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WO2025099197A1 - Lymphocytes cytotoxiques modifiés par smad4 pour thérapie cellulaire - Google Patents

Lymphocytes cytotoxiques modifiés par smad4 pour thérapie cellulaire Download PDF

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WO2025099197A1
WO2025099197A1 PCT/EP2024/081581 EP2024081581W WO2025099197A1 WO 2025099197 A1 WO2025099197 A1 WO 2025099197A1 EP 2024081581 W EP2024081581 W EP 2024081581W WO 2025099197 A1 WO2025099197 A1 WO 2025099197A1
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cells
cell
smad4
tgf
cytotoxic lymphocyte
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Aura Muntasell Castellvi
Anna REA
Marta SANVICENTE GARCÍA
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Fundacio Institut Hospital Del Mar D'investigacions Mediques
Universitat Autonoma de Barcelona UAB
Universitat Pompeu Fabra UPF
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Fundacio Institut Hospital Del Mar D'investigacions Mediques
Universitat Autonoma de Barcelona UAB
Universitat Pompeu Fabra UPF
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • TITLE SMAD4 engineered cytotoxic lymphocytes for cell therapy
  • the present invention relates to the field of cell therapy for human health, particularly of modified cytotoxic lymphocytes useful in the treatment of cancer.
  • Allogeneic NK cell adoptive transfer has been exploited as adjuvant therapy following hematopoietic stem cell transplantation, displaying a significant graft versus leukemia efficacy and good safety profile.
  • NK cell adoptive transfer as monotherapy or in combination with immunotherapy, chemotherapy, or targeted drugs, for the treatment of hematologic malignancies and solid tumors.
  • TEE immunosuppressive tumor microenvironment
  • the immunosuppressive TME refers to the cellular and molecular conditions that exist within and around a tumor, which collectively hinder the body's immune system from effectively recognizing and attacking the cancer cells. Main limitations include: i) enhancing the homing of intravenously infused NK cells into solid tumors/lesions and ii) enhancing NK cell cytotoxicity and persistence in the immunosuppressive TME. It has been shown that TGF-p, a pleiotropic suppressive cytokine produced by tumor cells and other regulatory cells (i.e.
  • Tumor NK cell infiltration is an important parameter controlled by chemokines and adhesion molecules.
  • Chemokine-chemokine receptor axis involved in NK cell homing to solid tumors include CCL5/CCR5, CXCL9-CXCL10/CXCR3, CX3CL1/CXC3CR1 , SDF-1/CXCR4 and CXCL8/CXCR1-2.
  • most solid tumors are poorly permeable to NK cells. It has been shown that TGF-p precludes immune cell infiltration.
  • TGF-p TGF-p type 2 receptor dimers
  • TGF-pRII TGF-p type 2 receptor dimers
  • TGF-pRI type 1 receptors
  • the canonical signaling is initiated by the phosphorylation of SMAD2 and SMAD3 receptor-SMADs (R-SMADs) which heterotrimerize with SMAD4 or, alternatively, TIF-1 y (also known as TRIM33), and translocate into the nucleus where they regulate the activity of cell type-specific transcription factors.
  • R-SMADs SMAD2 and SMAD3 receptor-SMADs
  • TIF-1 y also known as TRIM33
  • TGF-p limits NK cell IFN-y production and cytotoxicity by inhibiting the transcription of T-bet and granzyme B through SMAD2/SMAD3/SMAD4 canonical signaling while inducing the expression of tumor homing/ tissue-resident features such as Hobit, CD103, CD49a and TRAIL in murine NK cells, in a SMAD4-independent manner.
  • TGF-p has also been shown to reduce NK cell proliferation as well as the expression of NKp30 and NKG2D activating receptors yet the signaling mediators underlying these effects remain elusive.
  • TGF-p pathway inhibitors have been investigated in the preclinical setting, some of which are in clinical development (e.g., small molecules that inhibit TGF-p receptor kinase activity, TGF-p neutralizing antibodies, TGF-p ligand traps).
  • TGF-p receptor kinase activity e.g., TGF-p neutralizing antibodies, TGF-p ligand traps.
  • the effects of systemic TGF-p inhibitors are context/tumor- dependent and require their combination with cytotoxic treatments (chemotherapy or immunotherapy) which worsen the severity of adverse events.
  • Alternative strategies preclin ical ly tested to avoid TGF-p inhibition of NK cell anti-tumor function include the knocking out of TGF-pRII or the expression of a dominant-negative TGF-pRII by genetic engineering.
  • Patent application WO20074868A1 discloses cells which express a dominant-negative SMAD protein (dnSMAD), and a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • dnSMAD dominant-negative SMAD protein
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a dominant negative polypeptide involves the synthesis of a mutated form of a protein that interferes with the function of the wild-type protein.
  • the disclosure is directed to dnSMAD2, dnSMAD3 and dnS- MAD4, but only dnSMAD2 and dnSMAD3 were experimentally tested (EXAMPLE 1 , Figure 5), showing cytotoxicity to the target cells, both in the absence or presence of TGF-p.
  • Patent application WO2019243817A1 describes T cells expressing a CAR and a protein binder which is a blocking intrabody to e.g., SHP2, FADD, or SMAD4. Molecular interactions were tested by immunoprecipitation of cell lysate for SMAD4 followed by a western blot of SMAD2/3, but results are not shown.
  • NK cell homeostasis and function a TGFp-dependent and -independent role for SMAD4 on the regulation of NK cell homeostasis and function has been described in Wang Y et al., 2018. Deletion of the single gene Smad4 in innate lymphocytes, including NK and ILC1 cells, lead to impaired NK cell maturation, homeostasis and immune surveillance against melanoma metastases and cytomegalovirus infection. Smad4 deficient mNK cells were also less cytotoxic against b2m-Z- splenocytes in in vivo rejection experiments and ex vivo cytotoxicity assays.
  • Smac/4-deficient NK cells displayed reduced granzyme B levels at transcript and protein level, and produced less IFN-y in response to cytokines (IL-12 and IL-18) and target cells.
  • cytokines IL-12 and IL-18
  • TGFp-inhibition of IFN-y production in IL-12 and IL-18-stimulated mNK cells as well as IL-15-induced granzyme B and TRAIL up-regulation were partially dependent on SMAD4. Accordingly, these prior data in mice support that deletion of Smad4 gene negatively affects NK cell homeostasis as well as anti-tumor and anti-viral function.
  • TGF-p a pleiotropic suppressive cytokine produced by tumor cells and other regulatory cells present in the tumor microenvironment, prevents immune cell infiltration, reduces immune cell cytotoxicity and also immune cell proliferation, which collectively hinder the immune system from effectively recognizing and attacking the cancer cells.
  • the solution is based on the inhibition (e.g., by genetic inactivation) of SMAD4 biological activity in cytotoxic lymphocytes as a novel strategy for bypassing TGF-p inhibitory effects on the lymphocytes function, thereby exhibiting their cytotoxicity.
  • cytotoxic lymphocyte cells comprising an inactivating mutation in SMAD4 gene are resistant (i.e., less susceptible) to immunosuppressive effects of TGF-p, i.e., are capable of tolerating the tumor microenvironment. As a result, these cells are able to exert their biological functions such as their cytotoxicity, important for tumor cell killing.
  • cytotoxic effectors granzymes and perforin
  • IL15/IL2 receptor chains essential for NK cell proliferation in response to pro-proliferative cytokines
  • CD11 a, CD18 and CD49d integrins important for NK cell extravasation from blood vessels into tumors and required for tumor cell recognition and cytotoxic synapsis formation
  • anti-HER2 antibodies tumor antigen-targeted monoclonal antibodies therapy
  • CAR chimeric antigen receptor
  • NK SMAD4+/- cells were not reduced in the presence of TGF-p, compared to NK SMAD4+/+ cells.
  • NK SMAD4+/- cells exhibited a less severe downregulation of NKG2D and granzyme B, the maintenance of CD103, a higher degranulation, and a higher TNF-a secretion ( Figure 1).
  • SMAD4+/- cells had a decreased expression of SMAD4 ( Figure 3).
  • SMAD4 haploinsufficient NK cells exhibited cytotoxicity in the presence of TGF-p.
  • EXAMPLE 3 and EXAMPLE 4 show how SMAD4 K0 was successfully produced in NK92 and primary NK cells, respectively, evidencing the on-target efficiency and safety of the engineering approach, as well as resulting in NK cells with reduced levels of SMAD4.
  • SMAD4 K0 NK92 cells displayed enhanced cytotoxicity against the cancer cell line HCT116 upon TGF-p1 treatment, surpassing their baseline cytotoxicity and the cytotoxic capacity of NK92 parental cells with and without TGF-p treatment (see EXAMPLE 3, Figure 5(l, J)).
  • results provided herein evidence a complex relationship between TGF-p and SMAD4 in the regulation of NK cell proliferation and function.
  • Smad4-/- NK cells exhibited impaired homeostasis and maturation in in vivo mouse models, concluding that SMAD4 is included in the transcriptional complex that positively regulates homeostatic granzyme B expression along NK and T cell differentiation in mouse models, yet paradoxically contributing to the down-regulation of granzyme B expression upon TGF- p exposure.
  • TGF-p inhibits IL-2/15-dependent NK cell proliferation upon activation (Cabo M et al., 2021), i.e., the process in which the proliferation of NK cells is dependent on the presence of IL-2 and IL-15.
  • SMAD4 K0 NK cells showed preserved IL-2 and IL-15-dependent proliferation in the presence of TGF-p ( Figure 10 H-J).
  • SMAD4 K0 expanded human NK cells displayed enhanced cytotoxicity against tumor cells lines (EXAMPLE 6, Figure 10(D-F)) and enhanced penetrance in tumor spheroids (EXAMPLE 9), regardless of prior exposure to TGF-p.
  • TGF-p-dependent downregulation of adhesion molecules involved in cytotoxic synapse formation
  • activating receptors inquired for tumor cell recognition
  • cytotoxic effectors such as granzyme B and perforin
  • TGF-p-dependent induction of tissue-residency/retention molecules CD103, CD29 and CD49a
  • TRAIL and FasL ligands for death receptor pathways
  • TGF-p down-regulated the expression of several integrins/adhesion molecules (such as LFA-1 (CD11 a- CD18 or aLp2), MAC1 (CD11 b-CD18 or aMp2) and ICAM1) involved in NK cell extravasation and cytotoxic synapse formation in a SMAD4-dependent manner while inducing the expression of tissue- residency-associated integrins CD29 (p1), CD49a (a1), CD103 (aE) in a SMAD4-independent manner.
  • integrins/adhesion molecules such as LFA-1 (CD11 a- CD18 or aLp2), MAC1 (CD11 b-CD18 or aMp2) and ICAM1 involved in NK cell extravasation and cytotoxic synapse formation in a SMAD4-dependent manner while inducing the expression of tissue- residency-associated integrins CD29 (p1), CD49a (a1), CD103 (aE)
  • SMAD4 K0 NK cells treated with TGF-p showed increased migration towards SDF-1 gradients when combined with CCL5 and CXCL9 as well as increased penetrance in tumor spheroids supporting the advantage of reducing SMAD4 levels for simultaneously enhancing NK cell tumor-homing, tumor cell recognition and killing.
  • TIF1 y transcriptional intermediary factor 1
  • SMAD4 K0 NK cells would take advantage of those SMAD4-independent pathways regulated by TGF-p such as the acquisition of tissue-residency features, the expression of tumor-homing chemokine receptors and their enhanced cytotoxicity through death-receptor ligands.
  • SMAD4 K0 NK displayed enhanced cytotoxicity against tumor spheroids as compared to TGF-p-RI inhibitor SB-431542 treated control NK cells, indirectly evidencing the advantage of partially maintaining SMAD4-independent TGF-p-signaling on NK cell function (see EXAMPLE 10, Figure 14(A-D)).
  • SMAD4 K0 NK cells were also resistant to activin A, a suppressive cytokine produced by tumor associated fibroblasts that can redundantly inhibit NK cell effector function (see EXAMPLE 11 , Figure 14(G)).
  • SMAD4 K0 NK cells were also tested in a humanized in vivo mouse model of HER2-positive breast cancer (see EXAMPLE 12).
  • Mice treated with SMAD4 K0 NK cells showed superior tumor growth control as monotherapy as well as when combined with anti- HER2 therapeutic antibodies such as trastuzumab and pertuzumab, bypassing in both contexts the anti-tumor efficacy of control NK cells in combination with anti-HER2 antibodies ( Figure 18(B)).
  • results prove that SMAD4 K0 NK cells exhibit significantly enhanced anti-tumor activity both in vitro and in vivo, surpassing the efficacy of known treatments such as HER2 therapeutic antibodies.
  • EXAMPLE 13 demonstrates that knocking out SMAD4 in CD19-CAR transduced NK cells not only renders the cells resistant to the inhibitory effects of TGF-p, but also significantly enhances the cytotoxic activity of CAR-NK cells in the presence of TGF-p, underscoring an improvement in their anti-tumor efficacy (Figure 19).
  • SMAD4 K0 NK cells could be developed as a TGF-p and activin A resistant “off-the-shelf platform for cancer immunotherapy with superior anti-tumor function as compared to other NK cell products; suitable for CAR armoring as well as for combinatorial strategies including tumor-antigen targeted antibodies, bispecific antibodies, or immune checkpoint blockers.
  • a first aspect of the invention relates to an isolated modified human cytotoxic lymphocyte cell for adoptive immunotherapy, wherein SMAD4 biological activity is inhibited, and wherein the cell is resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity.
  • Another aspect refers to a method for producing a modified human cytotoxic lymphocyte cell for adoptive immunotherapy comprising genetically inactivating SMAD4 gene of a human cytotoxic lymphocyte cell, resulting in a modified cell with inhibited SMAD4 expression and resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity.
  • aspects of the present invention relate to a sgRNA targeting the SMAD4 gene of the cell as described herein (e.g., a sgRNA comprising SEQ ID NO: 1-11), or a kit comprising: (a) at least a targeted nuclease as described herein; and (b) at least a sgRNA targeting the SMAD4 gene of the cell as described herein.
  • a sgRNA targeting the SMAD4 gene of the cell as described herein e.g., a sgRNA comprising SEQ ID NO: 1-11
  • kits comprising: (a) at least a targeted nuclease as described herein; and (b) at least a sgRNA targeting the SMAD4 gene of the cell as described herein.
  • Another aspect refers to a modified human cytotoxic lymphocyte cell obtained by the methods provided herein.
  • An aspect of the present invention also relates to a method for producing modified human cytotoxic lymphocyte cells for adoptive immunotherapy, resulting in modified cells with inhibited SMAD4 expression and resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity, the method comprising: (a) optionally, expanding or propagating cells from a sample of a subject in need of treatment or a healthy donor; (b) optionally, separating or purifying human cytotoxic lymphocyte cells from the sample or from the cells obtained in (a); (c) genetically modifying the human cytotoxic lymphocyte cells by inactivating (knocking out) the SMAD4 gene as defined herein; (d) optionally, expanding or propagating the cells obtained in (c); and (e) optionally, separating or purifying the cells obtained in (c) or (d).
  • another aspect relates to a method of adoptive cell transfer comprising: (a) obtaining a sample (e.g., blood) from a subject in need of treatment or a healthy donor; (b) producing modified human cytotoxic lymphocyte cells with inhibited SMAD4 expression following the method for producing modified human cytotoxic lymphocyte cells as described above; (c) optionally, administering a conditioning or preconditioning treatment to the subject in need of treatment; and (d) administering an effective amount of the cells obtained in (b) or (c) to the subject in need of treatment.
  • This aspect can alternatively be formulated as a method of treating cancer in a subject in need thereof comprising the steps as described above.
  • aspects relate to a method of adoptive cell transfer and a method of treating cancer in a subject in need thereof, comprising: (a) thawing isolated modified SMAD4 human cytotoxic lymphocyte cells according to the invention; (b) optionally, administering a conditioning or preconditioning treatment to the subject in need of treatment; and (c) administering the cells obtained (a) to the subject in need of treatment.
