Engineered immune cell platform

Abstract

The present invention provides engineered immune cells, which contain gain-of-function mutations in Cav channels and / or overexpress L-type voltage-gated calcium (Cav) channels. Also provided are related engineered immune cells for use in medicine. Also provided are L-type Cav channel agonists for use in methods of stimulating cell killing activity of immune cells in subjects with proliferative diseases. Also provided are pharmaceutical compositions and methods.

Description

[Technical field]

[0001] The present invention relates to products and methods for modulating (including enhancing) the cytotoxicity of immune cells. In particular, enhancing the expression or activity of L-type Cav channels is disclosed for use in medicine. The present invention provides engineered immune cells, agents and methods for manipulating immune cells, and medical uses thereof. [Background technology]

[0002] Cytotoxic T lymphocytes (CTLs) play a key role in immune responses against cancer and intracellular pathogens. CTLs recognize tumor and infected cells via the T cell receptor complex (TCR), and this recognition induces a cascade of intracellular signaling pathways. One such pathway is the Hedgehog (Hh) signaling pathway. Induction of Hh enhances the expression of the Hh transcription factor Gli1 (de la Roche, 2013). Thus, GLI1 expression is a reliable reporter of Hh signaling. We previously showed that Gli1 induction is a Ca 2+ It is dependent on (Hanna, 2021) and CD8 + We have shown that this non-classical Hh signaling pathway is required for T cell killing (Hanna, 2021 and de la Roche, 2013). We show that this non-classical Hh signaling pathway mediates the L-type voltage-gated Ca 2+ It further shows that it works by channel.

[0003] There are four members of the L-type voltage-gated calcium channel family: Cav1.1, Cav1.2, Cav1.3 and Cav1.4. All four members are expressed in mouse T cells (Badou, 2013), whereas only Cav1.4, and to a lesser extent Cav1.3, are expressed in human CD8+ T cells (Hanna, 2021).

[0004] Cytotoxic lymphocytes are CD8 + T cells, CD4 +These include T cells and natural killer (NK) cells, all of which exert their effects against tumors and virus-infected cells by recognizing different targets. For example, CD8 + T cells recognize major histocompatibility complex class I (MHCI) antigens, whereas CD4 + T cells recognize MHC class II antigens. In contrast, NK cells recognize and kill MHC negative cells.

[0005] Immune cell therapy is the most advanced class of cell therapy and has already shown clear evidence of clinical benefit in cancer and infectious diseases. Immune cell types that can be used as therapeutic agents include alpha-beta T cells, NK cells, gamma-delta (gd) T cells, NK T (NKT) cells, and induced pluripotent stem cell (iPSC)-derived immune effectors. The applications of immune cell therapy are broad and include the treatment of cancer, infection, allogeneic transplantation, and autoimmunity.

[0006] T cell therapy has emerged as the most advanced of this therapeutic class. To treat cancer, T cell immunotherapy is deployed in a variety of ways: non-engineered tumor-infiltrating lymphocytes (TILs), T cells expressing engineered T cell receptors (TCRs), and chimeric antigen receptor (CAR) T cells.

[0007] The application of NK cells in cancer therapy is also being investigated: NK cells have cytotoxic potential when administered as an allogeneic transplant to patients, but a major advantage is that they do not mediate severe graft-versus-host disease (GVHD), potentially allowing the development of an "off the shelf" product.

[0008] There are currently four adoptive cell therapies approved by the FDA for the treatment of subtypes of lymphoma, leukemia, and myeloma. Despite these advances, there remains a need for methods and agents to improve the effectiveness of cell therapies for treating conditions such as cancer.

[0009] The present invention has been made in consideration of the above considerations. Summary of the Invention

[0010] The present inventors sought to provide a platform that enhances the cytotoxicity of immune cells by increasing the expression or activity of L-type Cav channels, which may be applied to adoptive cell therapy.

[0011] Thus, in a first aspect, the present invention provides engineered immune cells that overexpress L-type voltage-gated calcium (Cav) channels and / or contain gain-of-function mutations (GOFs) in L-type Cav channels.

[0012] In some embodiments, the engineered immune cells overexpress Cav1.4 compared to wild-type immune cells of the same type. In other embodiments, the engineered immune cells overexpress Cav1.3 compared to wild-type immune cells of the same type. In other embodiments, the engineered immune cells overexpress Cav1.2 compared to wild-type immune cells of the same type. In other embodiments, the engineered immune cells overexpress Cav1.1 compared to wild-type immune cells of the same type.

[0013] In some embodiments, the engineered immune cells comprise a mutation in a gene encoding an L-type Cav channel. In some embodiments, the engineered immune cells comprise a gain-of-function mutation in CACNA1F (the gene encoding Cav1.4). In some embodiments, the CACNA1F mutation comprises I745T (using EU numbering) (Hemara-Wahanui, 2005). This mutation is set forth in SEQ ID NO:5. This mutation is also referred to in the literature as I756T (Williams, 2020). Pore-forming Ca 2+ The protein structure of the channel α1 subunit is highly conserved and therefore gain of function in other L-type Cav channels can be expected to be conferred by the same I745T GOF mutation as in CACNA1F since this region is highly conserved.

[0014] Other gain-of-function mutations encompassed by the present invention are set forth in Table 1 below.

[0015] [Table 1]

[0016] The preferred mutations are not in the voltage sensor region of the L-type Cav channel, because the L-type Cav channel in T cells may not be voltage-gated (Kotturi, 2005). The preferred mutations are located in the activation gate, i.e., in the cytoplasmic region of the S6 helix; in the S5-S6 linker (Striessnig, 2021); or in the S5-S6 pore. 2+ GOF mutations that convert the channel into a nonselective cation channel are 2+ This is unfavorable as trafficking is essential for Gli1 induction (Hanna, 2021).

[0017] In another aspect, the present invention provides an engineered immune cell, comprising a mutation in a gene encoding a component of the Hh signaling pathway, which mutation results in increased GLI1 expression and / or activity that can be measured by a Gli1 reporter assay. For example, the mutation can be in the PTCH1 gene encoding protein patch homolog 1 (PTCH1). PTCH1 enhances classical Hh signaling.

[0018] In a related aspect, the invention provides engineered immune cells for use in medicine (e.g., for use in treating a mammalian proliferative disease), the engineered immune cells comprising a mutation in a gene encoding a component of the Hh signaling pathway, the mutation resulting in increased GLI1 expression and / or activity. In some embodiments, the mutation may be in the PTCH1 gene encoding protein patch homolog 1 (PTCH1) (e.g., a PTCH1 mutation found to be present in subjects with Gorlin syndrome, or a mutation artificially introduced into PTCH1 to cause silencing, downregulation, or otherwise reduction in the ability of PTCH1 to function as a negative regulator of GLI1).

