (20 ng/mL) for 2-3 weeks, following the manufacturer’s instructions.
  • PBMCs were enrich for iNKT cells using anti-iNKT microbeads (Miltenyi Biotech) and MACS-sortmg, followed by stimulation with donor-matched irradiated aGC-PBMCs at the ratio of 1: 1 and cultured in CIO medium supplemented with human recombinnat IL-7 (10 ng/ml) and IL-15 (10 ng/ml) for 2-3 weeks.
  • the resulting PBMC-iNKT cells could be further purified using Fluorescence-Activated Cell Sorting (FACS) via human iNKT TCR antibody (Clone 6B11; BD Biosciences) staining.
  • FACS Fluorescence-Activated Cell Sorting
  • HSC-iNKT cells were analyzed in comparison with PBMC-Tcon and PBMC- iNKT cells. Phenotype of these cells was studied using flow cytometry by analyzing cell surface markers including co-receptors (i.e., CD4 and CD8), NK cell receptors (i.e., CD161, NKG2D, and DNAM-1), and memory T cell markers (i.e., CD45RO).
  • cell surface markers including co-receptors (i.e., CD4 and CD8), NK cell receptors (i.e., CD161, NKG2D, and DNAM-1), and memory T cell markers (i.e., CD45RO).
  • cytokines i.e., IFN-y, TNF-a, IL-2, and IL-4
  • cytotoxic molecules i.e., Perforin and Granzyme B
  • Ganciclovir In Vitro and In Vivo Killing Assays, HSC-iNKT cells were cultured in CIO medium in the presence of titrated amount of GCV (0-50 pM) for 4 days; live HSC- iNKT cells were then counted using a hematocy tome ter (VWR) via Trypan Blue staining (Fisher Scientific).
  • GCV in vivo killing assay was performed using an NSG xenograft mouse model.
  • NSG mice received i.v. injection of 1 x 10 7 HSC-iNKT cells on day 0, followed by i.p. injection of GCV for 5 consecutive days (50 mg/kg per injection per day). On day 5, mice were terminated.
  • Multiple tissues i.e., blood, spleen, liver, and lung
  • tissueinfiltrating HSC-iNKT cells identified as iNKT TCR + CD45 + ), following established protocols (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
  • Tumor cells (1 x 10 4 cells per well) were co-cultured with HSC-iNKT cells (at ratios indicated in figure legends) in Coming 96-well clear bottom black plates for 24 hours, in CIO medium. At the end of culture, live tumor cells were quantified by adding D-luciferin (150 pg/ml; Caliper Life Science) to cell cultures and reading out luciferase activities using an Infinite M1000 microplate reader (Tecan).
  • D-luciferin 150 pg/ml; Caliper Life Science
  • MLR Mixed Lymphocyte Reaction
  • PBMCs of multiple healthy donors were irradiated at 2,500 rads and used as stimulators, and non-irradiated allogeneic PBMCs were used as responders.
  • HLA-A2 + responders and HLA-A2" stimulators were used in this study. Irradiated stimulators (2.5 x 10 5 cells/well) and responders (1 x 10 4 cells/well) were co-cultured with or without the addition of 3rd HSC-iNKT cells (1 x 10 4 cells/well) in 96-well round bottom plates in CIO medium for up to 4 days.
  • BLI was performed using a Spectral Advanced Molecular Imaging (AMI) HTX imaging system (Spectral instrument Imaging). Live animal imaging was acquired 5 minutes after intraperitoneal (i.p.) injection of D-Luciferin (1 mg per mouse). Imaging results were analyzed using an AURA imaging software (Spectral Instrument Imaging).
  • AMI Spectral Advanced Molecular Imaging
  • mice were pre-conditioned with 100 rads of total body irradiation (day - 1), followed by intravenous injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells.
  • Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
  • Various mouse tissues i.e., blood, spleen, liver, lung, bone marrow, skin, and salivary ligand) were harvested and processed for either flow cytometry or histologic analysis.
  • Human PBMC Xenograft NSG Mouse Model Studying CD14 + Myeloid Cell Modulation of GvHD
  • mice were pre-conditioned with 100 rads of total body irradiation (day - 1), followed by intravenous injection of 2 x 10 7 healthy donor PBMCs or 9 x 10 6 CD14-depleted donor PBMCs.
  • the amount of PBMCs given was normalized to contain the same number of T cells.
  • Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment).