  • the invention relates to a cell population comprising modified human cytotoxic lymphocyte cells as defined in the first aspect of the invention, wherein the cell population is characterized by: (a) an allelic frequency of between about 10% and 90% of modified SMAD4 gene cytotoxic lymphocyte cells; (b) a genotype frequency of between about 10% and 90% of SMAD4-/- and/or SMAD4+/- cytotoxic lymphocyte cells; and/or (c) an off-target rate of less than 1 %.
  • Another aspect of the invention is a method for producing a cell population comprising modified cytotoxic lymphocyte cells following the steps of the method for producing a modified cytotoxic lymphocyte cell.
  • Another aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cytotoxic lymphocyte cell as defined herein or a cell population as defined herein and pharmaceutically acceptable excipients.
  • modified cytotoxic lymphocyte cell for use as a medicament, for use as an immunotherapy agent, or for use in the treatment of a cancer (e.g., colorectal cancer).
  • the invention also encompasses a method for treating a cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a modified cytotoxic lymphocyte cell, a cell population, or a pharmaceutical composition as defined herein.
  • Figure 1 shows the effect of TGFp-canonical signaling and SMAD4 gene dose on NK cell phenotype and function.
  • Purified human NK cells from 3 independent individuals were activated in anti-CD16- coated plates for 5 days in the presence or absence ofTGF-p1 (10 ng/ml) priorto total RNA extraction and microarray analysis.
  • A, B, C Volcano plot and Heat Maps showing global and selected differential expressed genes (DEG) between anti-CD16 activated NK cells in the presence or absence of TGF-p (
  • DEG differential expressed genes
  • D-H PBMC from a healthy donor and two JPS patients were cultured in anti-CD16-coated plates in the presence or absence of TGF-p (10 ng/ml) for 6 days.
  • D-G NK cell numbers and expression of indicated markers in NK cells from two JPS patients (PT, SMAD4+/-) and from a healthy control (HD, SMAD4+/+) at day 6, as analyzed by multiparametric flow cytometry.
  • E-G Data normalized to NK cells cultured in the absence of TGF-p.
  • Figure 2 shows the results of purified human NK cells from 3 independent individuals that were activated in anti-CD16-coated plates for 5 days in the presence or absence of TGF-p1 (10 ng/ml) prior to total RNA extraction and microarray analysis.
  • Bar graph showing those biological pathways down- (A) or up-regulated (B) in TGF-p1 -treated NK cells.
  • Figure 3 shows the SMAD4 expression in PBMC from SMAD4 haploinsufficient patients and healthy individuals.
  • A,B SMAD4 and p-actin expression in fresh PBMC from two patients with juvenile polyposis syndrome and two healthy donors (HD#1 and HD#2) by western blot. Mutations for each JPS patient are indicated.
  • FIG. 4 shows TGF-p effect on NK92 cell cytotoxicity, phenotype and proliferation NK92 cells were overnight (ON) or 5 days-treated with TGF-p (5 ng/ml).
  • HCT116 cell death in each co-culture was monitored by detection of activated caspase 3 (aC3) by multiparametric flow cytometry.
  • D Fold change of NK92 cell counts after 5 day treatment with TGF-p. Data from 3-5 independent experiments. Statistical significance by paired Student t test (* ⁇ 0.05 ,** ⁇ 0.01 , *** ⁇ 0.001 ).
  • Figure 5 shows the effect of SMAD4 gene dose and TGF-p on the phenotype and function of NK92 cells.
  • SMAD4-engineered NK92 cell clones were obtained by CRISPR/Cas9 engineering, sorting of nucleofected cells and subsequent cloning by limiting dilution as detailed in methods.
  • A-D Baseline SMAD4 levels in two S/VWD4-engineered clones: NK92-G3 and NK92-F3, the parental NK92 cell line and in HT29 and HCT116 colorectal cancer cell lines as negative and positive controls, respectively, as determined by intracellular flow cytometry (A, B) and western blot (C, D).
  • E-F Cas9-targeted PAM sequence in SMAD4 exon 5 and exon 10 was amplified by PCR from G3 and F3 as well as from the parental NK92 cells and sequenced by Illumina. Allelic frequencies of the mutations introduced by CRISPR in exon 5 (E) and 10 (F) from the indicated cells.
  • NK92 parental cells and SMAD4-engineered NK92 cell clones were treated or not (NT) with TGF-p (5 ng/ml) for 5 days. Cell number fold change (G) and Granzyme B levels (H) for each NK92 cell clone in the indicated conditions. Data from 3 independent experiments.
  • I-J Parental and SMAD4-engineered NK92 cells treated or not with TGF-p were cocultured with HCT116 for 4h. Average percentage of active caspase 3 positive HCT116 cells at the indicated effectortarget ratios. Dashed and solid lines depict the cytotoxicity induced by TGF-p -treated and non-treated NK92 cells, respectively. Data from 3 independent experiments.
  • FIG. 6 shows TGF-p canonical signaling mediators in SMAD4 Knock out (SMAD4 K0 ') and control human primary NK cells.
  • CRISPR/Cas9 engineering of primary NK cells protocol scheme: PBMC from healthy donors were cocultured with irradiated 8866 feeder cells. At day 7 NK cells were isolated by negative selection and nucleofected with either SMAD4 exon 5 gRNA or control (CTRL) gRNA-
  • Figure 7 shows Cas9 RNP nucleofection efficiency and SMAD4 reduction on primary human expanded NK cells.
  • A-B NK cells were isolated at day 7 post expansion with 8866 and nucleofected with control gRNA or SMAD4 gRNA complexed to Cas9 and ATTO550-labeled sgRNA. Monitoriza- tion of nucleofection efficiency was analyzed after overnight culture by flow cytometry.
  • B Median nucleofection efficiency in 13 independent experiments.
  • Figure 8 shows TGFp-dependent and -independent effects of SMAD4 in human expanded NK cell transcriptome and phenotype.
  • A-F Total RNA was extracted from control and SMAD4 K0 NK cells expanded in the presence/absence of TGF-p1 as previously described and analyzed by RNAseq. Data correspond to average changes in gene expression from engineered NK cells from 3 different donors.
  • A) Number of differentially expressed genes (DEG) between the four indicated conditions (pval ⁇ 0,01).
  • DEG differentially expressed genes between control (CTRL) and SMAD4 K0 NK cells in the absence or presence of TGF-p. Red dots indicate significantly DEG.
  • C-E Heat Maps showing the expression of selected genes related to NK cell receptors and signaling adaptors (C), NK cell cytotoxic effector molecules (D) and soluble mediators (E) in the indicated cells. Asterisks label genes significant differential expression between control- and SMAD4-engineered NK cells treated with TGF-p.
  • GSEA Gene set enrichment analysis
  • G-H Expression of surface NKG2D, CD16, NKp30 and intracellular Granzyme B (GzmB) and perforin in control (CTRL) and SMAD4 K0 NK cells treated or not with TGF-p as previously indicated by flow cytometry.
  • G Histograms showing data from a representative experiment. Inset numbers indicate mean fluorescence intensity of the analyzed marker in each indicated condition.
  • H Mean SEM of each indicated parameter in the four conditions tested. Each dot corresponds to data from an independent experiment done with NK cells from different individuals. Data from 3-6 experiments. Statistical significance by ANOVA test for multiple comparisons.
  • Figure 9 shows the transcriptional profile of SMAD4 engineered primary NK cells in the absence or presence of TGF-p.
  • Total RNA was extracted from control and SMAD4 K0 NK cells expanded in the presence/absence of TGF-p1 as previously described and analyzed by RNAseq. Data correspond to average changes in gene expression from engineered NK cells from 3 different donors
  • Figure 10 shows that SMAD4 K0 NK cells showed enhanced anti-tumor function in the presence of TGF-p.
  • A-C Control- and SMAD4 K0 NK cells treated or not with TGF-p (5 ng/ml) were cocultured with HCT116 colorectal cancer cell line at the indicated effectortarget (E:T) ratios for four hours.
  • E:T effectortarget
  • E GFP intensity along time in each coculture. Data represents mean+/- SEM data of 5 spheroids for each condition analyzed.
  • F Luciferase activity of remaining alive HCT116 cells after 30h of coculture with NK cells. Statistical significance calculated by one way ANOVA test (*** p ⁇ 0.001 ; **p ⁇ 0.01 ; *p ⁇ 0.5).
  • G Control- and SMAD4 K0 NK cells treated or not with TGF-p (5 ng/ml) were cocultured with HCT116 colorectal cancer cell line at 0.75:1 effector:target (E:T) ratios for 2 hours in the presence of blocking antibodies for Fas ligand (anti- FASL), TRAIL (anti-TRAIL) or control lgG2.
  • H Expression of IL2 and IL15 Receptor transcripts in control and SMAD4 K0 NK cells by bulk RNAseq.
  • I CFSE-labeled control and SMAD4 K0 NK cells were cultured in anti-NKp46 coated plates with IL-2 (200U/ml) in the presence or absence of TGF-p (5ng/ml) for 6 days. Proliferation was analyzed as CFSE dilution by flow cytometry.
  • FIG 11 depicts the TNF-a secretion by control and SMAD4 K0 NK cells treated or not with TGF-p.
  • Statistical significance calculated by one way ANOVA test (*** p ⁇ 0.001 ; **p ⁇ 0.01 ; *p ⁇ 0.5).
  • Figure 12 shows the impact of SMAD4 and TGF-p in the integrin profile, tumor penetrance and transmigration of expanded NK cells.
  • HCT116 spheroids were cocultured with PKH26-labeled control- or SMAD4 K0 NK cells previously exposed or not to TGF-p. After 1 h coculture, spheroids and attached NK cells were fixed and processed for lightsheet imaging. HCT116 cells were labeled with an anti-Epcam-FITC antibody.
  • E Number of NK cells counted in spheroids in the indicated conditions.
  • F Quantification of the distance between the spheroid surface and each infiltrating NK cell.
  • Each dot represents the measurement of one infiltrating NK cell.
  • G Surface expression of CXCR3, CCR5 and CXCR4 in control and SMAD4 K0 NK cells treated or not with TGF-p as analyzed by flow cytometry. Bar graphs showing the mean+/- SEM of the fluorescence intensity for each indicated receptor. Data from 3-5 independent experiments.
  • H Transcript expression levels of chemokine receptors in control- and SMAD4 K0 NK cells exposed or not to TGF-p, according to RNAseq data from 3 independent donors.
  • NK cells Percentage of transmigrating control (gray bars) or SMAD4 K0 (empty bars) NK cells to CCL5 (50 ng/ml), CXCL9 (50 ng/ml), SDF- 1 (100 ng/ml) or the indicated chemokine combinations. Each dot indicates the results from an independent experiment including engineered NK cells from different individuals. Data normalized to number of cells migrating in the control condition (RPMI). In all assays statistical significance calculated by one-way ANOVA test (*** p ⁇ 0.001 ; **p ⁇ 0.01 ; *p ⁇ 0.5).
  • Figure 13 shows the impact of SMAD4 and TGF-p in the integrin profile, tumor penetrance and transmigration of expanded NK cells.
  • B) HCT116 spheroids were cocultured with PKH26-labeled control- or SMAD4 K0 NK cells previously exposed or not to TGF-p.
  • HCT116 cells were labeled with an anti- Epcam- FITC antibody. Image of a representative HCT116 spheroid for the indicated conditions. Inset numbers correspond to the number of NK cells counted in each spheroid.
  • Figure 14 shows that SMAD4 K0 NK cells display superior cytotoxicity than control NK cells treated with a TGFBR-I inhibitor and are resistant to Activin A suppression.
  • A-D SMAD4 K0 and control NK cells were expanded in the presence of TGF-p (5 ng/ml) for 7 days. Control NK cells were supplemented with the TGFBR-I inhibitor SB431542 (20 pmol) every day.
  • SMAD4 K0 and control NK cells treated with SB431542 were cocultured with HCT116-GFP+-Luc+ spheroids. Luciferase counts were analyzed at 24h of co-culture.
  • E Relative transcripts expression (zscores) of Activin Receptor genes in the indicated NK cells. Average expression in NK cells from 3 independent individuals as analyzed by RNAseq.
  • F-G SMAD4 K0 and control NK cells were incubated with TGF-p (5ng/ml) or Activin A (100 ng/ml) for the last 7 days of expansion.
  • F Mean +/-SEM Granzyme B levels as in SMAD4 K0 and control NK with the indicated treatments analyzed by intracellular staining by flow cytometry. Data from experiments with NK cells from two different individuals.
  • Figure 15 shows that SMAD4 K0 NK cells display superior cytotoxicity than control NK cells treated with a TGF-BRI inhibitor and are resistant to Activin A-mediated inhibition.
  • A) SMAD4 K0 and control NK cells were expanded in the presence of TGF-p (5 ng/ml) for 7 days. Control NK cells were supplemented with the TGFBR-I inhibitor SB431542 (20 pmol) every day. At day 7, expression of Granzyme B (GzmB), NKG2D and CD103 was analyzed by flow cytometry. Representative histograms of the indicated markers in each assayed condition.
  • SMAD4 K0 and control NK cells were incubated with TGF-p (5ng/ml) or Activin A (100 ng/ml) for the last 7 days of expansion.
  • TGF-p 5ng/ml
  • Activin A 100 ng/ml
  • expression of Granzyme B (GzmB) and perforin was analyzed by flow cytometry. Representative histograms of the indicated markers in each assayed condition.
  • Figure 16 shows the efficiency of SMAD4 knock-out by CRISPR/Cas9 RNP nucleofection in in vitro expanded NK cells.
  • Cas9-targeted PAM sequence in SMAD4 exon5 was amplified by PCR from engineered NK cells in 3 independent experiments and sequenced by Illumina. Sequences obtained were analyzed against de reference sequence.
  • FIG 17 shows TGF-beta signaling: Active TGF-p binds to TGF-p type 2 receptor dimers (TGF- pRI I) which recruit and activate type 1 receptors (TGF-pRI) to form a tetrameric receptor complex, unleashing a complex signaling cascade involving canonical as well as noncanonical signaling pathways.
  • the canonical signaling is initiated by the phosphorylation of SMAD2 and SMAD3 receptor- SMADs (R-SMADs) which heterotrimerize with SMAD4 or, alternatively, TIF-1 y (also known as TRIM33) or IkKa, and translocate into the nucleus where they regulate the activity of cell type-specific transcription factors.
  • Non canonical TGF-p signaling includes a variety of intracellular signaling pathways activated by TGF-p independently of SMAD2/3. Those pathways involve the activation of PI3K, ERK and MAPK kinases.
  • Figure 18 shows the SMAD4 K0 NK cells anti-tumor function evaluated in a humanized in vivo mouse model of HER2-positive breast cancer.
  • A-B HCC1954 cells (4x10 5 ) were subcutaneously implanted in NOD/Scid/yc-/- (NSG) mice.
  • mice were treated with either: i) trastuzumab (Tz)Zpertuzumab (Pt) (1 mg/Kg each, intraperitoneal); ii) control NK cells (2 x10 5 , intra- tumoral); iii) SMAD4 K0 NK cells (2 x10 5 , intratumoral); iv) control NK cells (1 x10 5 intratumoral) and trastuzumab/pertuzumab (1 mg/Kg each, intraperitoneal); or v) SMAD4 K0 NK cells (1 x10 5 intra- tumoral) and trastuzumab/pertuzumab (1 mg/Kg each, intraperitoneal).
  • mice were sacrificed at the end of treatment.
  • Figure 19 shows that knocking out SMAD4 enhanced the cytotoxicity of CAR19-NK cells in the presence of TGF-p.
  • Nalm6-Luc-GFP cells were co-cultured with CAR19-transuced control or SMAD4- KO NK cells for 4h.
  • Statistical significance by Student T test is indicated.