[0019] In some embodiments, the engineered immune cells are selected from the group consisting of T cells (including gamma delta (γδ) T cells), chimeric antigen receptor (CAR)-T cells, NK cells, and CAR-NK cells. The inventors have surprisingly found that calcium-dependent Gli1 signaling is conserved between T cells and NK cells. In some embodiments, the engineered immune cells are T cells. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + In some embodiments, the T cells are cytotoxic CD4 + In some embodiments, the T cells are γδ T cells.

[0020] In a second aspect, the present invention provides an engineered immune cell for use in medicine, the engineered immune cell overexpressing an L-type Cav channel and / or comprising a gain-of-function mutation in the L-type Cav channel. In some embodiments, the engineered immune cell is for use in a method for treating a chronic infection in a subject. For example, the engineered immune cell may be for use in a method for treating an opportunistic fungal infection such as SARS-CoV-II, HIV, Hepatitis C, Hepatitis B, human cytomegalovirus (CMV) or aspergillosis. In some embodiments, the engineered immune cell is for use in a method for treating an autoimmune disease in a subject. For example, the engineered immune cells may be for use in methods of treating rheumatoid arthritis (RA), type 1 diabetes, psoriasis, psoriatic arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease (IBD), autoimmune Addison's disease (AAD), Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, or autoimmune vasculitis.

[0021] In a third aspect, the present invention provides engineered immune cells for use in a method of treating cancer in a subject, the engineered immune cells overexpressing L-type Cav channel and / or comprising gain-of-function mutations in L-type Cav channel. Particularly interesting target cancers include cancers with high infiltration of cytotoxic lymphocytes. These include melanoma, lung cancer, colorectal cancer and breast cancer. Other interesting cancers are those that are susceptible to CAR T and NK cell therapy. These cancers include blood cancers, such as lymphoma, leukemia (e.g., ALL and CLL) and multiple myeloma; and solid tumors targeted by CAR, including lung cancer, liver cancer, gastric cancer (including EGFR) / CNS, pediatric glioma (HER2) / ovarian cancer, cervical cancer, pancreatic cancer, lung cancer (mesothelin), colon cancer, pancreatic cancer, prostate cancer, gastric cancer, liver cancer (EpCAM), and many others.

[0022] In some embodiments, the engineered immune cells overexpress Cav1.4 compared to wild-type immune cells of the same type. In some embodiments, the engineered immune cells comprise a gain-of-function mutation in CACNA1F. In some embodiments, the mutation in CACNA1F comprises I745T.

[0023] In some embodiments, the engineered immune cells are selected from the group consisting of T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells, and CAR-NK cells.

[0024] In some embodiments, the method of treating cancer in a subject is an in vivo method. Such methods can include, for example, adoptive transfer of T cells, as described herein. In these embodiments, the method can include removal of immune cells from a subject and subsequent processing. The processing can include genetic engineering to introduce gain-of-function mutations in L-type Cav channels or genetic engineering to overexpress L-type Cav channels. In some embodiments, the method can include administering the processed immune cells to a subject. The subject can be the same subject from which the immune cells were removed or a different subject, i.e., the method can be allogeneic or autologous. In some embodiments, the method of stimulating the killing activity of T cells is an ex vivo method, i.e., the method includes processing of immune cells outside the subject's body. It is contemplated that in such methods, the immune cells can be administered back to the subject after ex vivo processing.

[0025] In a fourth aspect, the present invention provides an L-type Cav channel agonist for use in a method of stimulating the cell killing activity of immune cells of a subject having a proliferative disease. In some embodiments, the method of stimulating the cell killing activity is an in vivo method. The immune cells can be T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells or CAR-NK cells. Such methods can include, for example, adoptive transfer of T cells as described herein. In these embodiments, the method can include removing immune cells from a subject and then processing. The processing can include treatment with an L-type Cav channel agonist. In some embodiments, the method can include administering the treated immune cells to a subject. The subject can be the same subject from which the immune cells were removed or a different subject, i.e., the method can be allogeneic or autologous. In some embodiments, the method of stimulating the cell killing activity is an ex vivo method, i.e., the method includes processing immune cells outside the subject's body. It is contemplated that in such a method, immune cells can be administered back to the subject after ex vivo treatment.In some embodiments, the L-type Cav channel agonist for use according to this aspect of the present invention shows immune cell Gli1 enhancing activity as determined by Gli1 reporter assay.For example, Gli1 reporter assay as defined herein.

[0026] In a fifth aspect, the present invention provides a pharmaceutical composition comprising immune cells treated with or admixed with an L-type Cav channel agonist. The immune cells may be T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells or CAR-NK cells. In some embodiments, the composition is for use in a method of stimulating cell killing activity against a target cell population (e.g., a diseased cell population, such as a cancer cell population). The composition may be for use in an in vivo, ex vivo, or in vitro method.

[0027] In a sixth aspect, the present invention provides an in vitro method for increasing the cell-killing activity of immune cells, comprising contacting an L-type Cav channel agonist with immune cells, wherein the Cav channel agonist can stimulate L-type Cav channel activity in a Gli1 reporter assay. In some embodiments, the L-type Cav channel agonist is incubated with immune cells for a period of time sufficient to cause an increase in cell-killing activity and / or cell-killing potential. For example, incubation can be for 10, 20, 30 minutes, or 1, 2, 5, 12, 24, 48 hours or more.

[0028] In some embodiments, according to any aspect of the present invention, the L-type Cav channel agonist is a compound disclosed in EP0230110A1 and EP0300688A1 (the contents of which are incorporated herein by reference). For example, the L-type Cav channel agonist may be a compound defined by the formula of claim 1 of EP0230110A1. For example, the L-type Cav channel agonist may be a compound defined by the formula of claim 1 of EP0300688A1. In some embodiments, the L-type Cav channel agonist is selected from the group consisting of FPL 64176 and Bay K8644.

[0029] In a seventh aspect, the present invention provides an in vitro method of engineering immune cells to increase cell killing activity, the method comprising genetically engineering immune cells to enhance or stimulate expression of L-type Cav channel activity in a Gli1 reporter assay.