  • mice were pre-conditioned with 100 rads of total body irradiation (day - 1), followed by intravenous injection of 9 x 10 6 CD14-depleted donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells. Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
  • GvHD body weight, activity, posture, skin thickening, diarrhea, and dishevelment
  • mice were pre-conditioned with 100 rads of total body irradiation (day - 1), followed by subcutaneous inoculation with 1 x 10 5 Raji-FG cells (day 0).
  • day - 1 the tumor-bearing experimental mice received intravenous (i.v.) injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells.
  • Tumor load were monitored over time using BLI.
  • Mice were also weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
  • mice were pre-conditioned with 175 rads of total body irradiation (day - 1), followed by intravenous inoculation with 2 x 10 5 HL60-FG (day 0).
  • day - 1 the tumor-bearing experimental mice received intravenous (i.v.) injection of 2 x 10 7 healthy donor PBMCs with or without the addition of 2 x 10 7 3rd HSC-iNKT cells.
  • Tumor load were monitored over time using BLI.
  • Mice were also weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
  • GvHD body weight, activity, posture, skin thickening, diarrhea, and dishevelment
  • Tissues i.e., liver, lung, salivary glands, and skin
  • Tissue sections were prepared and stained with Hematoxylin and Eosin (H&E) or anti-CD3 by the UCLA Translational Pathology Core Laboratory, following the Core’s standard protocols.
  • H&E-stained sections were imaged on a Zeiss Observer II upright microscope. All images were captured at either 100 x or 200 x and processed using Zen Blue software.
  • GvHD pathological score was calculated as follows: skin: epidermal changes (0-3), dermal changes (0-3), adipose changes (0-3); salivary: infiltration (0-4), follicular destruction (0-4); liver: duct infiltration (0-3), number of ducts involved (0- 3), liver cell apoptosis (0-3); lung: infiltrates (0-3); pneumonitis (0-3), overall appearance (0-3).
  • skin epidermal changes (0-3), dermal changes (0-3), adipose changes (0-3); salivary: infiltration (0-4), follicular destruction (0-4); liver: duct infiltration (0-3), number of ducts involved (0- 3), liver cell apoptosis (0-3); lung: infiltrates (0-3); pneumonitis (0-3), overall appearance (0-3).
  • CD3 surface area measurements the anti-CD3-stained sections were scanned in their entirety using Hamamatsu Nanozoomer 2.0 HT. The % CD3+ area was determined by CD3 + area divided by total tissue area, using an
  • iNKT cells are uniquely positioned at the crossroads of innate and adaptive immunity and have potent immunoregulatory functions in a variety of diseases (Brennan et al., 2013; Van Kaer et al., 2011). Research into harnessing iNKT cells to combat GvHD began decades ago (Lan et al., 2001), but clinical application of iNKT cells has been hindered by their scarcity in peripheral blood (Krijgsman et al., 2018).
  • HSC-iNKT cells do not cause GvHD themselves and are resistant to allorej ection due to their intrinsic low expression of HLA-I and II molecules (Li et al., 2021b; Y. R. Li et al., 2022), highlighting their potential for off-the-shelf anti- GvHD therapy.
  • GvHD prophylaxis is centered around calcineurin inhibitor (CNI)-based therapy and investigations into new methods, including those depleting T cells, modulating T cell co-stimulatory pathways (e.g., checkpoints), enhancing regulatory T cells, targeting T cell trafficking, and altering cytokine pathways (Gooptu and Antin, 2021).
  • CNI calcineurin inhibitor
  • acute GvHD is a common complication of allo-HSCT, occurring in 30-50% of patients, 14-36% of whom develop severe acute GvHD, and is a major cause of morbidity and mortality (Malard et al., 2020).
  • the current first-line treatment for acute GvHD is systemic steroid therapy, but almost half of patients will become refractory to treatment and there is no accepted standard- of-care treatment for steroid refractor -acute GvHD (Malard et al., 2020).
  • the dismal survival rate and poor quality of life in these patients highlight the urgent need for novel therapeutic and prophylactic agents against acute GvHD.
  • the driver of clinical acute GvHD is donor alloreactive T cells (Ball and Egeler, 2008). Following lymphodepletion and HSCT, host and donor antigen- presenting cells respond to host tissue damage and lead to the activation of donor T cells (Ramachandran et al., 2019). Although culpable for GvHD, HSCT-denved T cells are essential for antitumor effects, as their depletion from HSCT grafts precipitates increased relapse rates (Horowitz et al, 1990).