  • SMAD4- This term, also known as Mothers Against Decapentaplegic Homolog 4 (MADH4), DPC4, JIP or MYHRS, refers to a protein that plays a critical role in the transforming growth factor-beta (TGF-p) signaling pathway.
  • TGF-p transforming growth factor-beta
  • This pathway is a fundamental signaling cascade involved in various cellular processes, including cell growth, differentiation, proliferation, and embryonic development.
  • specific receptor-activated SMADs Upon activation of the TGF-p receptors by ligand binding, specific receptor-activated SMADs (R- SMADs), such as SMAD2 and SMAD3, are phosphorylated. These R-SMADs then form complexes with SMAD4 in the cytoplasm.
  • the R-SMAD/SMAD4 complexes translocate into the cell nucleus, where they act as transcription factors. Within the nucleus, these SMAD complexes bind to specific DNA sequences known as SMAD-binding elements (SBEs) or TGF-p-responsive elements (TREs) in the promoter regions of target genes. This binding either activates or represses the transcription of these target genes, depending on the context. Therefore, the primary function of SMAD4 is to act as a signal transducer ortranscription factor in the TGF-p signaling pathway.
  • transcription factor SMAD4 gene is registered in GenBank under accession number 4089 (date: 22.10.2023), corresponding to Ensemble accession number ENSG00000141646.
  • SMAD4 is coded by a gene located on chromosome 18.
  • the SMAD4 transcript is registered in Ensemble under accession number ENST00000342988.8 (release 110, date: July 2023).
  • the protein SMAD4 has 552 amino acid long (Uniprot: Q13485).
  • the term SMAD4 also encompasses polymorphic and natural variants.
  • biological activity as used herein, the terms “biological activity” and “biological function” are used interchangeably and refer to the ability of an agent (e.g., SMAD4 protein) to affect (e.g., stimulate or inhibit) one or more biological processes in a living organism.
  • agents e.g., SMAD4 protein
  • biological processes include biochemical pathways, physiological processes that contribute to the internal homeostasis of a living organism, developmental processes that contribute to the normal physical development of a living organism, and acute or chronic diseases.
  • SMAD4 The biological activity of SMAD4 primarily involves its role as a mediator in the TGF-p signaling pathway. Specifically, SMAD4 acts as a transcription factor that plays a role in transducing signals from the TGF-p family of ligands to regulate gene expression. The transcriptional regulation of target genes by SMAD4-containing complexes influences various biological processes, including the control of cell growth, differentiation, and tissue development. SMAD4's role in the TGF-p pathway is relevant for proper embryonic development, tissue repair, immune responses, and the maintenance of tissue homeostasis.
  • Inhibition of SMAD4 biological activity the terms “inhibition of SMAD4 biological activity/function”, “SMAD4 biological activity/function is inhibited” and grammatical variants thereof refer to total (i.e., blocking) or partial inhibition (i.e., reduction) of the biological activity of SMAD4 protein. Therefore, in the context of the present invention and for the sake of simplicity in the description, the term “inhibition” also encompasses “reduction" of SMAD4 biological activity.
  • the SMAD4 biological activity is inhibited by at least about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to the endogenous biological activity of SMAD4 that is not inhibited.
  • SMAD4 biological activity can be inhibited by gene editing (e.g., CRISPR-Cas9), RNA interference (RNAi) which specifically target the mRNA of SMAD4, small molecule inhibitors specific for SMAD4, dominant-negative mutations that interfere with the normal function of the endogenous SMAD4 protein, antibodies designed to specifically bind to SMAD4 protein, protein degradation technologies (e.g., PROTACs and degron systems), phosphorylation and post-translational modifications, competitive inhibition, protein expression knockdown (e.g., antisense oligonucleotides or ribozymes that reduce the expression at the mRNA level), or protein sequestration.
  • the SMAD4 biological activity is inhibited by gene editing, i.e., by modifying the DNA sequence of SMAD4 gene or regulatory sequence, leading to the loss of function or altered function of the protein encoded by the targeted gene.
  • expression refers to transcription of a polynucleotide from a DNA template, resulting in, e.g., a mRNA or other RNA transcript (e.g., non-coding, such as structural or scaffolding RNAs).
  • the term further refers to the process through which transcribed mRNA is translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be referred to collectively as "gene product”. Expression may include splicing the mRNA in a eukaryotic cell, if the polynucleotide is derived from genomic DNA.
  • Inhibition ofSMAD4 expression the terms “inhibition of SMAD4 expression”, “SMAD4 expression is inhibited” and grammatical variants thereof refer to total (i.e., blocking) or partial inhibition (e.g., reduction) of the expression (e.g., transcription and/or translation) of SMAD4 protein. Therefore, in the context of the present invention and for the sake of simplicity in the description, the term “inhibition” also encompasses “reduction” of SMAD4 expression.
  • the SMAD4 expression is inhibited by gene editing, i.e., by modifying the DNA sequence of SMAD4 gene or regulatory sequence, leading to the loss of function or altered function of the protein encoded by the targeted gene.
  • the SMAD4 expression is inhibited by a gene knockout (KO).
  • a gene knockout can be performed by introducing mutations or deletions in both alleles of the SMAD4 gene to completely block the expression of the protein, or by creating heterozygous knockouts by introducing mutations in only one of the two alleles, resulting in partial inhibition of SMAD4 expression.
  • the inhibition of SMAD4 expression in a cell can be determined by means of a range of assays that are available in the art. In some embodiments, the inhibition of SMAD4 expression is determined by flow cytometry.
  • Inactivation mutation in SMAD4 gene- refers to a type of genetic mutation that disrupts or impairs the normal function of the SMAD4 gene (or regulatory sequences), typically resulting in the loss or reduction of the gene's protein product or its activity. Inactivating mutations can lead to the inactivation or loss of function of the SMAD4 protein, which may have important roles in various cellular processes. These mutations can occur through various mechanisms, including point mutations, insertions, deletions, or large-scale genetic rearrangements. Inactivation mutations can be homozygous or heterozygous.
  • Gene this term as used herein refers to a polynucleotide sequence comprising exon(s) and related regulatory sequences.
  • a gene can further comprise intron(s) and/or untranslated region(s) (UTR(s)).
  • regulatory sequences are interchangeable and refer to polynucleotide sequences that are upstream (5’ non coding sequences), within, or downstream (3’ non-translated sequences) of a polynucleotide sequence to be expressed. Regulatory sequences influence, e.g., the timing of transcription, amount or level of transcription, RNA processing or stability, and/or translation of the related structural nucleotide sequence.
  • Regulatory sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences such as from bovine growth hormone (BGH) or Simian Virus 40 (SV40), promoters, repressor binding sequences, stem-loop structures, translational initia- tion sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like.
  • BGH bovine growth hormone
  • SV40 Simian Virus 40
  • Lymphocyte this term as used herein refers to a leukocyte (white blood cell) that is part of the vertebrate immune system. Also encompassed by the term “lymphocyte” is a hematopoietic stem cell that gives rise to lymphoid cells. Lymphocytes include T cells for cell-mediated, cytotoxic adaptive immunity, such as CD4+ and/or CD8+ cytotoxic T cells; alpha/beta T cells and gamma/delta T cells; regulatory T cells, such as Treg cells; natural killer (NK) cells that function in cell-mediated, cytotoxic innate immunity; and B cells, for humoral, antibody-driven adaptive immunity.
  • the lymphocyte can be a mammalian cell, such as a human cell.
  • lymphocyte also encompasses genetically modified T cells, modified to produce chimeric antigen receptors (CARs) on the T cell surface (CAR- T cells). These CAR-T cells recognize specific antigens on a target cell surface, such as a tumor cell surface.
  • the CAR comprises an extracellular ligand binding domain, a hinge region, a transmembrane region and an intracellular signaling region.
  • TCRs T cell receptor engineered T cells
  • TCRs T cell receptor engineered T cells
  • TILs Tumor infiltrating lymphocytes
  • lymphocyte TILs
  • TILs are immune cells that have penetrated the environment in and around a tumor (“the tumor microenvironment”). TILs are typically isolated from tumor cells and the tumor microenvironment and are selected in vitro for high reactivity against tumor antigens. TILs are grown in vitro under conditions that overcome the tolerizing influences that exist in vivo and are then introduced into a subject for treatment.
  • CARs can also be incorporated into TILs, NK cells or TCRs resulting in CAR TILs, CAR- NK cells, or TCR engineered CAR-T cells.
  • Lymphocyte activation occurs when lymphocytes are triggered through antigen-specific receptors on their cell surface. This causes the cells to proliferate and differentiate into specialized effector lymphocytes. Such “activated” lymphocytes are typically characterized by a set of receptors on the surface of the lymphocyte. Surface markers for activated T cells include CD3, CD4, CD8, PD1 , IL2R, and others. Activated cytotoxic lymphocytes can kill target cells (such as cancer cells) after binding cognate receptors on the surface of target cells.
  • Cytotoxicity refers to the the immune response mediated by cytotoxic immune cells, such as natural killer (NK) cells and cytotoxic T cells (CTLs), whereby these cells recognize and eliminate specific target cells, typically those that are infected with pathogens or abnormal cells, including cancer cells, primarily by inducing apoptosis (programmed cell death) in the target cells. Cytotoxicity plays a vital role in the immune system's defense mechanisms against infections and cancer. This immune response begins with the recognition of markers or antigens on the surface of the target cell that indicate infection, stress, or abnormality.
  • cytotoxic immune cells Upon recognition, cytotoxic immune cells become activated, leading to the release of cytotoxic molecules like perforin and granzymes, which create pores in the target cell's membrane, triggering apoptotic pathways. Additionally, cytotoxic lymphocytes may employ the Fas ligand (FasL)-Fas interaction or other death receptor signaling mechanisms to induce apoptosis in the target cell. As a result of these interactions, the target cell undergoes programmed cell death, characterized by nuclear fragmentation and other cellular changes. Ultimately, the immune system's goal is to eliminate these apoptotic target cells, often through phagocytosis by macrophages, thus preventing the spread of pathogens or the growth of cancer cells and maintaining overall health and immunity.
  • FasL Fas ligand
  • Cytotoxicity can be determined by means of a range of assays that are available in the art.
  • cytotoxicity is determined by analysis of cytotoxic cell degranulation (e.g., by monitoring CD107a mobilization by flow cytometry); by analysis of activated caspase 3 in target cells by intracellular staining; by measuring luciferase activity remaining in alive Luc-transfected tumor cells upon co-culture with NK cells; by chromium release assay; by calcein release assay; by LDH cytotoxicity assay; or by annexin V and propidium iodide uptake.
  • Cytotoxic lymphocytes refers to lymphocytes with the ability to exert cytotoxicity.
  • Examples of cytotoxic lymphocytes are cytotoxic T lymphocytes (CTLs) and NK cells.
  • isolated can refer to a nucleic acid or polypeptide that, by the hand of a human, exists apart from its native environment and is therefore not a product of nature. Isolated means substantially pure. An isolated nucleic acid or polypeptide can exist in a purified form and/or can exist in a non-native environment such as, e.g., in a recombinant cell.
  • Allelic frequency refers to the frequency or proportion of a particular allele (gene variant) within a cell population.
  • a knockout in the SMAD4 gene in a cell population can result in different alleles within the cells, obtaining e.g. cells with a specific deletion in SMAD4 gene, others with a specific insertion in SMAD4 gene, and some remaining unaltered.
  • a cell population can have the following allelic frequencies: 30% of cells with deletion A, 20% of cells with deletion B, 30% of cells with insertion C, and 20% of wild type cells.
  • the allelic frequency indicates the percentage of mutated cells with an out- of-frame (OUT) mutation (e.g., an insertion or deletion) which will not lead to a functional protein.
  • Genotype frequency refers to the frequency or proportion of specific genotypes (combinations of alleles) within a population. Considering a single gene with two alleles, A and B, a genotype frequency of 10% AA, 40% AB and 50% BB in a cell population of 1000 cells implies that 100 cells are AA, 400 cells are AB and 500 cells are BB.
  • a cell population can have a genotype frequency of 60% of SMAD4-/- cells, 35% of SMAD4+/- cells and 5% of SMAD4+/+ cells (unaltered).
  • Estimated allelic frequency and genotype frequency can be calculated by assessing the editing outcomes at the target site or locus of interest and then compute the average or mean value. This can involve techniques such as DNA sequencing, PCR, or other molecular assays known in the art. The frequencies are often expressed as a percentage, indicating the proportion of edited alleles or targets within a cell population. In some embodiments, the estimated allelic frequency or genotype frequency is calculated by PCR using at least one of the primers with SEQ ID NO: 13-16 and 23-24 (listed in Table 4 and Table 5 of herein).
  • Off-target rate- this term refers to the frequency or proportion of unintended genetic modifications that occur at genomic sites other than the intended target site when using genome-editing technologies like CRISPR/Cas9. These unintended genetic alterations can potentially lead to undesired effects or mutations in non-target genes or genomic regions.
  • the off-target rate is typically expressed as a percentage or ratio and quantifies the proportion of edited cells or organisms that exhibit genetic modifications at off-target sites relative to the total number of edited cells or organisms.
  • Off-target indel rate can be determined by e.g., DNA sequencing (such as high-throughput sequencing), PCR assays, or other molecular methods.
  • the off-target rate is determined by PCR using at least one of the primers with SEQ ID NO: 17-22 (listed in Table 4 of herein).
  • Treatment refers to a clinical intervention to prevent (e.g., suppress or inhibit) a disease or condition; cure the disease or condition (e.g., cancer); delay the onset of the disease or condition; reduce the seriousness or severity of the disease or condition (e.g., cancer); ameliorate or eliminate one or more symptoms or sequelae associated with a disease or condition; or the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • a disease or condition e.g., cancer
  • cure the disease or condition e.g., cancer
  • delay onset of the disease or condition
  • reduce the seriousness or severity of the disease or condition e.g., cancer
  • ameliorate or eliminate one or more symptoms or sequelae associated with a disease or condition e.g., cancer
  • the term refers to a clinical intervention to improve one or more symptoms; improve one or more sequelae; prevent (e.g., suppress, inhibit or delay) one or more symptoms; prevent (e.g., suppress, inhibit or delay) one or more sequelae; delay one or more symptoms; delay one or more sequelae; ameliorate one or more symptoms; ameliorate one or more sequelae; shorten the duration one or more symptoms; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more sequelae; improve the quality of life; increase survival; prevent (e.g., suppress, inhibit or delay) a recurrence of the disease or condition; delay a recurrence of the disease or condition; reduce the severity of the disease; or any combination thereof, e.g., with respect to what is expected in the absence of the treatment with the cells of the present invention.
  • treatment also includes prophylaxis or prevention (e.g., suppression, inhibition or delay) of a disease or condition or its symptoms or sequelae thereof.
  • Prophylaxis refers to a therapeutic or course of action used to prevent, inhibit, suppress, reduce the risk, reduce the occurrence or delay the onset of a disease or condition, or to prevent, inhibit, suppress, or delay a symptom associated with a disease or condition.
  • an effective amount of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied.
  • an effective amount of an agent is an amount sufficient to reduce or decrease e.g., the size of a tumor, inhibit tumor growth, induce tumor regression, alleviate cancer-related symptoms in a subject, or any combination thereof as compared to the response obtained without administration of the agent.
  • the term "effective amount” can be used interchangeably with “effective dose”, “therapeutically effective amount”, or “therapeutically effective dose.”
  • the present invention is directed to cytotoxic lymphocyte cells with inhibited SMAD4 function for adoptive immunotherapy.
  • the working examples herein demonstrate how these modified cells are capable of bypassing the immunosuppressive effects of TGF-p present in the tumor microenvironment, enabling them to exhibit their cytotoxicity and their anti-tumoral function.
  • an aspect of the present invention relates to an isolated modified human cytotoxic lymphocyte cell for adoptive immunotherapy, wherein SMAD4 biological activity is inhibited, and wherein the cell is resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity (i.e., in a TGF-p microenvironment).