[0030] In some embodiments, the manipulation comprises overexpressing Cav1.4. In some embodiments, the method of overexpression comprises using a vector for stable expression of Cav1.4. In some embodiments, the vector is a lentiviral vector, a retroviral vector, or a transposon-based vector.

[0031] In some embodiments, the engineering comprises mutating Cav1.4 to introduce a gain-of-function mutation, hi some embodiments, the Cav1.4 mutation comprises I745T. In other embodiments, the manipulation comprises nuclease-based editing. In some embodiments, the manipulation comprises CRISPR-based editing. In some embodiments, the manipulation comprises prime editing (Anzalone, 2019).

[0032] In some embodiments, the immune cells are selected from the group consisting of T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells, and CAR-NK cells. In another related aspect, the invention provides a method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune cells, wherein the immune cells overexpress an L-type Cav channel and / or comprise a gain-of-function mutation in the L-type Cav channel.

[0033] In some embodiments, the immune cells are selected from the group consisting of T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells, and CAR-NK cells. In another related aspect, the present invention provides a method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an L-type Cav channel agonist.

[0034] In another aspect, the present invention provides a method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of immune cells treated with an L-type Cav channel agonist. In some embodiments, the immune cells are derived from the subject in need thereof, i.e., this is a method of autologous therapy. In some embodiments, the immune cells are not derived from the subject in need thereof, i.e., this is a method of allogeneic therapy. In a preferred embodiment, the immune cells are NK cells.

[0035] In some embodiments, the L-type Cav channel agonist is selected from the group consisting of FPL 64176 and Bay K8644.

[0036] [ka]

[0037] In another aspect, the present invention provides a method for determining the cytotoxicity suitability of immune cells from a mammalian subject, comprising measuring the level of GLI1 expression of immune cells from a mammalian subject. The method can be performed in vitro or in vivo. In particular, GLI1 expression can be measured using a Gli1 reporter assay as defined herein. The cytotoxicity suitability refers to the level of cytotoxicity of immune cells. In this way, GLI1 expression serves as a biomarker for the cytotoxicity capacity of the immune system of the subject or the immune cells of the subject.

[0038] In some embodiments, the immune cells are engineered immune cells. In some embodiments, the method includes administering a test agent to the immune cells. In some embodiments, the test agent is an L-type Cav channel agonist. In some embodiments, the engineered immune cells are CAR T cells, CAR NK cells, T cells (including gamma delta (γδ) T cells), or NK cells. Thus, the present invention provides GLI1 as a biomarker for determining the efficacy of cytotoxicity of cells for use in adoptive cell therapy.

[0039] In another aspect, the invention provides an engineered immune cell for use in a method of treating a proliferative disease in a subject, the engineered immune cell comprising a loss-of-function mutation in PTCH1, and / or PTCH1 is silenced or downregulated, and / or the PTCH1 gene product is antagonized. In some embodiments, the loss-of-function mutation in PTCH1 is a mutation found to be present in human patients with Gorlin's syndrome. In some embodiments, the engineered immune cell for use is selected from the group consisting of T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells, and CAR-NK cells. In some embodiments, the proliferative disease comprises cancer, and optionally the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, breast cancer, lymphoma, leukemia, multiple myeloma, liver cancer, gastric cancer, central nervous system (CNS) tumors, pediatric glioma, ovarian cancer, cervical cancer, colon cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0040] In another aspect, the invention provides a method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune cells, wherein the engineered immune cells comprise a loss-of-function mutation in PTCH1, and / or PTCH1 is silenced or downregulated, and / or the PTCH1 gene product is antagonized. In some embodiments, the loss-of-function mutation in PTCH1 is a mutation found to be present in human patients with Gorlin syndrome. In some embodiments, the engineered immune cells are selected from the group consisting of T cells (including gamma delta (γδ) T cells), CAR-T cells, NK cells, and CAR-NK cells. In some embodiments, the proliferative disease comprises cancer. For example, the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, breast cancer, lymphoma, leukemia, multiple myeloma, liver cancer, gastric cancer, central nervous system (CNS) tumors, pediatric glioma, ovarian cancer, cervical cancer, colon cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0041] The present invention includes combinations of the described embodiments and preferred features unless such combinations are expressly disallowed or explicitly avoided. array Cav1.1 (CACNA1S) The amino acid sequence of human Cav1.1 (encoded by CACNA1S) is set forth in SEQ ID NO:1: Cav1.2 (CACNA1C) The amino acid sequence of human Cav1.2 (encoded by CACNA1C) is set forth in SEQ ID NO:2: Cav1.3 (CACNA1D) The amino acid sequence of human Cav1.3 (encoded by CACNA1D) is set forth in SEQ ID NO:3: Cav1.4 (CACNA1F) The amino acid sequence of human Cav1.4 (encoded by CACNA1F) is set forth in SEQ ID NO:4: Cav1.4 (CACNA1F) mutation I745T The amino acid sequence of the human CACNA1F I745T mutant is set forth in SEQ ID NO: 5. This mutation is also referred to in the literature as I756T (e.g., Williams, 2020). The mutated residues are highlighted in bold and underlined. MSESEGGKDTTPEPSPANGAGPGPEWGLCPGPPAVEGESSGASGLGTPKRRNQHSKHKTVAVASAQRSPRALFCLTLANPLRRSCISIVEWKPFDILILLTIFANCVALGVYIPFPEDDSNTANNHNLEQVEYVFLVIFTVETVLKIVAYGLVLHPSAYIRNGWNLLDFIIVVVGLFSVLLEQGPGRPG DAPHTGGKPGGFDVKALRAFRVLRPLRLLVSGVPSLHIVLNSIMKALVPLLHIALLVLFVIIIYAIIGLELFLGRMHKTCYFLGSDMEAEEDPSPCASSGSGRACTLNQTECRGRWPGPNGGITNFDNFFFAMLTVFQCVTMEGWTDVLYWMQDAMGYELPWVYFVSLVIFGSFFVLNLVLGVLSGEFSK EREKAKARGDFQKQREKQQMEEDLRGYLDWITQAEELDMEDPSADDNLGSMAEEGRAGHRPQLAELTNRRRGRLRWFSHSTRSTHSTSSHASLPASDTGSMTETQGDEDEEEGALASCTRCLNKIMKTRVCRRLRRANRVLRARCRRAVKSNACYWAVLLLVFLNTLTIASEHHGQPVWLTQIQEYANK VLLCLFTVEMLLKLYGLGPSAYVSSFFNRFDCFVVCGGILETTLVEVGAMQPLGISVLRCVRLLRIFKVTRHWASLSNLVASLLNSMKSIASLLLLLFLFIIIFSLLGMQLFGGKFNFDQTHTKRSTFDTFPQALLTVFQILTGEDWNVVMYDGIMAYGGPFFPGMLVCIYFIILFICGNYILLNVFLA T BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention are now discussed with reference to the accompanying drawings. [Brief description of the drawings]