  • HSC- iNKT cells show low response to IL-12/IL-18 innate signaling in vitro (Data not shown)
  • Chaidos and colleagues conducted a comprehen sieve analysis of all immune populations in allogeneic HSCT grafts, and found that only CD4" iNKT cells were correlated with reduced acute GvHD occurrence (Chaidos et al, 2012).
  • Rubio and colleagues also revealed that only pre-transplant donor CD4" iNKT cells predicted clinical acute GvHD following HSCT (Rubio et al., 2017).
  • iNKT cells can also play a direct role in tumor killing. Through CDld dependent and independent means, iNKT cells have been shown to lysis a variety of tumor cells (King et al., 2018; Li et al., 2021b; Zhu et al., 2019). Furthermore, in hematological and solid tumor models, adoptive transfer of iNKT cells reduces tumor burden and enhances overall survival (Fujii et al., 2013).
  • HSC-iNKT cells do not recognize mismatched MHCs and thus pose no risk of inducing GvHD; furthermore, due to their intrinsic low expression of HLA-I and II molecules, these cells are resistant to allorejection (Li et al., 2021b; Y. R. Li et al., 2022). These features of HSC-iNKT cells make them suitable for allogeneic cell therapy.
  • Allo-HSCT is an established, effective treatment for hematological malignancies, but GvHD is common and debilitating adverse event for many allo- HSCT recipients.
  • the reported ex vivo HSC-iNKT cell culture is robust and of high yield and purity, with the potential of being scaled for further translation and clinical development. From one cord blood donor, over 10,000 doses of third-party HSC-iNKT cells can be manufactured and cryopreserved for ready distribution to allo-HSCT patients; MHC matching is not needed. This study highlights the potential of 3rd HSC-iNKT cells to address a critical unmet medical need and warrants further investigations of this promising off-the-shelf cell product.
  • Predominant mouse models studying GvHD typically employ transplantation of T cell-depleted bone marrow and donor-derived T cells into lethally irradiated recipients; these are paramount to advance the forefront of knowledge regarding the incidence of GvHD within allo-HSCT therapeutics (Schroeder and DiPersio, 2011).
  • healthy donor T cells were used to generate a PBMC-xenograft NSG mouse model, producing a construct where T cell-mediated GvHD could be studied and manipulated in vivo.
  • limitations to this model preclude its ability to fully reflect GvHD pathology in allo-HSCT.
  • EXPERIMENTAL MODEL AND SUBJECT DETAILS o Mice o Cell Lines and Viral Vectors o Human Periphery Blood Mononuclear Cells (PBMCs) o Media and Reagents
  • HSC-iNKT HSC-Engineered iNKT
  • PBMC-Tcon PBMC-Derived Conventional T
  • PBMC-iNKT PBMC-iNKT
  • Ganciclovir Ganciclovir
  • MLR Mixed Lymphocyte Reaction
  • iNKT Invariant natural killer T
  • HSC Hematopoietic stem cell
  • GvHD Graft versus host disease
  • GvL Graft versus leukemia/lymphoma
  • Bone marrow transplantation for patients with chronic myeloid leukaemia T-cell depletion with Campath-1 reduces the incidence of graft-versus-host disease but may increase the risk of leukaemic relapse. Bone Marrow Transplant. 1, 53-66.
  • Yamasaki S., Henzan, H., Ohno, Y., Yamanaka, T., lino, T., Itou, Y., Kuroiwa, M., Maeda, M , Kawano, N., Kinukawa, N., Miyamoto, T., Nagafuji, K , Shimoda,

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Abstract

Nous avons découvert que les cellules iNKT (3rdHSC-iNKT) humaines modifiées par HSC allogéniques présentent des fonctions anti-GvHD puissantes, en éliminant des cellules myéloïdes présentatrices d'antigène in vitro et dans des modèles de xénogreffe, sans affecter négativement l'éradication tumorale par des lymphocytes T allogéniques dans des modèles précliniques de lymphome et de leucémie. Les cellules 3rdHSC-iNKT ressemblent étroitement aux sous-ensembles CD4-CD8-/+ de cellules iNKT humaines endogènes dans le phénotype et la fonctionnalité. Des modes de réalisation de l'invention portent sur ces découvertes dans de nouveaux procédés et matériaux destinés à soulager la maladie du greffon contre l'hôte.
EP23855611.2A 2022-08-16 2023-08-15 Atténuation de la maladie du greffon contre l'hôte à l'aide de cellules inkt modifiées Pending EP4572775A2 (fr)

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