  • the SMAD4 biological activity is inhibited by gene editing (e.g., CRISPR- Cas9), RNA interference (RNAi) which specifically target SMAD4 mRNA, small molecule inhibitors specific for SMAD4, dominant-negative mutations that interfere with the normal function of the endogenous SMAD4 protein, antibodies designed to specifically bind to SMAD4 protein, protein degradation technologies (e.g., PROTACs and degron systems), phosphorylation and post-translational modifications, competitive inhibition, protein expression knockdown (e.g., antisense oligonucleotides or ribozymes that reduce the expression at the mRNA level), protein sequestration or any other methodology.
  • gene editing e.g., CRISPR- Cas9
  • RNAi RNA interference
  • small molecule inhibitors specific for SMAD4 small molecule inhibitors specific for SMAD4, dominant-negative mutations that interfere with the normal function of the endogenous SMAD4 protein
  • the SMAD4 biological activity is inhibited by gene editing, i.e., by modifying the DNA sequence of SMAD4 gene or regulatory sequence, leading to the loss of function or altered function of the protein encoded by the targeted gene.
  • the invention relates to an isolated modified human cytotoxic lymphocyte cell for adoptive immunotherapy comprising an inactivating mutation in SMAD4 gene, wherein the cell has inhibited SMAD4 expression and is resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity.
  • the term “inhibited SMAD4 expression” also includes “reduction” of SMAD4 expression.
  • the modified cytotoxic lymphocyte cell comprises a homozygous inactivating mutation in the SMAD4 gene (i.e., SMAD4-/-). In another embodiment, the modified cytotoxic lymphocyte cell comprises a heterozygous inactivating mutation in the SMAD4 gene (i.e., SMAD4+/-).
  • the modified cytotoxic lymphocyte cell comprises an inactivating mutation in SMAD4 gene and express a truncated form of SMAD4 protein.
  • the modified cytotoxic lymphocyte cell is a natural killer (NK) cell or a T-cell.
  • the T-cell is selected from the group consisting of a CD4+ T-cell, a CD8+ T-cell, a regulatory T-cell and a helper T-cell.
  • the modified cytotoxic lymphocyte cell is a tumor infiltrating lymphocyte (TIL).
  • TIL tumor infiltrating lymphocyte
  • the modified cytotoxic lymphocyte cell is a NK cell.
  • the modified cytotoxic lymphocyte cell is a T cell with an engineered T cell receptor (TCR). Cytotoxic lymphocyte cells can be allogeneic (or "off-the-shelf cytotoxic lymphocytes cells) or autologous.
  • Allogeneic cytotoxic lymphocytes cells are cells that are derived from a donor source, typically isolated from healthy donors, and can be used for therapeutic purposes, such as cancer immunotherapy.
  • Autologous cytotoxic lymphocytes cells are patient's own cells that are isolated, manipulated (e.g., expanded or engineered), and then reinfused into the same individual. This approach is used in certain immunotherapy strategies.
  • the modified cytotoxic lymphocyte cells are differentiated cells derived from pluripotent stem cells or induced pluripotent stem cells (iPSCs), said differentiated cells having a NK or T cell phenotype (i.e., iPSC-NK or iPSC-T cells).
  • iPSC-NK or iPSC-T cells a NK or T cell phenotype
  • Commercial iPSC lines are available and can be used to generate de iPSC-NK or iPSC-T cells described herein.
  • the modified cytotoxic lymphocyte cell provided herein comprises an additional mutation or modification.
  • Non-limiting mutations or modifications that enhance the anti-tumor activity of cytotoxic lymphocytes can be chimeric antigen receptors (CAR), such as CAR-T or CAR- NK cells; B-cell maturation antigen (BCMA) targeting, CD19 targeting, CD20 targeting, CD30 targeting, HER2 targeting, EGFR targeting, IL-13Ro2 targeting, checkpoint inhibitors (e.g., PD-1 knockout), T-cells redirected for universal cytokine killing (TRUCKS); or combinations thereof (e.g., CAR-T-19, which is a CAR-T cell targeting CD19).
  • CAR chimeric antigen receptors
  • BCMA B-cell maturation antigen
  • CD19 targeting CD20 targeting
  • CD30 HER2 targeting
  • HER2 targeting e.g., EGFR targeting
  • IL-13Ro2 targeting IL-13Ro2 targeting
  • checkpoint inhibitors e
  • CAR can be incorporated into TILs, NK cells or TCRs resulting in CAR TILs, CAR-NK cells, or TCR engineered CAR-T cells.
  • EXAMPLE 13 demonstrates the efficient incorporation of a CAR into SMAD4 K0 NK cells (CAR19-SMAD4-KO NK cells) and its enhanced anti-tumor efficacy.
  • the modified cytotoxic lymphocyte cell comprises a CAR.
  • CAR constructs can include, but are not limited to, receptors targeting CD19, CD20, CD22, BCMA (B-cell maturation antigen), CD319 (SLAMF7), CD38, CD138, NY-ESO-1 , CEA, PSMA, MUC1 , CD276 (B7-H3), GD2, FAP, VEGFR-2, Mesothelin, EG- FRvlll, CD30, HER2, GPRC5D, CD33, IL-13Ra2, and EGFR.
  • the CAR is CD19-CAR (CAR19).
  • the modified cytotoxic lymphocyte cell shows higher cell killing ability than unmodified cells.
  • Cell killing ability can be determined by incubating the modified cells (and unmodified control cells) with target cells and measuring the disappearance of the target cells using e.g., a fluorescent marker.
  • the modified cytotoxic lymphocyte cell comprising an inactivating mutation in SMAD4 gene kills target cells with an efficiency that is about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of cells not comprising an inactivating mutation in SMAD4 gene.
  • the modified cytotoxic lymphocyte cell comprising an inactivating mutation in SMAD4 gene kills target cells with an efficiency that is about: 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than that of cells not comprising an inactivating mutation in SMAD4 gene.
  • the modified cytotoxic lymphocyte cell results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of:
  • cytotoxic effectors e.g., granzymes and perforin
  • IL15/IL2 receptor chains essential for NK cell proliferation in response to pro-proliferative cytokines
  • CD11 a, CD18 and CD49d integrins important for NK cell extravasation from blood vessels into tumors and required for tumor cell recognition and cytotoxic synapsis formation
  • anti-HER2 antibodies tumor antigen-targeted monoclonal antibodies therapy
  • CAR chimeric antigen receptor
  • the cytotoxic lymphocyte cell of the present invention can be modified by means of genetic engineering to render the SMAD4 gene non-functional or impaired.
  • This genetic manipulation can be achieved through various methods and techniques known in the art, such as CRISPR-Cas9, homologous recombination, RNAi, zinc finger nucleases (ZFNs), or TAL effector nucleases (TALENs).
  • An exemplary method is detailed in EXAMPLE 4.
  • another aspect of the present invention relates to a method for producing a modified human cytotoxic lymphocyte cell for adoptive immunotherapy comprising genetically inactivating SMAD4 gene of a human cytotoxic lymphocyte cell, resulting in a modified cell with inhibited SMAD4 expression and resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity.
  • SMAD4 gene includes SMAD4 gene (e.g., SMAD4 gene coding region, or SMAD4 exons, introns or untranslated regions - UTRs) and related regulatory sequences (e.g., SMAD4 promoter, SMAD4 enhancer, SMAD4 regulator).
  • SMAD4 gene e.g., SMAD4 gene coding region, or SMAD4 exons, introns or untranslated regions - UTRs
  • related regulatory sequences e.g., SMAD4 promoter, SMAD4 enhancer, SMAD4 regulator.
  • the regulatory sequences can be upstream polynucleotide sequences (5’ non coding sequences), within, or downstream (3’ non-translated sequences) of SMAD4 gene, activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translational initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like.
  • the genetic inactivation of SMAD4 gene is in a SMAD4 coding region, SMAD4 intron, SMAD4 UTRs, SMAD4 regulatory sequences, SMAD4 promoter, SMAD4 enhancer or SMAD4 regulator.
  • the genetic inactivation is in a SMAD4 coding region. More particularly, the SMAD4 coding region is in exon 5 or exon 10, more particularly exon 5.
  • the genetic inactivation is performed by DNA cleavage.
  • the DNA cleavage is performed using at least one targeted nuclease.
  • the DNA cleavage is performed by introducing into the cell at least: i) a targeted nuclease; and ii) a single-guide ribonucleic acid (sgRNA) targeting the SMAD4 gene of the cell.
  • a targeted nuclease e.g., a single-guide ribonucleic acid (sgRNA) targeting the SMAD4 gene of the cell.
  • sgRNA single-guide ribonucleic acid
  • the genetic inactivation can be performed by introducing a knock-in into a suitable position in the SMAD4 amino acid sequence, by introducing into the cell at least: i) a targeted nuclease; ii) a single-guide ribonucleic acid (sgRNA) targeting the SMAD4 gene of the cell; and iii) a donor DNA sequence.
  • a knock-in into a suitable position in the SMAD4 amino acid sequence, by introducing into the cell at least: i) a targeted nuclease; ii) a single-guide ribonucleic acid (sgRNA) targeting the SMAD4 gene of the cell; and iii) a donor DNA sequence.
  • sgRNA single-guide ribonucleic acid
  • Genetic inactivation can be achieved through knock-in, e.g., by adding a sequence with a stop codon or a sequence that induces a reading frame shift in the gene. Therefore, in a particular embodiment, the donor DNA sequence encodes a stop codon. Particularly, the genetic inactivation is performed by homology-directed repair (HDR).
  • HDR homology-directed repair
  • Genetic inactivation can also be achieved through prime editing (PE), using e.g., Cas9 and reverse transcriptase, or base editing (BE), using e.g., a base editor.
  • PE prime editing
  • BE base editing
  • the targeted nuclease is selected from the group consisting of a CRISPR/Cas nuclease system, a TAL-effector domain nuclease (TALEN), a zinc finger nuclease (ZFN), a meganuclease, or a variant thereof.
  • the targeted nuclease is a CRISPR/Cas nuclease system.
  • the CRISPR-Cas system has been used in genome editing in prokaryotic and eukaryotic cells.
  • an expression vector or a combination of vectors comprising a DNA fragment encoding for a single guide RNA (sgRNA) and a CRISPR-associated (Cas) protein (e.g., Cas9) or a gene encoding the protein or the protein are introduced into host cells.
  • the single guide RNA (sgRNA) functions to guide Cas protein to a specific genomic location enabling targeted genome editing.
  • a "CRISPR-Cas system” is meant any of the various CRISPR-Cas classes, types and subtypes.
  • the targeted nuclease is a CRISPR-associated protein.
  • the targeted nuclease can be in its natural form (e.g., spCas9) or a form modified through genetic engineering (e.g., xCas9, enCas12, SpC, SpRY).
  • the targeted nuclease is a Cas protein.
  • Cas protein or “Cas enzyme” refers to a Cas protein derived from any species, subspecies, or strain of bacteria that encodes the Cas protein of interest, as well as variants and orthologs of the particular Cas protein in question.
  • the Cas proteins and their cognate crRNAs or tracrRNAs can either be directly isolated and purified from bacteria, or synthetically or recombinantly produced, or can be delivered using a construct encoding the protein.
  • the targeted nuclease can be selected from the group consisting of Cas9, spCas9, Cas12, Cas12a, Cas13, Cas14, CasX, C2c6, Cas3, Cas8, Cas6, Cas7, Cas5, Cas11 and Csm/Cmr.
  • the targeted nuclease is selected from the group consisting of Cas9, spCas9, and Cas12a.
  • Cas9 protein refers to wild-type proteins derived from Type II CRISPR- Cas9 systems, modifications of the Cas9 proteins, variants of Cas9 proteins, Cas9 orthologs, and combinations thereof.
  • Cas9 proteins can be derived from any of various bacterial species having genomes that encode such proteins, such as Streptococcus pyogenes Cas9 protein, termed “Spy- Cas9”.
  • the targeted nuclease is Cas9.
  • the Cas9 gene can be from Streptococcus pyogenes, or other organisms.
  • the Cas9 comprises SEQ ID NO: 12.
  • the sgRNA guides the Cas enzyme to the target location within the DNA.
  • the sgRNA is a synthetic RNA molecule that is engineered to have a sequence complementary to the target DNA site, this target DNA site known as the "protospacer".
  • the complementary part of the sgRNA that aligns with the protospacer is called the "spacer”.
  • the sgRNA is mainly composed of two sequences, the crRNA (CRISPR RNA) and the tracrRNA (trans-activating CRISPR RNA).
  • the crRNA provides target recognition since it contains the spacer sequence, while the tracrRNA facilitates the processing and functional binding with the Cas enzyme.
  • the sgRNA when the sgRNA is introduced into a cell along with Cas, the sgRNA guides the Cas protein to the specific protospacer site in the DNA. At that location, the Cas enzyme induces a double-strand break, initiating DNA repair that can lead to genetic modifications.
  • the sgRNA sequence can be optimized to reduce the risk of off-target mutations.
  • the protospacer sequence comprises the SMAD4 coding region.
  • the SMAD4 coding region comprises a sequence from exon 5, exon 6, exon 7, exon 9, exon 10, or exon 12.
  • the SMAD4 coding region comprises a sequence from exon 5 or exon 10, and more particularly from exon 5.
  • the protospacer sequence comprises a sequence that corresponds to the DNA binding domain of the SMAD4 protein. In another embodiment, the protospacer sequence comprises a sequence of SMAD4 that corresponds to the SMAD2/3 binding domain of the SMAD4 protein.
  • the sgRNA targeting the SMAD4 gene of the cell comprises a sequence (i.e., spacer or crRNA) which is complementary to a target SMAD4 gene (i.e., complementary to the protospacer, which can be part of a SMAD4 gene or regulatory sequence).
  • the sgRNA targeting the SMAD4 gene of the cell is configured to target one SMAD4 gene selected from a SMAD4 coding sequence, a SMAD4 enhancer region, or a SMAD4 promoter region.
  • the sgRNA targeting the SMAD4 gene of the cell can be about 20 nucleotides in length and selected to have at least 80% of sequence identity to the SMAD4 gene based on the total length of the sgRNA sequence.
  • the sgRNA targeting the SMAD4 gene of the cell is configured to provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of the target SMAD4 gene.
  • the sgRNA targeting the SMAD4 gene of the cell comprises a crRNA comprising a sequence selected from SEQ ID NO: 1-10, particularly SEQ ID NO: 1 .
  • Exemplary crR- NAs that can be used for the inactivation of SMAD4 gene are shown in Table 1. Therefore, in an embodiment, the sgRNA targeting the SMAD4 gene of the cell comprises a sequence selected from SEQ ID NOs: 1-10, or a sequence having at least 80% of sequence identity to any of the sequences SEQ ID NOs: 1-10.
  • the sgRNA targeting the SMAD4 gene of the cell comprises SEQ ID NO: 1 or SEQ ID NO: 2, and more particularly SEQ ID NO: 1.
  • crRNA sequences (spacer sequence) targeting the SMAD4 gene.
  • the sgRNA targeting the SMAD4 gene of the cell comprises a tracrRNA comprising a sequence which is complementary to a targeted nuclease (e.g., Cas9 enzyme).
  • the tracrRNA sequence is SEQ ID NO: 11. Therefore, in an embodiment, the sgRNA targeting the SMAD4 gene of the cell comprises SEQ ID NO: 11 , or a sequence having at least 80% of sequence identity to the sequence SEQ ID NO: 11 . In a particular embodiment, the sgRNA targeting the SMAD4 gene of the cell comprises SEQ ID NO: 11 .
  • the sgRNA targeting the SMAD4 gene of the cell comprises the crRNA and tracrRNA sequences as described herein (e.g., SEQ ID NO: 1-11).
  • the sgRNA targeting the SMAD4 gene of the cell comprises a sequence selected from SEQ ID NOs: 1-11 , or a sequence having at least 80% of sequence identity to any of the sequences SEQ ID NOs: 1-11.