[0042] [Figure 1-1] L-type Cav channel agonists increase Gli1 expression and enhance specific cell killing by mouse CD8+ CTLs. CD8+ T cells were isolated from spleens and inguinal lymph nodes of Rag2- / - OT-I mice and CTLs were generated. (A) Cells were restimulated for 3 h on day 9 / 10 with plate-bound anti-CD3ε in the presence of the indicated concentrations of FPL 64176 or carrier control. RNA was extracted and Gli1 expression was assessed by qRT-PCR. Data are from n=3 independent experiments. us=unstimulated cells. (B) LDH cytotoxicity assays were performed using OT-I CTLs. On day 7, CTLs were co-cultured with EL-4 cells pulsed with Ova peptide in the presence of 10 μM FPL 64176 (squares) or carrier control (circles). Data are from n=4 independent experiments. (C) Experiment updated from (A). Data are from n=4 independent experiments. Data are normalized to Tbp as a reference gene. 0h=unstimulated cells. (D) The same data as in (B) but shown in different representations. Left panel shows a representative killing assay. Right panel shows quantification of n=4 independent experiments normalized to killing by vehicle-treated cells at an effector:target ratio of 10:1. Error bars indicate SD. p values ​​were calculated using unpaired two-tailed Student's t-test (C) or two-way ANOVA (B / D). * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Figure 1-2]L-type Cav channel agonists increase Gli1 expression and enhance specific cell killing by mouse CD8+ CTLs. CD8+ T cells were isolated from spleens and inguinal lymph nodes of Rag2- / - OT-I mice and CTLs were generated. (A) Cells were restimulated for 3 h on day 9 / 10 with plate-bound anti-CD3ε in the presence of the indicated concentrations of FPL 64176 or carrier control. RNA was extracted and Gli1 expression was assessed by qRT-PCR. Data are from n=3 independent experiments. us=unstimulated cells. (B) LDH cytotoxicity assays were performed using OT-I CTLs. On day 7, CTLs were co-cultured with EL-4 cells pulsed with Ova peptide in the presence of 10 μM FPL 64176 (squares) or carrier control (circles). Data are from n=4 independent experiments. (C) Experiment updated from (A). Data are from n=4 independent experiments. Data are normalized to Tbp as a reference gene. 0h=unstimulated cells. (D) The same data as in (B) but shown in different representations. Left panel shows a representative killing assay. Right panel shows quantification of n=4 independent experiments normalized to killing by vehicle-treated cells at an effector:target ratio of 10:1. Error bars indicate SD. p values ​​were calculated using unpaired two-tailed Student's t-test (C) or two-way ANOVA (B / D). * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Diagram 2] FPL 64176, but not (R)-baclofen or nefiracetam, enhances Gli1 expression in mouse CD8+ CTLs. Naive CD8+ T cells were isolated from the spleens of OT-I Rag2- / - mice and CTLs were generated. Cells were restimulated using anti-CD3ε mAb in the presence of the indicated concentrations of (A) FPL 64176, (B) (R)-baclofen, or (C) nefiracetam (or carrier control). CTLs were preincubated with the relevant pharmacological agent for 40–50 min prior to stimulation (allowing sufficient time for the agent, e.g., FPL 64176, to enter the cells). Gli1 expression was assessed by qRT-PCR. Data are from n=2 independent experiments. Error bars indicate SD. [Figure 3-1] L-type Cav channel agonists increase the expression of GLI1 and enhance specific cell killing by human CD8+ CTLs. Naive human CD8+ T cells were purified from the buffy coat layer of blood isolated from healthy donors, stimulated using ImmunoCult™ CD3 / CD28 / CD2 human T-Cell Activator, and restimulated again on day 10 for 3 days. (A) Human CTLs were restimulated with plate-bound anti-CD3 for 24 hours on days 15-20 in the presence of 10 μM FPL64176 or carrier control, after which RNA was extracted for qRT-PCR analysis. n=1 healthy donor. TBP is used as the reference gene. (B) Updated graph of (A), showing n=3 independent experiments / healthy donor. Gli1 expression is normalized to the carrier case. (C) Between days 16 and 24, CD8+ T cells were co-cultured with P815 target cells at the indicated effector-to-target ratios in the presence of 10 μM FPL64176 (squares) or carrier control (circles) and subjected to VITAL cytotoxicity assays. (D) Same data as in (C) but presented differently. Left panel shows a representative killing assay. Right panel shows quantification of n=4 experiments with 4 different healthy donors normalized to killing by carrier-treated cells at an effector:target ratio of 10:1. Error bars indicate SD. p values ​​were calculated using unpaired two-tailed Student's t-test (B) or two-way ANOVA (C / D). * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Figure 3-2]L-type Cav channel agonists increase the expression of GLI1 and enhance specific cell killing by human CD8+ CTLs. Naive human CD8+ T cells were purified from the buffy coat layer of blood isolated from healthy donors, stimulated using ImmunoCult™ CD3 / CD28 / CD2 human T-Cell Activator, and restimulated again on day 10 for 3 days. (A) Human CTLs were restimulated with plate-bound anti-CD3 for 24 hours on days 15-20 in the presence of 10 μM FPL64176 or carrier control, after which RNA was extracted for qRT-PCR analysis. n=1 healthy donor. TBP is used as the reference gene. (B) Updated graph of (A), showing n=3 independent experiments / healthy donor. Gli1 expression is normalized to the carrier case. (C) Between days 16 and 24, CD8+ T cells were co-cultured with P815 target cells at the indicated effector-to-target ratios in the presence of 10 μM FPL64176 (squares) or carrier control (circles) and subjected to VITAL cytotoxicity assays. (D) Same data as in (C) but presented differently. Left panel shows a representative killing assay. Right panel shows quantification of n=4 experiments with 4 different healthy donors normalized to killing by carrier-treated cells at an effector:target ratio of 10:1. Error bars indicate SD. p values ​​were calculated using unpaired two-tailed Student's t-test (B) or two-way ANOVA (C / D). * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Figure 4]Gli1 is important in specific tumor cell killing by mouse NK cells. (A) NK cells were isolated from WT C57BL / 6 mice and treated with either the Gli1 / Gli2 inhibitor GANT61 (squares) or carrier control (circles). LDH killing assays (left panel) and VITAL assays (right panel) were performed to assess specific cell killing of NK-sensitive RMA / S tumor cells. Left panel: representative data from n=3 independent experiments. Right panel: data from two individual mice per genotype. (B) To investigate the contribution of Gli1 to NK cell effector function in detail, LDH killing assays (left panel) and VITAL assays (right panel) were performed using NK cells isolated from both WT mice (circles) and Gli1 knockout (KO, squares) mice. Left panel: representative data from n=2 independent experiments. Right panel: data from two individual mice per genotype. [Diagram 5] L-type Cav channel agonists increase GLI1 expression in the human NK cell line NK-92 and promote specific tumor cell killing. (A) NK-92 cells were stimulated with anti-NKp46 mAb in the presence of 10 μM FPL 64176 for 4 h and GLI1 expression was assessed using qRT-PCR. Data are from n=4 independent experiments. (B) NK-92 cells were stimulated with anti-NKp46 mAb in the presence of 100 μM Bay K8644 for 4 h and GLI1 expression was assessed using qRT-PCR. Data are from n=3 independent experiments. (C) LDH assays were performed at the indicated effector-to-target ratios in the presence of 10 μM FPL 64176 (right bars) or carrier control (left bars) to assess specific cell killing of K562 target cells. Error bars indicate SD. Data are from n=3 independent experiments. [Figure 6]Mutations resulting in increased GLI1 expression and a gain-of-function Hh phenotype with enhanced specific tumor cell killing by human NK cells. NK cells were purified from the buffy coat layer of blood isolated from patients with Gorlin syndrome and healthy control individuals and cultured for 2 days in complete RPMI + 20ng / ml hIL-15. (A) On day 2, NK cells were analyzed by qRT-PCR for expression of GLI1. (B) A VITAL assay was performed to assess specific killing of K562 target cells. Data are shown in replicates and represent NK cells from three individual Gorlin syndrome patients (squares) and three healthy control individuals (circles). [Figure 7] NK-92 cells overexpressing the I756T Cav1.4 gain-of-function mutation show enhanced tumor cell killing. NK-92 cells (parental cells) were engineered to stably express empty vector (EV), Cav1.4 (WT), Cav1.4 with the gain-of-function mutation I756T and loss-of-function mutation S229P, respectively. K562 tumor cell killing was assessed after 24 hours by LDH killing assay. Stable expression of WT, I756T (GOF) or S229P (LOF) Cav1.4 in NK-92 cells does not affect cell survival and proliferation. I756T (GOF) Cav1.4 can also be successfully expressed in human CD8 T cells. [Figure 8] Gli1 mediates specific cell killing by murine cytotoxic CD4+ T cells. Cytotoxic CD4+ T cells were generated from OT-II mice and treated with 5 μM Gli1 / Gli2 inhibitor GANT61 (gray) or carrier control (black) for 24 h, after which an LDH assay was performed to assess specific cell killing. Data are from n=3 independent experiments. [Figure 9]GzmB content and IFNγ secretion are unaffected by Gli inhibition in cytotoxic CD4+ T cells. Cytotoxic CD4+ cells were generated from OT-II mice and treated with 5 μM GANT61 or carrier control for 24 h on day 7, followed by flow cytometry analysis of granzyme B (GzmB) and IFNγ levels on day 8. (A) Representative flow cytometry plots of unstimulated (upper panel) or restimulated CD4+ T cells (lower panel). (B) Quantification of steady-state GzmB and IFNγ levels upon restimulation. Data are from n=3 independent experiments. [Figure 10] GzmB content and IFNγ secretion are unaffected by Gli inhibition in NK cells. NK cells were isolated from C57BL / 6 mice and treated overnight (~18 h) with 10 μM GANT61 or carrier control (EtOH), followed by analysis of intracellular granzyme B (GzmB) and IFNγ expression by flow cytometry. (A) Representative flow cytometry plots. (B) Quantitation of flow cytometry plots. Data are from n=2 mice (one independent experiment). [Figure 11-1]FPL 64176 treatment does not impair viability or proliferation of naive and cytotoxic CD8+ T cells, does not affect memory phenotype, and enhances killing by CTLs via extracellular Ca2+. Naive CD8+ T cells were stimulated with plate-bound anti-CD3 / CD28 antibodies in the presence of 10 μM FPL64176 or carrier control for 24 h, followed by flow cytometry analysis (Figure 11A-D). (A) Quantitative analysis of viable cell numbers. (B) Percentage of cells positive for the apoptosis marker Apotracker™ Green. (C) Percentage of cells positive for the cell viability dye DAPI. (D) Analysis of T cell subsets based on CD44 and CD62L expression: CD44- CD62L+ (naive), CD44+ CD62L+ (central memory), and CD44+ CD62L- (effector memory). (FIG. 11E-G) CTLs were restimulated with plate-bound anti-CD3 antibody on day 10 for 24 h in the presence of 10 μM FPL64176 or carrier control, followed by flow cytometry analysis. (E) Quantitative analysis of viable cell counts. (F) Percentage of cells positive for the apoptosis marker Apotracker™ Green. (G) Percentage of cells positive for the cell viability dye DAPI. (H) On days 7-8 post-stimulation, mouse CTLs were co-cultured with ovalbumin-pulsed EL-4 target cells at the indicated effector-to-target ratios in the presence of 10 μM FPL64176 and / or 1.25 mM BAPTA and respective carrier control, and subjected to LDH cytotoxicity assay for 3 h. n=4 independent experiments normalized to killing by carrier-treated cells at an effector:target cell ratio of 10:1. Error bars indicate SD. [Figure 11-2]FPL 64176 treatment does not impair viability or proliferation of naive and cytotoxic CD8+ T cells, does not affect memory phenotype, and enhances killing by CTLs via extracellular Ca2+. Naive CD8+ T cells were stimulated with plate-bound anti-CD3 / CD28 antibodies in the presence of 10 μM FPL64176 or carrier control for 24 h, followed by flow cytometry analysis (Figure 11A-D). (A) Quantitative analysis of viable cell numbers. (B) Percentage of cells positive for the apoptosis marker Apotracker™ Green. (C) Percentage of cells positive for the cell viability dye DAPI. (D) Analysis of T cell subsets based on CD44 and CD62L expression: CD44- CD62L+ (naive), CD44+ CD62L+ (central memory), and CD44+ CD62L- (effector memory). (FIG. 11E-G) CTLs were restimulated with plate-bound anti-CD3 antibody on day 10 for 24 h in the presence of 10 μM FPL64176 or carrier control, followed by flow cytometry analysis. (E) Quantitative analysis of viable cell counts. (F) Percentage of cells positive for the apoptosis marker Apotracker™ Green. (G) Percentage of cells positive for the cell viability dye DAPI. (H) On days 7-8 post-stimulation, mouse CTLs were co-cultured with ovalbumin-pulsed EL-4 target cells at the indicated effector-to-target ratios in the presence of 10 μM FPL64176 and / or 1.25 mM BAPTA and respective carrier control, and subjected to LDH cytotoxicity assay for 3 h. n=4 independent experiments normalized to killing by carrier-treated cells at an effector:target cell ratio of 10:1. Error bars indicate SD. [Figure 12]GLI1 is important for tumor cell killing of human Vγ9Vδ2 gamma delta T cells, and treatment with the L-type voltage-gated Ca2+ receptor agonist FPL 64176 increases killing activity. PBMCs were isolated from buffy coats of healthy donors using SepMate PBMC Isolation Tubes. Vγ9Vδ2 expansion was induced by plating PBMCs at a concentration of 1×106 with 1 μM zoledronate in the presence of 100 IU / mL IL-2 and 120 IU / mL IL-15. Cells were split every 2–3 days from day 6 to day 20. Killing assays were performed between days 16–20. (A) Vy9V52 T lymphocytes were incubated overnight with Gli1 / 2 inhibitors GANT61 or EToH (carrier control) and then co-cultured with K562 target cells at the indicated effector to target ratios for 24 h and subjected to flow cytometry-based killing assays. n=1 donor. (B) Vy9V52 cells were pre-incubated with 10 μM FPL 64176 or EToH (carrier control) and then co-cultured with K562 target cells at the indicated effector to target ratios in the presence of 10 μM FPL 64176 or EToH (carrier control) and subjected to flow cytometric cytotoxicity assays. Killing was assessed after 24 h. Tumor cell killing normalized to carrier-treated cells at an effector:target ratio of 10:1. n=4 individual healthy donors from two independent experiments. [Figure 13]Treatment of tumor-specific CD8 T cells with the Cav channel agonist FPL 64176 followed by adoptive transfer into tumor-bearing mice increases tumor cell death in vivo. Cytotoxic OTI T cells were adoptively transferred via tail vein injection into MC38-Ova tumor-bearing Rag2 KO mice after in vitro restimulation in the presence of FPL 64176 or carrier control. 24 h after adoptive transfer, tumors were excised and analyzed for cleaved caspase 3 by immunohistochemistry (A). 3 mice per condition, % of CC3 across the entire tumor area for all mice. (B) Experiments repeated with 3-4 mice per condition. Each data point represents the average CC3 staining of 3 separate tumor sections. Overall CC3 staining (upper panel), as well as weak, moderate, and strong CC3 staining (lower row), respectively, are shown. FPL treatment does not appear to affect effector and central memory phenotypes in tumors, spleen and draining lymph nodes in vivo. [Figure 14] The L-type voltage-gated Ca2+ channel agonist FPL 64176 promotes the initiation of Ca2+ flux after TCR cross-linking and specifically increases Cav-mediated Ca2+ flux in mouse CD8+ cytotoxic T lymphocytes. Mouse CD8+ CTLs were loaded with Calcium Sensor Dye eFluor™ 514, anti-CD3ε antibodies and calcium channel inhibitors / agonists. Other calcium channels expressed in CD8 T cells were blocked: "Block" = BTP2 inhibits CRAC channels downstream of the TCR, and BCTC inhibits TRPV channels. All inhibitors are non-toxic to T cells at the concentrations used (data not shown). T cell receptor (TCR) cross-linking was induced by adding antibodies to cross-link the TCR 30 seconds after the start of sample acquisition. The arrow indicates the initiation of calcium flux after TCR cross-linking. The line indicates the baseline fluorescence of Calcium Sensor Dye eFluor™ 514. Data shown are representative of two independent experiments. [Figure 15]The GLI inhibitor GANT61 reduces tumor cell killing of human CD8+ cytotoxic T lymphocytes. PBMCs were isolated from buffy coats of healthy donors using SepMate PBMC Isolation Tubes. CD8+ T cells were isolated using negative selection (Miltenyi Biotec, CD8+ T Cell Isolation Kit) and plated in TexMACS cell culture medium supplemented with 100 IU / mL human IL-2 and 100 U / ml penicillin / streptomycin with 25 μl / mL ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator. Human CTLs were incubated overnight with the Gli1 / 2 inhibitor GANT61 or EToH (carrier control) and then cocultured with anti-CD3ε-pulsed P815 target cells at the indicated effector-to-target ratios for 3 h and subjected to LDH cytotoxicity assays. (A) Representative graph, (B) Quantification of N=3 donors normalized to killing by vehicle-treated cells at an effector:target ratio of 10:1. Error bars indicate SD. From two independent experiments with n=3 donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in the text are incorporated herein by reference.