  • the targeted nuclease is able to selectively inactivate by DNA cleavage, by effecting a single-strand break and/or double-strand break (DBS) within the SMAD4 gene, resulting in at least one permanent deletion or insertion of the SMAD4 gene, and thereby reducing or inhibiting the expression of SMAD4.
  • DBS double-strand break
  • a particular embodiment relates to a method for producing a modified human cytotoxic lymphocyte cell for adoptive immunotherapy comprising genetically inactivating SMAD4 gene of a human cytotoxic lymphocyte cell by DNA cleavage, by introducing into the cell at least: i) a targeted nuclease; and ii) a single guide ribonucleic acid targeting the SMAD4 gene of the cell, wherein the targeted nuclease is able to selectively inactivate by DNA cleavage, by effecting a singlestrand break and/or double-strand break within the SMAD4 gene, resulting in at least one permanent deletion or insertion of the SMAD4 gene, and thereby reducing or inhibiting the expression of SMAD4, and wherein the cell results in a modified cell with inhibited SMAD4 expression and resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity.
  • the various components for use in the methods can be produced by synthesis or e.g., using expression cassettes encoding the targeted nuclease and/or the sgRNA targeting the SMAD4 gene of the cell.
  • the various components can be produced recombinantly in a host cell. These components can be present on a single cassette or multiple cassettes, in the same or different constructs.
  • Suitable replicating vectors will contain a replicon and control sequences derived from species compatible with the intended expression host cell.
  • polynucleotides encoding one or more of the various components are operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter.
  • Transformed host cells are cells that have been transformed or transfected with the vectors constructed using recombinant DNA techniques. General methods for construction of expression vectors are known in the art.
  • the targeted nuclease (e.g., Cas9) is introduced into the cell as a polynucleotide (i.e., DNA or mRNA) or as a protein, particularly as a protein.
  • the sgRNA targeting the SMAD4 gene of the cell is introduced into the cell as a plasmid (i.e., DNA) or as RNA, particularly as RNA.
  • the targeted nuclease and the sgRNA targeting the SMAD4 gene of the cell are introduced into the cell as a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the ribonucleoprotein complex is prepared by combining the targeted nuclease as protein (e.g., Cas9) and the prepared sgRNA targeting the SMAD4 gene of the cell at 1 :3 molar ratio for about 20 min at room temperature.
  • the ribonucleoprotein complex comprises at least one crRNA selected from the group consisting of SEQ ID NO: 1-10, a tracrRNA comprising SEQ ID NO: 11 , and a Cas9 comprising SED ID NO: 12.
  • Such methods include, e.g., viral or bacteriophage infection, transfection, conjugation, electroporation, calcium phosphate precipitation, polyethyleneimine-mediated transfection, DEAE-dextran mediated transfection, protoplast fusion, lipofection, liposome-mediated transfection, particle gun technology, direct microinjection, and nanoparticle-mediated delivery.
  • the targeted nuclease and the sgRNA targeting the SMAD4 gene of the cell are introduced by transfection (e.g., nucleofection or electroporation), microinjection, or by means of viral vectors (e.g., lentivirus), particularly by nucleofection.
  • viral vectors e.g., lentivirus
  • Viral vectors can be selected from the group consisting of lentivirus, retrovirus, plasmids, and adeno-associated viruses (AAV).
  • the method for producing modified cytotoxic lymphocyte cells as defined above further comprises the steps of separation or purification of cells.
  • Cell purification can occur either before or after cell modification, depending on the specific therapy.
  • purification is performed before cell modification, the aim is to isolate the desired cell type from a mixed population, ensuring a highly enriched starting cells.
  • purification is conducted after cell modification, it allows for the selection of cells with desired traits after they have been processed (e.g., through genetic engineering). The choice between these approaches depends on the specific treatment strategy and clinical requirements, with each method tailored to the particular goals of the therapy.
  • Cell separation or purification can be performed using different techniques known in the art, such as, immunomagnetic separation, flow cytometry sorting, density gradient centrifugation, or positive and negative selection.
  • the cell separation is performed by immunomagnetic negative selection.
  • the cytotoxic lymphocyte cells e.g., NK cells
  • the cytotoxic lymphocyte cells are purified by negative selection using e.g., a NK-cell isolation kit.
  • the obtained cells are substantially purified for T cell content by selecting cells for expression of a marker selected from the group consisting of: a) CD3; b) CD4; c) CD8; and d) CD90.
  • T cells are selected based on the expression of the CD3 marker and optionally the expression of the CD8 and CD4 markers.
  • the obtained cells are substantially purified for NK cell content by selecting cells for expression of a marker selected from the group consisting of: a) CD56; b) CD57; c) KIR; and d) CD16.
  • NK cells are selected based on the expression of the CD56 marker and the lack of expression of the CD3 marker.
  • the process of cell separation or purification involves the use of two separate methods to ensure the isolation of the desired cells. This approach is adopted to enhance the purity and enrichment of the selected cells within the population. For example, negative selection can be employed to isolate the desired cell type, which can then be subsequently expanded to increase their numbers and prominence within the population.
  • a second step can involve positive selection, such as the use of specific antibodies to isolate the desired cell type with high purity.
  • the cell separation or purification comprises the use of two separation methods.
  • the method for producing modified cytotoxic lymphocyte cells as defined above comprises separation or purification of cells following cell modification.
  • Cell separation or purification can be done using different techniques known in the art, particularly, the cells are separated by selective pressure.
  • Selective pressure as an isolation technique after cell modification, involves subjecting the modified cells to an environment that favors the survival and growth of cells with specific traits or genetic modifications. By exposing the cells to conditions that selectively benefit the desired characteristics, such as resistance to a drug or the ability to target specific antigens, it becomes possible to isolate and expand cells that possess the intended properties.
  • EXAMPLE 4 and Figure 6 shows that from day 7 to day 14 the cells were cultured with TGF-p1 as a selective pressure for reducing the proliferation of non-engineered while enriching SMAD4 K0 NK cells. Therefore, in a particular embodiment, the separation step involves the use of TGF-p as a selective pressure. In a particular embodiment, the cells can be enriched by cultivation in the presence of TGF-p.
  • the method for producing the modified cytotoxic lymphocyte cells as defined above further comprises the step of expanding or propagating the cells.
  • Cell expansion can be applied in different stages, e.g., post-collection to expand the immune cells obtained from the patient or donor before modification, during the modification phase, or before patient infusion.
  • the cells of the present invention can be propagated in an in vitro culture system supported by growth factors IL- 2, IL-15, feeder cells, genetically modified feeder cells containing IL-15 transmembrane, CD137 ligand, or a combination thereof.
  • the cells can be expanded for about 10 4 folds.
  • an embodiment of the present invention relates to a method for producing a modified human cytotoxic lymphocyte cell for adoptive immunotherapy comprising genetically inactivating SMAD4 gene of a human cytotoxic lymphocyte cell, resulting in a modified cell with inhibited SMAD4 expression and resistant to immunosuppressive effects of TGF-p, thereby exhibiting cytotoxicity, the method further comprising: a) expanding or propagating cells from a sample of a subject in need of treatment or a healthy donor before genetically inactivating the SMAD4 gene; b) separating or purifying human cytotoxic lymphocyte cells from the sample or from the cells obtained in (a) before genetically inactivating the SMAD4 gene; c) expanding or propagating the cells obtained after genetically inactivating the SMAD4 gene; and/or d) separating or purifying the cells obtained after genetically inactivating the SMAD4 gene or obtained in (c) (e.g., with TGF-01 as a selective pressure).
  • the method for producing the modified cytotoxic lymphocyte cells does not involve modifying the germ line genetic identity of human beings, and it is not a method for treatment of the human or animal body by therapy.
  • a sgRNA targeting the SMAD4 gene of the cell as described herein e.g., a sgRNA comprising SEQ ID NO: 1-11.
  • the sgRNA targeting the SMAD4 gene of the cell comprises a sequence selected from SEQ ID NOs: 1-11 , or a sequence having at least 80% of sequence identity to any of the sequences SEQ ID NOs: 1-11.
  • kits comprising: a) at least a targeted nuclease as described herein (e.g., Cas9 nuclease or the polynucleotide encoding the Cas9 nuclease); and b) at least a sgRNA targeting the SMAD4 gene of the cell as defined herein (e.g., a sgRNA comprising SEQ ID NO: 1-11).
  • a targeted nuclease as described herein e.g., Cas9 nuclease or the polynucleotide encoding the Cas9 nuclease
  • a sgRNA targeting the SMAD4 gene of the cell as defined herein (e.g., a sgRNA comprising SEQ ID NO: 1-11).
  • Another aspect of the present invention relates to a modified human cytotoxic lymphocyte cell obtained by the methods provided herein.
  • the cytotoxic lymphocyte cell is modified by the methods described herein.
  • the cytotoxic cell is modified by genetic inactivation of SMAD4 gene, by introducing into the cell: i) at least a targeted nuclease; and ii) at least a sgRNA targeting the SMAD4 gene of the cell.
  • the cytotoxic lymphocyte cell is modified by genetic inactivation of SMAD4 gene, by introducing into the cell a Cas9 ribonucleoprotein complex as described herein.
  • the method of adoptive cell transfer is a therapeutic approach in immunotherapy that involves the isolation, manipulation, and subsequent infusion of a patient's own immune cells or engineered immune cells into the patient's body to treat a disease, typically cancer.
  • Adoptive cell transfer can be autologous or allogeneic (e.g., "off-the-shelf).
  • immune cells are collected from the patient's own body, typically from their blood or tumor tissue. These collected immune cells are modified, activated and expanded outside the patient's body, and reintroduced into the patient.
  • Allogeneic adoptive cell transfer involves the use of immune cells that are not derived from the patient's own body. Instead, these immune cells are typically obtained from healthy donors, which can be modified and expanded to enhance their effectiveness in targeting the patient's disease.
  • the modified cells can be stored by freezing them and preserving the viability of the cells until its use in a patient, known as "off-the-shelf therapy, which offers potential advantages in terms of accessibility and reduced turnaround time for treatment.
  • NK cell therapy is one area of adoptive cell transfer where the "off-the-shelf approach is particularly attractive, because NK cells have unique properties that make them relatively well- suited for allogeneic or "off-the-shelf use, as they e.g., have a lack of antigen-specific receptors, unlike T cells (i.e., TCRs), a limited expression of HLA molecules, and are part of the innate immune system with more general and immediate response to abnormal cells.
  • T cells i.e., TCRs
  • the method for producing the modified cytotoxic lymphocyte cells as defined above further comprises obtaining a sample (e.g., blood) from a subject in need of treatment or a healthy donor.
  • a sample e.g., blood
  • the sample is obtained through peripheral blood (e.g., standard venous blood draw), leukapheresis (to collect large quantities of white blood cells), umbilical cord blood (to collect hematopoietic stem cells and other immune cells), bone marrow aspiration (to collect hematopoietic stem cells and other immune cells), or induced pluripotent stem cell reprogramming.
  • the sample is obtained through leukapheresis.
  • the sample is from a subject in need of treatment (i.e., autologous treatment).
  • the sample is from a healthy donor (i.e., allogenic treatment or "off-the-shelf).
  • the sample is histological compatible to the subject in need of treatment (e.g., cells from a donor whose human leukocyte antigens (HLA) are acceptable matches to the patient's).
  • the sample is selected from the group consisting of: blood, peripheral blood, lymph organs, lymph fluids, an organ, a tissue (e.g., tumor infiltrating NK cells), a tumor, or a combination thereof.
  • Another aspect relates to a method of adoptive cell transfer comprising: a) obtaining a sample (e.g., blood) from a subject in need of treatment or a healthy donor; b) producing modified human cytotoxic lymphocyte cells with inhibited SMAD4 expression following the method for producing modified human cytotoxic lymphocyte cells as described above; c) optionally, administering a conditioning or preconditioning treatment to the subject in need of treatment (e.g., IL-2 treatment); and d) administering an effective amount of the cells obtained in (b) to the subject in need of treatment.
  • a conditioning or preconditioning treatment e.g., IL-2 treatment
  • This aspect can alternatively be formulated as a method of treating cancer in a subject in need thereof comprising the steps as described above.
  • the process can be simplified, primarily involving the defrosting of stored modified cells and subsequent cell administration. Initially, immune cells are collected from healthy donors and modified, after which they are cryopreserved, ensuring long-term storage and accessibility for future therapeutic use. When a patient requires treatment, the frozen cells are defrosted and directly administered, typically via intravenous infusion or a suitable delivery method. The expansion step may not be necessary in "off-the-shelf treatments, as the donor-derived cells are already available in sufficient quantities, streamlining the process and eliminating the complexities associated with extensive cell expansion and modification procedures.
  • a method of adoptive cell transfer and a method of treating cancer in a subject in need thereof comprising: a) thawing isolated modified SMAD4 human cytotoxic lymphocyte cells according to the invention; b) optionally, administering a conditioning or preconditioning treatment to the subject in need of treatment (e.g., IL-2 treatment); and c) administering the cells obtained in (a) to the subject in need of treatment.
  • a conditioning or preconditioning treatment e.g., IL-2 treatment
  • the cells produced in any of aforementioned processes can be preserved (e.g., cryo preserved) for future use in the event of e.g., cancer recurrence in the subject.
  • the methods of adoptive cell transfer or treating cancer as defined above comprise administering a conditioning or preconditioning treatment to the subject to prepare the subject for receiving the engineered cells.
  • the conditioning or preconditioning treatment can be administered to the subject before, during and/or after the administration of modified or expanded immune cells. This treatment aims to establish a more conducive internal environment within the patient's body, creates space within the patient's immune system, and can also improve the efficacy of the therapeutic cells.
  • Non-limiting examples of preconditioning or conditioning treatment are lymphocyte depletion, total body irradiation, cytokine treatment (e.g., IL-2 that helps activate and expand NK or T cells), targeted antibodies (e.g., rituximab), or tumor microenvironment modulators (e.g., checkpoint inhibitors).
  • the conditioning treatment is a cytokine treatment.
  • the cytokine treatment consists on the administration of IL-2.
  • the methods of adoptive cell transfer or treating cancer as defined above comprise administering an effective amount of the obtained cells (that can be in the form of a pharmaceutical composition) to the subject in need thereof.
  • the effective amount of the cells is between about 1x10 5 to about 1x10 7 cells/kg of the human subject.
  • the administration can take various forms as described above, such as intravenous infusion, intratumoral injection, or intraperitoneal injection, depending on the specific therapy and its intended site of action.
  • the cells are administered by intravenous infusion to a subject.
  • the cells are directly injected into a tumor or to a specific tissue or area surrounding a tumor of a subject. The combination of injection and infusion can be useful to treat an original tumor at one location, such as its primary site and one or more metastatic tumors in different locations in a subject.
  • the subject is administered a cell population that encompasses both modified cells as described herein, as well as non-modified cells.
  • a cell population that encompasses both modified cells as described herein, as well as non-modified cells.
  • a cell population that encompasses both modified cells as described herein, as well as non-modified cells.
  • a fraction of cells e.g., 30%
  • the resulting cell population can exhibit enhanced therapeutic effects by harnessing the synergistic interactions and functions of the entire cell population. Therefore, the method for producing the modified cytotoxic lymphocyte cells described in the present invention can result in a cell population wherein a significant proportion of cells comprise an inactivating mutation in SMAD4 gene.
  • modified human cytotoxic lymphocyte cell has been defined to modify a cell but applies likewise to modify a set of cells derived from a sample.
  • product obtained from the methods described above is defined as modified cell(s), or as a cell population comprising modified and non-modified cells. Therefore, another aspect of the invention is a method for producing a cell population comprising modified cytotoxic lymphocyte cells following the steps described above.
  • another aspect of the present invention relates to a cell population comprising modified human cytotoxic lymphocyte cells as defined in the first aspect of the invention, wherein the cell population is characterized by: a) an allelic frequency of between about 10% and 90% of modified SMAD4 gene cytotoxic lymphocyte cells; b) a genotype frequency of between about 10% and 90% of SMAD4-/- and/or SMAD4+/- cytotoxic lymphocyte cells; and/or c) an off-target rate of less than 1 %.
  • EXAMPLE 4 and Figure 16 of the present invention show the allelic and genotype frequencies and safety evaluation of SMAD4 knock out in human NK cells performed by CRISPR/Cas9 nucleofection, and detail the obtained allelic and genotype frequencies.
  • allelic frequencies of edited sequences in exon 5 were of 71 %, 57% and 74% in the absence of TGF-p treatment, respectively increasing to 79%, 72% and 79% when TGF-p was included along the expansion of nucleofected NK cells.
  • the allelic frequency indicates the percentage of mutated cells with an out-of-frame (OUT) mutation (e.g., an insertion or deletion) which will not lead to a functional protein.
  • OUT out-of-frame
  • the cell population is characterized by an allelic frequency of between about: 10% and 90%, or 20% and 90%, or 30% and 90%, or 40% and 90%, or 50% and 90%, or 60% and 90%, or 70% and 90%, or 80% and 90%, or 40% and 80%, or 50% and 80%, or 70% and 80% of modified SMAD4 gene cytotoxic lymphocyte cells.
  • the cell population comprises about: 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of cytotoxic lymphocyte cells comprising an inactivating mutation in SMAD4 gene.
  • NK cells were SMAD4-/- 30%, 46% and 26% of NK cells were SMAD4+/- and only 3%, 10% and 2% of NK cells remained SMAD4+/+ upon engineering.
  • TGF-p along NK cell expansion reduced to almost inexistent SMAD4+/+ NK cells while increasing the proportion of SMAD4- /- NK cells.
  • the cell population is characterized by a genotype frequency of at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of SMAD4-I- cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of SMAD4+/- cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of at least about: 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of SMAD4-I- cytotoxic lymphocyte cells; at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of SMAD4+I- cytotoxic lymphocyte cells; and at least about: 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of between about: 10% and 90% of SMAD4-I-, 10% and 90% of SMAD4+I-, and 1 % and 90% of SMAD4+/+ cytotoxic lymphocyte cells; or 20% and 80% of SMAD4-/-, 20% and 80% of SMAD4+/-, and 1 % and 50% of SMAD4+/+ cytotoxic lymphocyte cells; or 40% and 75% of SMAD4-/-, 25% and 50% of SMAD4+/-, and 1 % and 15% of SMAD4+/+ cytotoxic lymphocyte cells; or 43% and 72% of SMAD4-/-, 26% and 46% of SMAD4+/-, and 2% and 10% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of between about: 10% and 90%, or 20% and 90%, or 30% and 90%, or 40% and 90%, or 50% or 90%, or 40% and 70% of SMAD4-/- and/or SMAD4+/- cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of between about 10% and 90% of SMAD4- /-, and between about 1 % and 90% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of between about 10% and 90% of SMAD4+/-, and between about 1 % and 90% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of at least about 30% of SMAD4-/-, at least about 20% of SMAD4+/- and up to about 10% of SMAD4+/+ cytotoxic lymphocyte cells.
  • the cell population is characterized by a genotype frequency of at least about 40% of SMAD4-/- and at least about 30% of SMAD4+/- cytotoxic lymphocyte cells.
  • the cell population is characterized by an off-target rate of less than 1 %.
  • the off-target events are in CREBBP, DCTN5 and NUFIP2 genes.
  • the cell population is characterized by: a) a genotype frequency of between about 10% and 90% of SMAD4-/- and/or SMAD4+/- cytotoxic lymphocyte cells; and b) an off-target rate of less than 1 %.
  • the cell population is characterized by: a) a genotype frequency of between about 20% and 70% of SMAD4-/- and/or SMAD4+/- cytotoxic lymphocyte cells; and b) an off-target rate of less than 1 %.
  • the cell population is typically administered to the subject through intravenous infusion or intra- tumoral injection.
  • the cell population is mixed with pharmaceutically acceptable excipients to ensure safe and effective delivery.
  • these excipients help maintain cell viability during the storage, handling, and delivery process and also, in the context of an off-the-shelf treatment, excipients are employed to preserve the cells, such as cryoprotectants that enable freezing and long-term storage, thus preserving the cells' viability until they are needed for subject treatment.
  • compositions comprising a modified cytotoxic lymphocyte cell or a cell population as defined herein and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable is art-recognized, and includes excipients, compounds, materials, compositions, carriers, vehicles and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable ben- efit/risk ratio.
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.
  • Non-limiting examples of pharmaceutically acceptable excipients that a solution comprising the cell population of herein can include are: buffering agents to maintain pH such as phosphate-buffered saline (PBS) or 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES); saline solutions for isotonicity such as normal saline (0.9% sodium chloride solution) or Ringer's solution; cryoprotectants for freezing and thawing such as dimethyl sulfoxide (DMSO) or glycerol; anticoagulants to prevent clotting such as heparin or citrate-phosphate-dextrose (CPD); preservatives for shelf-life extension such as gentamicin or thimerosal; stabilizers for maintaining cell integrity such as human serum albumin (HSA) or polyvinylpyrrolidone (PVP); diluents for concentration adjustment such as cell culture media or sterile water for injection; carrier proteins to aid in the
  • the pharmaceutically acceptable excipient is a cryoprotectant selected from DMSO or glycerol. In another particular embodiment, the pharmaceutically acceptable excipient is serum.
  • the pharmaceutical composition is cryopreserved. It can be cryopreserved for about: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 2 years, or more than 3 years before thawing and its use.
  • the viability of the cells is at least: 30%, 40%, 50%, 60%, or 70% as determined by a suitable assay known in the art. Viability can be determined, e.g., using trypan blue exclusion.
  • the pharmaceutical composition can be administered to a subject by means of any suitable administration route, e.g., via injection, such as subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intraperitoneally, intramedullary, intratumorally, intranodally, or by infusion.
  • the pharmaceutical composition is administered intravenously.
  • the pharmaceutical composition is directly injected into a tumor or to a specific tissue or area surrounding a tumor of a subject.
  • the combination of injection and infusion can be useful to treat an original tumor at one location, such as its primary site and one or more metastatic tumors in different locations in a subject.
  • the pharmaceutical composition is administered by intravenous infusion over about: 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, or 5 hours.
  • the rate of infusion can vary with the number of cells being infused to the subject.
  • the pharmaceutical composition can be in the form of a liquid solution or suspension.
  • the pharmaceutical composition can also be in the form of powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • Solutions or suspensions used for intravenous administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzoyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made glass or plastic.
  • the dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and particular lymphocytes being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.
  • the pharmaceutical composition comprises a an effective amount of the obtained cell population.
  • the total number of cells can be administered in a single bolus dose, or can be administered in two or more doses, such as one or more days apart.
  • the amount of compound administered will depend on the potency of the specific cell composition, the disease being treated and the route of administration.
  • one or more doses of the pharmaceutical composition are administered. If two or more doses of the pharmaceutical composition are administered, the duration between the administrations should be sufficient to allow time for propagation of the cells in the individual. In specific embodiments the duration between doses is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 or more weeks.
  • the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the present invention is an immunotherapy agent.
  • the immunotherapy agent is used for adoptive cell therapy.
  • the examples of the present invention provide evidence of the anti-tumor activity of the modified cytotoxic lymphocyte cells and the cell population described herein. Further, it has been demonstrated that these cells or population are effective against a wide range of cancer types, particularly TGF-beta-rich cancers, including solid and hematological cancers such as colorectal cancer, breast cancer and leukemia.
  • Another aspect relates to a modified cytotoxic lymphocyte cell, a cell population, or a pharmaceutical composition as defined herein, for use as a medicament.
  • Another aspect relates to a modified cytotoxic lymphocyte cell, a cell population, or a pharmaceutical composition as defined herein, for use as an immunotherapy agent.
  • An aspect of the present invention also relates to a modified cytotoxic lymphocyte cell, a cell population, or a pharmaceutical composition as defined herein, for use in the treatment of a cancer.
  • the invention also encompasses a method for treating a cancer (e.g., colorectal cancer, breast cancer or leukemia) in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a modified cytotoxic lymphocyte cell, a cell population, or a pharmaceutical composition as defined herein.
  • a cancer e.g., colorectal cancer, breast cancer or leukemia
  • the cancer is a TGF-beta-rich cancer.
  • the cancer can be, but it is not limited to breast cancer, lung cancer, colorectal cancer, colon cancer, rectal cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, stomach cancer, esophageal cancer, thyroid cancer, brain tumors, skin cancer (melanoma, squamous cell carcinoma, basal cell carcinoma), sarcomas, osteosarcoma, testicular cancer, adrenal gland tumors, neuroendocrine tumors, head and neck cancers, gastrointestinal stromal tumors, mesothelioma, thymoma, Wilms tumor, retinoblastoma, rhabdomyosarcoma, teratoma, multiple myeloma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndromes, myeloprolifer
  • the cancer is a solid tumor.
  • solid tumors include, but are not limited to, breast cancer, lung cancer, colorectal cancer, colon cancer, rectal cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, stomach cancer, esophageal cancer, thyroid cancer, brain tumors, skin cancer (melanoma, squamous cell carcinoma, basal cell carcinoma), sarcomas, osteosarcoma, testicular cancer, adrenal gland tumors, neuroendocrine tumors, head and neck cancers, gastrointestinal stromal tumors, mesothelioma, thymoma, Wilms tumor, retinoblastoma, rhabdomyosarcoma, teratoma, and multiple myeloma.
  • the cancer is an hematologic cancer.
  • hematologic cancer includes, but is not limited to, leukemia, lymphoma, multiple myeloma, myelodysplastic syndromes, myeloproliferative neoplasms, erythroleukemia, pure red cell aplasia, and hemophagocytic lympho- histiocytosis.
  • the cancer is a lymphoma (e.g., Hodgkin's lymphoma or Non-Hodgkin lymphoma).
  • the cancer is leukemia.
  • the leukemia is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia.
  • the leukemia is acute lymphoblastic leukemia.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, primary melanoma, glioblastoma, leukemia, and lymphoma.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, breast cancer, and leukemia.
  • the cancer is colorectal cancer.
  • the cancer is colon cancer.
  • the cancer is breast cancer.
  • the breast cancer is selected from the group consisting of ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), inflammatory breast cancer (IBC), hormone receptor-positive breast cancer (HR+) (e.g., estrogen receptor (ER)+/progesterone receptor (PR)+, ER+/PR-, ER- /PR+), HER2-positive breast cancer (HER2+ or ERBB2+) (e.g., ER+/PR+/HER2+, ER-/PR-/HER2+), triple-negative breast cancer (TNBC) (i.e., ER-/PR-/HER2-), Paget's disease of the breast, angiosarcoma, phyllodes tumor, and metastatic breast cancer.
  • the breast cancer is HER2-posi- tive breast cancer.
  • TGF-p tumor growth and suppressing the immune system's ability to target cancer cells.
  • Some cancers that are known to express high levels of TGF-p include, but are not limited to, pancreatic cancer, lung cancer, breast cancer, colorectal cancer, glioblastoma, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), and skin cancer (e.g., melanoma).
  • the cancer is metastatic or advanced cancer.
  • the cancer is characterized by high levels of TGF-p expression.
  • the cancer is characterized by the presence of a tumor microenvironment with a high abundance of NK cells.
  • the cancer is selected from the group consisting of pancreatic cancer, lung cancer, breast cancer, colorectal cancer, glioblastoma, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), and skin cancer (e.g., melanoma).
  • the cancer is selected from the group consisting of pancreatic cancer, breast cancer, colorectal cancer, glioblastoma, ovarian cancer, prostate cancer, liver cancer, and kidney cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, glioblastoma, ovarian cancer, and liver cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, glioblastoma, ovarian cancer, liver cancer, and leukemia (e.g., acute lymphoblastic leukemia).
  • the treatment of cancer is characterized by the administration of the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the invention which are resistant to TGF-p inhibitory effects present in the tumor microenvironment, thereby preserving their anti-tumor function.
  • the administration of the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the invention results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of:
  • the administration of the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the invention results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of: - Tumor regression;
  • the invention is focused on human applications, but the subject can also be a different mammalian subject for veterinary purposes, e.g., domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like). Therefore, in an embodiment, the subject is a human. In another embodiment, the subject is a mammal. In a particular embodiment, the mammal is a domestic animal, a farm animal or a laboratory animal.
  • the modified cytotoxic lymphocyte cell, the cell population and the pharmaceutical composition described herein can be administered in combination with one or more other therapeutic or biologically active agents or treatments (e.g., chemotherapy, a cancer drug, a checkpoint inhibitor, a NK cell engager, a cytotoxic agent, a cytokine, or radiotherapy). It is understood that the administration of the combination can be formulated for a separate, sequential or concomitant administration, or in a mixture in a single pharmaceutical composition.
  • EXAMPLES 12 and 13 demonstrate that the combination of SMAD4 K0 NK cells with other therapeutic treatments, such as anti-HER2 antibodies and CAR cells, respectively, exhibits an enhanced anti-tumor effect.
  • the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the present invention is combined with one or more therapeutic agent or treatment.
  • therapeutic agents are described hereinafter.
  • Another aspect relates to a combination
  • a combination comprising the modified cytotoxic lymphocyte cell, the cell population or the pharmaceutical composition of the present invention, and one or more therapeutic agent or treatment for a separate, sequential or concomitant use, or in a mixture in a single pharmaceutical composition.
  • the combination is for use as a medicament, particularly, for use in the treatment of a cancer.
  • the other therapeutic agent or treatment is an anti-tumor agent, e.g., a chemotherapeutic agent, a targeted therapy agent (e.g., monoclonal antibody such as trastuzumab or bevacizumab), an immunotherapy agent (e.g., anti-PD1 , anti-PDL1 , anti-CTLA4 checkpoint inhibitors), a tumor antigen-specific therapeutic antibody, an antibody drug conjugate, a cytotoxic agent, a NK engager, a cytokine treatment, radiotherapy, bone marrow transplantation, gene therapy, surgery, or a combination thereof.
  • a chemotherapeutic agent e.g., a targeted therapy agent such as trastuzumab or bevacizumab
  • an immunotherapy agent e.g., anti-PD1 , anti-PDL1 , anti-CTLA4 checkpoint inhibitors
  • a tumor antigen-specific therapeutic antibody e.g., an antibody drug conjugate, a cytotoxic agent,
  • the anti-tumor agent is a targeted therapy agent.
  • the targeted therapy agent is a monoclonal antibody.
  • monoclonal antibodies are: trastuzumab, pertuzumab, rituximab, cetuximab, bevacizumab, nivolumab, pembroli- Kursab, ipilimumab, daratumumab, elotuzumab, atezolizumab, tositumomab, conatumumab, ta- fasitamab, and margetuximab.
  • the monoclonal antibody is trastuzumab, pertuzumab, or a combination thereof.
  • the other therapeutic agent is a NK cell engager (NKCE).
  • NKCEs such as BiKE (bispecific killer cell engager) or TriKE (trispecific killer cell engager) are a class of antibodybased therapeutics. By bridging NK and tumor cells, NKCEs activate NK cells and lead to tumor cell lysis.
  • Non-limiting examples of NKCE are those targeting CD16a, NKG2D, Nkp30, Nkp46, Nkp80, NKG2C/CD94, KIR2DS/KIR3DS, CD160, DNAM-1 , 2B4, IL-2/IL-15, PD-1 , NKG2A, TIGIT, TIM-3, KIR2DL/KIR3DL, or CD96.
  • the NKCE is an NKp46 NK cell engager.