[0044] Cav Channel Voltage-gated calcium channels (Ca V ) form a family of 10 members that are classified according to the expression of the pore-forming α1 subunit. These 10 members are grouped into 3 subfamilies: Ca V 1. Ca V 2 and Ca V 3. The Cav1 subfamily is also known as the L-type Cav family and is V 1.1(α 1S ), Ca V1.2(α 1C ), Ca V 1.3(α 1D ) and Ca V 1.4(α 1F The "L" in this subfamily stands for "long-lasting currents." The present invention relates to L-type Ca V Regarding.

[0045] immune cells The present invention provides a platform for engineered immune cells. It is envisioned that the engineered immune cells can be any cytotoxic lymphocyte. For example, the engineered immune cells can be CD8+ T cells, CD4+ T cells, or NK cells. The T cells can be gamma delta T cells. The inventors hypothesize that all cytotoxic lymphocytes rely on Gli1 for cell killing function. Cytotoxic lymphocytes exert their action on target cells by releasing granzymes, serine proteases that induce apoptosis in target cells. Granzymes are packaged in cytotoxic granules with perforin, a pore-forming protein that allows granzymes to enter target cells. Humans have five granzymes: granzyme A, granzyme B, granzyme H, granzyme K, and granzyme M.

[0046] operation The engineered immune cell platform can use any suitable genetic engineering approach, which can be nuclease-based.