  • Non-limiting examples of chemotherapeutic agents are: a bifunctional alkylator (e.g., cyclophosphamide, mechlorethamine, chlorambucil or melphalan); a monofunctional alkylator (e.g., dacarbazine (DTIC), nitrosoureas or temozolomide); an anthracycline (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone or valrubicin); a taxane (e.g., paclitaxel, docetaxel, Nab-paclitaxel or taxotere); an epothilone (e.g., patupilone, sagopilone or ixabepilone); deacetylase inhibitor (e.g., vori- nostat or romidepsin); an inhibitor of topoisomerase I (e.g.,
  • Suitable checkpoint inhibitors can comprise anti-PD1 antibodies (such as pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab), anti-PD-L1 antibodies, anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen, also known as CD152) antibodies, anti-LAG3 (lymphocyte activation gene-3) antibodies, anti-TIM-3 (T cell immunoglobulin and mucin domain-3) antibodies, or a combination thereof.
  • anti-PD1 antibodies such as pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab
  • anti-PD-L1 antibodies anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen, also known as CD152) antibodies
  • anti-LAG3 lymphocyte activation gene-3) antibodies
  • anti-TIM-3 T cell immunoglobulin and mucin domain-3) antibodies, or a combination thereof.
  • Suitable therapeutic agents can comprise anti-CD19 antibodies, anti-CD20 antibodies such as tositumomab, cytokines such as interleukins, interferon-a 2a (INF-a 2a), interferon-a (INF-a), granulocyte colony stimulating factor (G-CSF) (also known as filgrastim), T cell receptor (TCR), chimeric antigen receptor or chimeric antigen T cell receptor (CAR-T), chimeric antigen NK cell receptor (CAR- NK) or a combination thereof.
  • the therapeutic agent is a chimeric antigen NK cell receptor (CAR-NK), particularly CAR19-NK cells.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • Patient #1 was diagnosed as harboring a c.1349_1376 (p.Ala460Glyfs*43) duplication in heterozygosis which induce a premature stop codon in SMAD4 gene.
  • Patient #2 carried a c.403C>T (p.Arg135*) (rs377767326) SMAD4 mutation in heterozygosis which induce a premature stop codon, predicted to accelerate mRNA decay.
  • HCT116 (CCL-247): this cell line encodes for wt SMAD4 genes.
  • HT-29 (HTB-38): this cell line encodes for mutated SMAD4 genes.
  • - NK92 cell line (PTA-6967): a model for NK cell-based cellular immunotherapy in clinical development.
  • the human colorectal cancer cell lines HCT116 and HCT116-GFP + -Luciferase + stable transfectant and HT-29 were maintained in complete DMEM medium (Sigma-Aldrich) supplemented with penicil- lin/streptomycin (100 U/mL and 100 pg/mL, respectively, Invitrogen), sodium pyruvate (1 mM, Gibco) and 10% FBS (Gibco).
  • the B lymphoblastoid cell line, 8866 and the HLA class I -negative human erythroleukemic cell line, K562 were cultured in complete RPMI 1640 GlutaMAX.
  • the NK92 cell line was cultured in alpha-MEM (Sigma) supplemented with 10% fetal calf serum (Gibco), 10% Horse serum (Sigma), glutamine (2 mM, Gibco), sodium pyruvate (1 mM, Gibco), non-essential amino acids 1 x (Gibco), penicillin/streptomycin (100 U/mL and 100 pg/mL, respectively, Invitrogen), IL-2 (200 U/ml, Proleukin), 0.2 mM myoinositol, 0.002 mM folic acid, and 0.1 mM beta-mercaptoethanol.
  • SMAD4 KO was performed using ribonucleoprotein (RNP) complex in both NK92 and primary NK cells.
  • RNP ribonucleoprotein
  • PCR polymerase chain reaction
  • Protospacer sequences targeting SMAD4 gene were identified using the CRISPRscan algorithm and synthesized by IDT. Two gRNAs targeting SMAD4 exon 5 and 10 were used, respectively: crRNAI- Exon 5: GTCGATGCACGATTACTTGG (SEQ ID NO: 1), crRNA2-Exon 10: AACTCTGTACAAA- GACCGCG (SEQ ID NO: 2). Cas9 ribonucleoprotein (RNP) complexes were prepared according to the manufacturer's instructions.
  • sgRNAs were ensembled by incubating the specific crRNA and a fluorescently-labeled tracrRNA-ATTO550: 5'-ATTO-AGCAUAGCAAGUUAAAAU- AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU 3' (SEQ ID NO: 11), (IDT) at 1 :1 molar ratio at 95°C for 5 min.
  • Cas9-gRNA RNP Prior to each nucleofection, Cas9-gRNA RNP were prepared by combining Cas9 enzyme (IDT) (Uniprot accession number Q99ZW2, SEQ ID NO: 12) and the prepared sgRNA at 1 :3 molar ratio for 20 min at room temperature.
  • IDTT Cas9 enzyme
  • NK92 cell line a mixture of sgRNA targeting SMAD4 exon 5 and 10 was used for RNP preparation.
  • NK92 cell engineering was done by nucleofection and sequential cloning. 1 million NK92 cells per cuvette were electroporated following the Neon® Transfection System (Invitrogen) protocol (Pulse voltage: 1250 V, 10 ms, 3 pulses, including an enhancer (IDT)). After 24h culture in medium without antibiotics, ATTQ550 positive cells (corresponding to sgRNAs) were sorted at the FACSAria III Cell Sorter (BD biosciences), cloned at 2 cells/well in 96 well plates and kept in complete culture media. SMAD4 reduction in selected clones was assessed by flow cytometry and western blot.
  • PBMC from healthy donors were co-cultured with irradiated 8866 cell line (40 Gy) at 3:1 ratio in complete RPMI 1640 GlutaMAX (Gibco).
  • NK cells were isolated by negative selection and electroporated at 1900 V, 20 ms, 1 pulse using the Neon® Transfection System kit. Electroporated NK cells were left 1 h at room temperature and cultured for 7 days in complete RPMI 1640 GlutaMAX (Gibco) complemented with IL-2 (200 U/ml) or IL-2 and TGF-01 (5 ng/ml) combination. Nucleofection efficiency (ATTO550 positive cells) was checked after 24h by Fortessa flow cytometer (BD Biosci- ence).
  • Anti-NKG2A (clone Z199, kindly provided by Dr. A. Moretta (University of Genoa) and anti-TNF- a infliximab (Remicade®, Janssen) were labelled with CF-Blue.
  • NK cells were stained for extracellular markers and then fixed and permeabilized with InvitrogenTM eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher). Cells were subsequently incubated with the unlabeled anti-SMAD4 antibody (Cell Signaling) for 1 hour at room temperature followed by an incubation with a polyclonal donkey anti-rabbit IgG Alexa Fluor®488. Flow cytometry data was acquired in a FACS-Fortessa flow cytometer (BD Bioscience) and analyzed with FlowJo X software (FlowJo LLC).
  • NK cell functional assays NK cell activation, cytokine production, degranulation, cytotoxicity by activated Caspase 3 assays were used as described below.
  • NK cell activation in anti-CD16-coated plates Purified NK cells were cultured in flat-bottom 96 well plates pre-coated with purified anti-CD16 mAb (5 pg/ml, clone KD1). TGF-01 (10 ng/ml, Peprotech) was added at 8 hours post activation. NK cells were maintained in culture for 5-6 days. In some experiments, the TGF-p-RI inhibitor SB-431542 (20 pM, Sigma-Aldrich) was added prior to activation and maintained along the culture.
  • NK cells were cultured in the presence of target cells (i.e. K562) at the indicated E:T ratio.
  • NK cell degranulation was analyzed by monitoring CD107a mobilization by flow cytometry after 4-hour-co- culture including the anti-CD107a-FITC (clone H4A3, BD Bioscience) and monensin (5 pg/ml, Sigma- Aldrich).
  • TNF-a infliximab-CF Blue
  • NK92 cells or primary expanded NK cells were cultured with HCT116 cells (at the indicated target: effector ratios) for 2 or 4h.
  • HCT116 Activated caspase 3 in HCT116 was analyzed by intracellular staining. HCT116 cells were gated based on forward and size scatter (FSC/SSC) and by CD45 exclusion. Basal levels of active caspase 3 were subtracted in each other condition.
  • FSC/SSC forward and size scatter
  • HCT 116 GFP-Luc cells were plated in ultra-low attachment 96 well plates (Costar), centrifuged (1000g x 10 minutes) and cultured for 72 hours.
  • Control- and SMAD4-eng' ⁇ - neered NK cells treated or not with TGF-01 were labeled with PKH26 dye (Sigma-Aldrich), according to the manufacturer’s instructions.
  • NK cells (100.000 cells) were co-cultured with spheroids. Images of the green (HCT116, GFP), red (NK; PKH26) and transmitted light channels were acquired at 6 and 24 hours on a Zeiss Cell Observer HS microscop, Z-slices 13.5 pm, total magnification 5x.
  • Control- and SMAD4-engineered NK cells were co-cultured with HCT116 spheroids for 1 hour.
  • Spheroid processing was done according to Miltenyi manufacturer’s instructions for "Immunostaining and clearing of stem cell-derived cerebral organoids for 3D imaging analysis”.
  • Fixed spheroids were stained with anti-CD45-Vio® R667 (10 pg/mL, Miltenyi Biotec), anti-Epcam-AlexaFluor® 488 (5 ug/mL, abeam) and Propidium Iodide (PI, 0.2 ug/mL, Miltenyi Biotec).
  • Specimens were cleared using tissue Clearing Kit and Imaging Solution (Miltenyi Biotec) and were scanned with the UltraMicroscope Blaze (Miltenyi Biotec). Samples were scanned using an objective lense 12x, 150 ms exposure, 2 pm step size in the transverse plane. 3D spheroid reconstruction was done using the Imaged software and the distance between the spheroid surface and each NK cell was analyzed with Imaris® software.
  • PBMC, NK cells, NK92, HCT1 16 or HT29 cell pellets were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitors [10mM p-glycerol-phosphate, 2mM Na3VO4, 5mM NaF, and 1 mM protease inhibitor cocktail Sigma (P8340, Sigma- Aldrich)] for 20 minutes on ice. Protein extracts were separated in a 10% polyacrylamide gel (SDS-PAGE) and transferred to PVDF membranes (Immobilon, Millipore).
  • Membranes were blocked with 5% milk in tris-buffered saline + 0.05% Tween 20 (TBST) or 10% bovine serum albumin for 1 hour and then incubated with the primary antibody O/N at 4 °C.
  • Specific antibodies used were: anti-Smad4 (46535, Cell Signaling, 1 :1000), anti-Smad7 (MAB2029, R&D Systems, 1 :1000), anti-Phospho-Smad2 (Ser465/467, 138D4, Cell Signaling, 1 :1000), anti-TGFp-RII (sc-17791 , Santa Cruz Biotechnology, 1 :100), anti-TIF1 gamma (A301-060A, 1 :1000, Bethyl Laboratories, Inc) and anti-p-actin (A5441 , Sigma-Aldrich).
  • Membranes were washed in TBST and incubated with a secondary antibody horseradish peroxidase (HRP) labelled anti-rabbit or anti-mouse antibodies (GE Healthcare, 1 :2500 except for p-actin 1 :20000) at RT for 1 hour.
  • HRP horseradish peroxidase
  • Membranes were developed with ECL substrate (SuperSignal West Pico or Femto Chemiluminescent Substrate, Thermofisher) and signal quantified by Imaged software.
  • RNA-Seq sample processing and analysis were performed by Pompeu Fabra University Genomic Core Facility, Barcelona.
  • Gene set enrichment analysis was performed by Mar Genomics Facility at IMIM, Barcelona.
  • RNA samples were pooled in equimolar proportions, amplified by qPCR with specific primers and sequenced in a NextSeq High output 2x75 cycles run (Illumina).
  • GSEA was performed by R package “clusterProfiler” package version 4.0.0 against the molecular signature database (MSigDB) collection “C7” version 7.5.1 .
  • CRISPRroots tool was used for off-target assessment in CRISPR-Cas9 edited cells.
  • DNA was isolated by QIAquick Gel Extraction Kit (Qiagen). A 2-step PCR was conducted on control- and SMAD4-engineered NK cells treated or not with TGF-01 .
  • a second PCR for introducing barcodes was done with general primers encoding barcodes, NEB P/N E7335S (Table 5). Protocol: initial denaturation for 3 minutes at 95 °C followed by 8 cycles of 98 °C for 20 seconds, 60 °C for 15 seconds, and 72 °C 20 seconds and a final elongation for 1 minute at 72 °C. PCR products were purified by QIAquick PCR Purification Kit (Qiagen).
  • NK cells were injected intratumorally once per week over three weeks, along with recombinant human IL-2 (rhlL-2, 200 lU/mice) and sustained by intraperitoneal rhlL-2 (20.000 lU/mice) every 3-4 days ( Figure 18 (A)).
  • rhlL-2 recombinant human IL-2
  • Figure 18 (A) 1.14 CD19 CAR-NK cells generation and cytotoxicity assay
  • CD19-CAR NK cell generation was performed at the Immunotherapy Department of the Hospital Clinic-IDIBAPS, Barcelona (Herrera L et al., 2019).
  • ScFv of anti-CD19 A3B1 antibody was cloned in frame with 4-1 BB and CD3z signaling domains in the pCCL lentiviral Vector (Castella M et al., 2018).
  • Lentivirus supernatant was generated by transient transfection of HEK293T cells, as previously described (Herrera L et al., 2019).
  • Control and SMAD4 K0 NK cells were restimulated by coculture with irradiated (40Gy) K562-CD137L-IL-15tmb-IL21tmb cell line (Oberoi P et al., 2020) at 1 :1 ratio.
  • NK cells were transduced with lentivirus supernatant in the presence of 6 pg/ml polybrene and MOI 20.
  • Cells were centrifuged at 2000rpm for 1 h at 37°C. After 24h, cells were cultured with 200 U/ml IL-2 in the presence or absence of TGF-p (1 OOng/ml) for 6 days.
  • Nalm6 cells is a B cell precursor leukemia cell line derived from the peripheral blood of a patient with acute lymphoblastic leukemia. Luciferase activity remaining, proportional to Luc+ alive cells, was measured by incubating with luciferin substrate (10 pg/ml, Sigma) for 5 minutes. Luminescence was measured at FB12 Tube Luminometer (Berthold Technologies).
  • EXAMPLE 2 Analysis of SMAD4 role in NK cells from healthy donors (NK SMAD4+/+ cells) and NK SMAD4 haploinsufficient cells from patients with juvenile polyposis syndrome (JPS) (NK SMAD4+/- cells) in the presence or absence of TGF-p.
  • JPS juvenile polyposis syndrome
  • TGF-p transforming growth factor beta
  • TGF-p1 up-regulated transcripts were grouped in biological pathways related to cell adhesion, chemotaxis and leukocyte differentiation ( Figure 2).
  • transcripts of NK cell-related transcription factors (BOMBS, PRDM1, TBX21) and effector molecules (GZMB, PRF1, IFNG, TN FA) were significantly down-regulated ( Figure 1 (B)) while transcripts associated to tumor homing (CXCR3, CXCR4) and tissue resident lymphocytes (ZNF683, ITGAE) were increased in TGF-p1- treated NK cells ( Figure 1 (C)).
  • TGF-p binds to TGF-pRII in NK cells unleashing a complex signaling cascade involving canonical as well as noncanonical signaling pathways.
  • Down-stream of canonical TGFp-RI, SMAD4- dependent and -independent (e.g. involving TIF-1 y or IkKa) transcriptional programs have been described.
  • SMAD4- dependent and -independent e.g. involving TIF-1 y or IkKa
  • JPS juvenile polyposis syndrome
  • Decreased expression of SMAD4 in their PBMC samples was confirmed by western blot analysis (Figure 3). Both patients displayed normal NK cell proportions and CD56bright/dim subset distribution.
  • SMAD4 act as the molecular switch separating TGF-p inhibition of NK cell antitumor function from TGF-p-induced acquisition of a tissue residency program.
  • EXAMPLE 3 Cytotoxicity of SMAD4-engineered (S/WAD4 eng ) NK92 cells against colorectal cancer cell line HCT116 in the presence of TGF-p: SMAD4 regulates NK92 cytotoxicity in a dose- and context-dependent manner.