[0047] For example, engineered immune cells can be engineered using CRISPR / Cas9 gene editing. This technique is based on the bacterial antiviral defense system CRISPR-Cas9. The Cas9 nuclease forms a complex with a synthetic guide RNA (gRNA), which guides the nuclease to the desired DNA site where it is cleaved.

[0048] Alternatively, engineered immune cells can be engineered using prime editing (Anzalone, 2019). The genome editing method uses a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, which simultaneously identifies the target site and carries a prime editing guide RNA (pegRNA) that encodes the desired edit. In this way, novel genetic information is written directly into the target DNA site.

[0049] Function acquisition "Gain-of-function" (or "gain of function" or "GOF"), as used herein, refers to a modification, e.g., a mutation, that enhances one or more recognized functions of a gene product. In particular, a gain-of-function mutation (GOF) in an L-type Cav channel may be a mutation that produces the following changes in the L-type Cav channel: a greater Ca 2+ Flux, Faster opening, Longer opening, Ca 2+ Enhanced flux initiation, slower inactivation kinetics, or other changes that result in greater calcium channel activity in vitro or in vivo compared to non-mutated L-type Cav channels tested under similar conditions. Specific examples of L-type Cav channel GOF mutations are described above (see, e.g., Table 1).

[0050] PTCH1 The protein "protein patch homolog 1" has a human amino acid sequence disclosed in UniProt Q13635 (UniProt Release 2023_01 on February 22, 2023) and is encoded by the PTCH1 gene (NCBI GeneID: 5727). Several variants of PTCH1 have been reported to be present in human patients with Gorlin syndrome. For example, rs1843916565. All of the PTCH1 mutations associated with Gorlin syndrome disclosed in UniProt Q13635 (in the section entitled "Disease & Variants") are expressly incorporated herein by reference in their entirety. PTCH1 mutations that give rise to Gorlin syndrome are considered herein to be PTCH1 loss-of-function mutations.

[0051] Chimeric Antigen Receptor Chimeric antigen receptor (CAR) is a recombinant receptor molecule that has both antigen binding and T cell activation functions.The structure and operation of CAR are, for example, outlined in Dotti et al., Immunol Rev (2014) 257 (1), which is incorporated herein by reference in its entirety.

[0052] CAR comprises an antigen-binding domain linked to a transmembrane domain and a signaling domain. An optional hinge domain can separate the antigen-binding domain and the transmembrane domain and can act as a flexible linker.

[0053] The antigen-binding domain of a CAR may be based on the antigen-binding region of an antibody specific for the antigen that the CAR targets. For example, the antigen-binding domain of a CAR may comprise the amino acid sequence of the complementarity determining region (CDR) of an antibody that specifically binds to a target protein. The antigen-binding domain of a CAR may comprise or consist of the variable region amino acid sequence of the light and heavy chains of an antibody that specifically binds to a target protein. The antigen-binding domain may be provided as a single-chain variable fragment (scFv) that comprises the sequence of the variable region amino acid sequence of the light and heavy chains of an antibody. The antigen-binding domain of a CAR may target antigens based on other protein:protein interactions, such as ligand:receptor binding; for example, an IL-13Rα2-targeting CAR has been developed using an antigen-binding domain based on IL-13 (see, e.g., Kahlon et al., 2004 Cancer Res 64(24):9160-9166).

[0054] The transmembrane domain is provided between the antigen binding domain and the signal transduction domain of the CAR. The transmembrane domain fixes the CAR to the cell membrane of the cell expressing the CAR, with the antigen binding domain in the extracellular space and the signal transduction domain in the intracellular space. The transmembrane domain of the CAR can be derived from the transmembrane region sequence of CD3-zeta, CD4, CD8 or CD28.

[0055] The signaling domain allows activation of T cells. The CAR signaling domain may contain amino acid sequences from the intracellular domain of CD3-ζ that provide an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signaling domains containing sequences from other ITAM-containing proteins, such as the domain containing the ITAM-containing region of FcγRI (Haynes et al., 2001 J Immunol 166(1):182-187), have also been used in CARs. CARs containing signaling domains derived from the intracellular domain of CD3-ζ are often referred to as first generation CARs.

[0056] The signal transduction domain of CAR can also contain a costimulatory sequence derived from the signal transduction domain of a costimulatory molecule to promote the activation of CAR-expressing T cells when binding to target protein.Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS and CD27.CARs with a signal transduction domain that contains additional costimulatory sequences are often called second-generation CARs.

[0057] In some cases, CARs are engineered to costimulate different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, while 4-1BB-mediated signaling is via the TNF receptor-associated factor (TRAF) adaptor protein. Thus, the signaling domain of a CAR may contain multiple costimulatory sequences derived from the signaling domains of two or more costimulatory molecules. CARs that contain a signaling domain with multiple costimulatory sequences are often referred to as third-generation CARs.

[0058] The optional hinge region can provide separation between the antigen-binding domain and the transmembrane domain and can function as a flexible linker. The hinge region can be a flexible domain that allows the binding sites to face in different directions. The hinge region can be derived from the IgG1 or CH2CH3 regions of immunoglobulins.

[0059] Adoption In an embodiment of the present invention, the method of treatment or prevention may include adoptive transfer of immune cells, particularly T cells. Adoptive T cell transfer generally involves a process of obtaining T cells from a subject, typically by drawing a blood sample and isolating T cells from the sample. The T cells are then typically treated or altered in some way, optionally expanded, and then administered to either the same subject or a different subject. Treatment typically aims to provide a subject with a T cell population with certain desired characteristics, or to increase the frequency of T cells with such characteristics in the subject. Adoptive transfer of CAR-T cells is described, for example, in Kalos and June 2013, Immunity 39(1): 49-60, which is incorporated herein by reference in its entirety.

[0060] Embodiments of the present invention may also include adoptive transfer of NK cells. In some embodiments, the subject from which the T cells are isolated is the subject to which the modified T cells are administered (i.e., the adoptive transfer is of autologous T cells). In some embodiments, the subject from which the T cells are isolated is a different subject than the subject to which the modified T cells are administered (i.e., the adoptive transfer is of allogeneic T cells).

[0061] T cell therapy T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation.

[0062] T cells for immunotherapy can be obtained from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumor. In addition, T cells can be obtained from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using many techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Further methods of isolating T cells for T cell therapy are disclosed in US2013 / 0287748, which is incorporated herein by reference in its entirety.