  • NK92 a model for NK cell-based cellular immunotherapy in clinical development, could enhance their resistance to TGF-p-inhibition.
  • TGF-p canonical signaling was preserved in NK92 cell line ( Figure 4(A)) yet TGF-p exposure only partially reproduced the effects on primary NK cells.
  • treatment with TGF-p1 reduced granzyme B levels in NK92 cells, leading to decreased cytotoxicity against the colorectal cancer cell line HCT116 ( Figure 4(B, C)); as well as enhanced the expression of the tumor homing receptor CXCR3 ( Figure 4(B)).
  • TGF-p1 did not induce CD103 in NK92 cells nor inhibited cell proliferation ( Figure 4(D)), consistent with the leukemic origin of this cell line.
  • NK92 cell clones with reduced SMAD4 expression were generated by CRISPR/Cas9 ribonucleoprotein (RNP) transfection, sorting of RNP(ATTO550)-positive cells and sequential cloning by limiting dilution. Two different gRNA targeting SMAD4 exon 5 and 10 were simultaneously used. Clones NK92-F3 and NK93-G3 were selected for further study based on their decreased SMAD4 levels, as determined by intracellular staining ( Figure 5(A, B)) and western blot ( Figure 5(C, D)).
  • RNP CRISPR/Cas9 ribonucleoprotein
  • the colorectal cancer cell lines HCT116 and HT29 encoding for wt and mutated SMAD4 genes were used as a positive and negative control for SMAD4 expression, respectively.
  • the NK92 cell line has a tetrapioid karyotype with multiple chromosomal rearrangements, one of them [der(10)t(10; 18)] affecting chromosome 18 where SMAD4 is located (18 q21 .2), resulting in 4 potentials SMAD4 loci.
  • NK92-F3 cells 20% of SMAD4 exon 5 sequences showed a deletion of 39 nucleotides while no mutations were detected in exon 10 ( Figure 5(E-F)).
  • NK92-G3 cells 20% of SMAD4 exon 5 sequences showed a deletion of 1 thymidine (position 117), and 20% of the sequences a deletion of 19 nucleotides (position 111).
  • NK92 cells were treated for 5 days with TGF-01 and subsequently cocultured for 4h with the colorectal cancer cell line HCT1 16.
  • both SMAD4 en9 NK92 clones displayed enhanced cytotoxicity against HCT116 cells upon TGF-01 treatment, surpassing their baseline cytotoxicity and the cytotoxic capacity of NK92 parental cells with and without TGF-p treatment ( Figure 5(l, J)).
  • the influence of SMAD4 in NK92 cell cytotoxicity was context dependent. In homeostatic conditions, reduced SMAD4 expression correlated with Granzyme B levels and cytotoxic capacity against tumors cells. However, reduction of SMAD4 in NK92 cells associated with enhanced cytotoxicity upon TGF-p treatment, indicating the acquisition of alternative cytotoxic mechanisms.
  • EXAMPLE 4 Efficiency and safety evaluation of SMAD4 knock out (SMAD4 K0 ) in in vitro expanded human NK cells by CRISPR/Cas9 nucleofection.
  • the aim of this study was to generate primary human NK cells with reduced SMAD4 levels.
  • the experimental strategy for the generation of primary SMAD4 K0 NK cells is shown in Figure 6(A) and involved: i) an initial NK cell expansion with irradiated 8866 lymphoblastoid cell line, ii) the isolation and nucleofection of NK cells with Cas9/trRNA-ATTO550 (CONTROL) or Cas9/trRNA- ATTO550/ SMAD4 exon 5 gRNA, on day 7 and iii) the use of TGF-p1 as a selective pressure in the second phase of the expansion (from day 7 to day 14), for reducing the proliferation of non-engi- neered while enriching in SMAD4 K0 NK cells.
  • the average Cas9/gRNA RNP nucleofection efficiency was of 86% as analyzed at 24h post-nucleofection by flow cytometry ( Figure 7(A, B)), inducing a homogeneous reduction of 40 to 70% of SMAD4 amount in expanded NK cells as determined by flow cytometry and western blot, respectively ( Figure 6(B-D)).
  • SMAD4 levels detected in control NK cells were not significantly affected by the addition of TGF-p along the second expansion phase whereas tended to decrease in SMAD4 K0 NK cells ( Figure 6(B-D), Figure 7(C)).
  • RNA-Seq data and CRISPR-roots tool we investigated possible off-targets editing events mediated by CRISPR-Cas9 RNP complexes in engineered NK cells. No off-target events were found considering differentially expressed genes, yet three putative off-target events were identified considering genomic variants in genes containing the canonical PAM sequence 5'-NGG-3'.
  • EXAMPLE 5 Transcriptom ic profile of SMAD4 KO human NK cells: the knockout of SMAD4 in in vitro expanded human NK cells does not impact on their maturation status or functional profile yet partially prevents TGF-p inhibition.
  • SMAD4 K0 NK cells To gain a broad view on TGF-p-dependent and -independent changes in SMAD4 K0 NK cells, inventors performed bulk RNAseq analysis with engineered NK cells from three independent donors. In the absence of TGF-p, the global transcriptomic profile of control and SMAD4 K0 NK cells was remarkably similar, with only 53 DEG ( Figure 8(A)). SMAD4 K0 NK cells presented lower levels of SMAD4, CD160 and CD226 (DNAM-1) transcripts and higher levels of IL9R and of ZNF683 (Hobit) (Figure 8(B)).
  • GSEA Gene Set Enrichment Analysis against the C5 data collection in GO Biological Process ontology using DEG between TGF-p-treated SMAD4 K0 as compared to TGF-p-treated control NK cells identified NK cell cytotoxicity, myeloid leukocyte activation, leukocyte migration, cytokine mediated signaling, leukocyte proliferation, and inflammatory response as biological pathways upregulated in SMAD4 K0 NK cells ( Figure 9(B)).
  • the transcriptomic profile of SMAD4 K0 NK cells was more akin to that of activated NK cells while TGF-p treated control NK cells resembled ILC1 ( Figure 8(F)).
  • some examples of genes with higher expression in control- as compared to SMAD4 K0 NK cells treated with TGF-p were VEGFA and CD9 ( Figure 8(B)).
  • SMAD4-engineer-ing did not significantly impact on the expression and distribution of HLA-l-specific inhibitory receptors (KIR, NKG2A) and maturation markers (NKG2C, CD57 and KLRG1) in primary expanded NK cells (Figure 8(l)).
  • EXAMPLE 6 Evaluation of anti-tumor function and serial killing in SMAD4 K0 NK cells in the presence of TGF-p.
  • the aim of this study was to evaluate the anti-tumor activity of SMAD4 K0 expanded NK cells in in vitro cytotoxicity assays against the colorectal cancer cell line HCT1 16.
  • control- and SMAD4 K0 NK cells showed comparable cytotoxicity against HCT116 cells.
  • control NK cells displayed reduced cytotoxicity upon TGF-p1 treatment while SMAD4 K0 NK cells maintained their killing capacity ( Figure 10(A)).
  • NK cell serial killing was subsequently assessed by coculturing PKH26-labeled NK cells with HCT116-GFP+Luc+ spheroids.
  • Control NK cells treated with TGF-p1 accumulated in the spheroid periphery and displayed reduced spheroid growth control.
  • SMAD4 K0 NK cells treated with TGF-p1 exhibited enhanced spheroid killing, reaching similar levels to non-TGF-p treated cells as evidenced by GFP and luciferase analysis ( Figure 10(D-F, K)).
  • TGF-p Another detrimental effect of TGF-p on NK cell function is the inhibition of proliferation upon activation.
  • Analysis of RNAseq data showed that expression of IL15RA, IL2RG and IL2RA was higher in SMAD4 K0 as compared to control NK cells exposed to TGF-p ( Figure 10(H)).
  • SMAD4 K0 NK cells displayed enhanced proliferation in response to IL-2 and TGF-p combination upon NKp46-re- directed activation (Figure 10(1)).
  • expansion of SMAD4 K0 NK cells with K562-CD137L- IL15tm-IL-21 tm feeders was maintained in the presence of TGF-p while significantly reduced in control NK cells ( Figure 10(d)).
  • knocking out SMAD4 in human expanded NK cells prevented TGF-p-inhibition of cytotoxic granule-dependent killing, displaying enhanced serial killing in 3D tumor models.
  • EXAMPLE 7 Analysis of cytokines of SMAD4 K0 NK cells.
  • SMAD4 knocking out SMAD4 in human expanded NK cells prevented TGF-p-inhibition of cytokine production preserving their capacity for secreting CCL5, IFN-y and TNF-a.
  • SMAD4 K0 NK cells would also acquire the capacity to simultaneously produce CSF1 , XCL1 and FLT3L cytokines involved in dendritic cell recruitment and activation.
  • EXAMPLE 8 TRAIL and FasL activity of SMAD4 K0 NK cells.
  • SMAD4 K0 NK cells acquired the expression of TGF-p-induced TRAIL and FasL enabling the possibility of killing by those death receptor pathways, which represent an alternative mechanism to granzyme.
  • EXAMPLE 9 SMAD4 K0 NK cells exposed to TGF-p display enhanced spheroid penetrance and tumor-homing potential.
  • SMAD4 K0 NK cells were partially resistant to the inhibitory effect of TGF-p on the expression of multiple adhesion molecules [ITGA2 (CD49b), ITGB2 (CD18), ITGAL (CD11 a), ITGAM (CD11 b), ICAM1 (CD54), ICAM2 (CD102), PECAM1 (CD31)] ( Figure 12(A)) while preserving the sensitivity to TGF-p-dependent up-regulation of transcripts for tissue residency-associated integrins [ITGB1 (CD29), ITGA1 (CD49a), ITGAE (CD103)] ( Figure 12(A)).
  • SMAD4 K0 NK cells were partially resistant to the downregulation of CD11 a, CD18 and CD49d expression induced by TGF-p1 , while acquiring or maintaining the expression of CD103 and CD29 ( Figure 12(B- C)).
  • chemokine receptor expression was also modulated by TGF-p.
  • TGF-p reduced CCR5 expression in both control- and SMAD4 K0 NK cells while inducing the expression of CXCR4 ( Figure 12(G, H)).
  • TGF-p1 treatment reduced CXCR3 transcript in control NK cells yet these changes were not detected at protein levels ( Figure 12(G, H)).
  • Transmigration assays were performed with control and SMAD4 K0 NK cells expanded in the presence of TGF-p. Transmigration to CCL5, CXCL9 and CCL5+CXCL9 combination was modest in both control and engineered NK cells. However, SMAD4 K0 NK cells treated with TGF-p1 showed enhanced migration towards SDF-1 and all combination of SDF-1 with CCL5 and CXCL9 chemokines, as compared to TGF-p1 -treated control NK cells ( Figure 12(1)).
  • SMAD4 K0 NK cells exposed to TGF-p displayed enhanced spheroid penetrance and tumor-homing potential.
  • EXAMPLE 10 Comparison of cytotoxicity between SMAD4 K0 NK cells and NK cells treated with a TGF- -inhibitor: SMAD4 K0 NK cells display superior cytotoxicity.
  • TGF-p i.e. neutralizing antibodies, blocking antibodies for TGF-p receptor chain 2 (TGFBR2), TGF-p inhibitors as well as genetically engineered NK/T cells with dominant negative TGF-B Receptor II
  • TGFBR2 TGF-p receptor chain 2
  • SB-431542 activity was also corroborated by the lack of Granzyme B and NKG2D down-regulation as well as CD103 acquisition by TGF-p-treated control cells ( Figure 15(A)).
  • SMAD4 K0 NK cells treated with TGF-p showed superior cytotoxicity against HCT116-Luc+GFP+ spheroids than control NK cells treated with SB-431542 and TGF-p combination ( Figure 14(C-D)).
  • EXAMPLE 11 Analysis of SMAD4 ⁇ ° NK cells in the presence of Activin A: resistance of Ac- tivin A suppression.
  • SMAD4 is a signaling hub that also participates in the transcriptional changes induced by other members of the TGF-p family.
  • Activin A is also produced in the tumor microenvironment and contribute to attenuate NK-cell effector function.
  • Activin A shares the canonical SMAD2/3 pathway with TGFp yet signaling is initiated by specific Type II receptors II (ActRIIA, ACtRIIB) which promiscuously couple to different Type I receptors (mainly ActRIB, but also ActRIA and ActRIC). Consequently, Activin A could contribute to NK cell inhibition, also in situations of TGF-p or TGFp- RII blockade.
  • RNAseq data showed expression of Activin A type I and type II receptors in NK cells.
  • Both control and SMAD4 K0 NK cells expressed ACVR2A and ACVR1 at basal conditions, while switching to ACVR2B and ACVR1B upon TGF-p exposure ( Figure 14(E)).
  • the reduction in GzmB and perforin levels induced by Activin A treatment was less pronounced that the one induced by TGF-p both in control and SMAD4 K0 NK cells ( Figure 14(F) and Figure 15(B)).
  • SMAD4 K0 NK cells treated with Activin A showed comparable cytotoxicity against HCT1 16 spheroids than non-treated NK cells in contrast to the modest decrease in cytotoxicity detected in control NK cells treated with activin A ( Figure 14(G)).
  • EXAMPLE 12 Evaluation of SMAD4 K0 NK cell anti-tumor function in a humanized in vivo mouse model of HER2-positive breast cancer.
  • the aim of this study was to evaluate the anti-tumor activity of SMAD4 K0 NK cells in a humanized in vivo model of HER2-positive breast cancer.
  • HCC1954 xenografts were subcutaneously implanted in NOD/Scid/yc-/- (NSG) mice.
  • NSG NOD/Scid/yc-/- mice.
  • mice were treated with either: control or SMAD4 K0 NK cells (0.2 x10 6 cells) for assessing tumor growth control upon direct NK cell recognition, or control, or SMAD4 K0 NK cells (0.1 x10 6 cells) in combination anti-HER2 antibodies (trastuzumab and pertuzumab) for assessing tumor growth control upon antibody-dependent NK cell-mediated cytotoxicity.
  • a control a group of mice was treated with anti-HER2 antibodies.
  • NK cells were intratumorally injected once a week, anti-HER2 antibodies were intraperitonially administered every 3-4 days, together with IL-2 to promote in vivo NK cell survival (Figure 18(A)).
  • mice treated with SMAD4 K0 NK cells showed superior tumor growth control as monotherapy as well as when combined with anti-HER2 therapeutic antibodies, bypassing in both contexts the anti-tumor efficacy of control NK cells in combination with anti-HER2 antibodies ( Figure 18(B)).
  • knocking out SMAD4 in human expanded NK cells prevented TGF-p-inhibition of NK cell cytotoxicity and cytokine production endowing them with a superior in vitro and in vivo anti-tumor function.
  • EXAMPLE 13 CD19-CAR SMAD4 K0 NK cells exhibit sensitivity to the inhibitory effects of TGF- P and enhance the cytotoxic activity of CAR-NK cells in the presence of TGF-p in a leukemia cell line.
  • CRISPR-Analytics A platform for precise analytics and simulations for gene editing.

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Abstract

L'invention concerne une cellule lymphocytaire cytotoxique humaine modifiée pour l'immunothérapie adoptive, comprenant une mutation inactivatrice dans le gène SMAD4, ce qui rend la cellule résistante aux effets immunosuppresseurs du TGF-β et lui permet de maintenir sa cytotoxicité. De plus, l'invention concerne une population cellulaire et une composition pharmaceutique comprenant la cellule lymphocytaire cytotoxique humaine modifiée, qui sont utiles dans le traitement du cancer. En particulier, l'invention concerne des cellules NK modifiées pour un traitement standard.
PCT/EP2024/081581 2023-11-08 2024-11-07 Lymphocytes cytotoxiques modifiés par smad4 pour thérapie cellulaire Pending WO2025099197A1 (fr)

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