[0063] Gli1 reporter assay Any suitable Gli1 reporter assay can be used as described herein. Gli1 reporter assay can be used to determine the stimulation of L-type Cav channel activity. For example, Gli1 reporter assay can be any assay that detects binding to Gli1 consensus sequence. Such assay can use fluorescent probe or can be colorimetric assay. Gli1 reporter assay can detect Gli1 mRNA by qRT-PCR. Gli1 reporter assay can detect Gli1 protein level and nuclear translocation by immunofluorescence.

[0064] cancer "Cancer" may include any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenal cortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer of the female reproductive system, cancer of the male reproductive system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia disease, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, cancer of the nervous system, neuroblastoma, non-Hodgkin's lymphoma, cancer of the oral cavity, cancer of the oropharynx, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumors, plasma cell neoplasms, primary CNS lymphoma, prostate cancer, rectal cancer, cancer of the respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, cancer of the stomach, cancer of the stomach pouch, testicular cancer, thyroid cancer, cancer of the urinary tract system, uterine sarcoma, vaginal cancer, cancer of the vascular system, Waldenström's macroglobulinemia, and Wilms' tumor.

[0065] The cancer may be of a particular type. Exemplary cancer types include astrocytoma, carcinoma (e.g., adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g., angiosarcoma, chondrosarcoma, osteosarcoma).

[0066] In a preferred embodiment, the cancer is melanoma, lung cancer, colorectal cancer or breast cancer. Preferably, the cancer may also be lymphoma, leukemia (including acute lymphoblastic leukemia and chronic lymphocytic leukemia) or multiple myeloma.

[0067] Therapies for cancer, such as the use of the compositions and / or cells of the invention, may be advantageously combined with other anti-cancer therapies, such as checkpoint inhibitors (e.g. anti-PD-1, anti-PD-L1, anti-CTLA4 antibodies such as nivolumab, atezolizumab or pembrolizumab).

[0068] autoimmune disease Autoimmune disease is an inflammatory disease resulting from the abnormal activation of immune response.Without being bound by theory, it is believed that autoimmune disease may result from the failure of immune control process (e.g., including failure of thymic selection) or from the persistent activation of immune response (e.g., through the persistent release of damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs)).Autoimmune disease is often associated with the production of autoreactive autoantibodies, and / or autoreactive lymphocytes, such as autoreactive T cells and B cells.

[0069] It is widely known that autoimmune diseases can affect all aspects of the body. The term "autoimmune disease" may refer to any one or more of the following diseases: acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) (including fulminant antiphospholipid syndrome (CAPS)), autoimmune Addison's disease (AAD), autoimmune autonomic ganglionopathy (AAG), autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis (including acute disseminated encephalomyelitis (ADEM)), autoimmune gastritis, autoimmune hemolytic anemia (AIH ... encephalitis (including acute disseminated encephalomyelitis (ADEM)), autoimmune encephalitis (including acute disseminated encephalomyelitis (ADEM)), autoimmune encephalitis (including acute disseminated encephalomyelitis (ADEM)), autoimmune encephalitis (including acute A), autoimmune hepatitis (AIH), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), autoimmune polyglandular syndromes (including types I, II, and III), autoimmune progesterone dermatitis, Barro's disease, Behçet's disease, birdshot uveitis, bullous pemphigoid, celiac disease, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, Cogan's syndrome, cold agglutinin disease, CREST syndrome, Crohn's disease (CD), cutaneous lupus erythematosus, dermatitis herpetiformis, dermatomyositis, Devic's disease, 1 Type 2 diabetes, discoid lupus, eosinophilic fasciitis, Evans syndrome, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, Henoch-Schönlein purpura, Hurst disease, Berger's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP), inflammatory bowel disease (IBD), juvenile idiopathic arthritis, juvenile myositis, Lambert-Eaton myasthenic syndrome (LEMS), linear immunoglobulin A disease (LAD), lupus nephritis, Meniere's disease, mixed kidney disease Multiple sclerosis (MS), myasthenia gravis (MG), ocular cicatricial pemphigoid, relapsing rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, paroxysmal nocturnal hemoglobinuria (PNH), Parsonage-Turner syndrome, herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis (PBC), psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), Raynaud's syndrome, Reiter's syndrome, relapsing polychondritis, rheumatic fever, rheumatoid arthritis (RA),Sarcoidosis, Schmidt syndrome, scleroderma, Sjögren syndrome, small fiber sensory neuropathy, systemic lupus erythematosus (SLE), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD) and vitiligo.

[0070] Preferably, the autoimmune disease is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) or type 1 diabetes.

[0071] infectious disease Infectious diseases are characterized by infection of a patient with an infectious agent, which typically involves activation of an immune response. Infectious agents can include microorganisms, such as viruses, bacteria, protozoa, or fungi. Small animals can also be considered infectious agents, and are usually referred to as "parasites." Additionally, the term "infectious agent" can also refer to prions.

[0072] Viruses can include hepatitis viruses, adenoviruses, rabies viruses, papilloma viruses, rotaviruses, herpes viruses, cytomegaloviruses, and bacteriophages. In some embodiments of the present disclosure, the term "virus" can also refer to human retroviruses, such as HIV. The term "virus" can also refer to pandemic coronaviruses, including SARS-CoV-II viruses.

[0073] Pathogenic bacteria include species of Staphylococcus, Streptococcus, Neisseria, Clostridium, Bacillus, Listeria, Escherichia, Salmonella, Vibrio, Campylobacter, Bordetella, Haemophilus, Legionella, Helicobacter, Mycobacterium, Treponema, Borrelia, Chlamydia, and Rickettsia. Pathogenic protozoa include species of Entamoeba, Trichomonas, Leishmania, Chilomonas, Giardia, Isopora, Sarocystis, Nosema, Balantidium, Histomonas, Trypanosoma, Plasmodium, Babesia, Haemoproteus, and Eimeria. Pathogenic fungi include species of Candida, Pneumocystitis, Histoplasma, Blastomyces, Malassezia, and Cryptococcus. Parasites can include intracellular or extracellular parasites such as species of Ancylostoma, Ascaris, Enteroblus, Paragonimus, Schitosoma, Taenia, Trichuris, Wuchereria, Balantidium, Entamoeba, Giardia, Leishmania, Plasmodium, Trichomonas, and Trypanosoma. Infectious diseases caused by prions can include Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (CJD), variably protease-sensitive prion disease (VPSPr), Gerstmann-Sträussler-Scheinker disease (GSS), kuru, and fatal insomnia (FI).

[0074] Preferably, the infectious disease is infection with HIV, SARS-CoV-II, Hepatitis C, Hepatitis B, human cytomegalovirus or an opportunistic fungal infection such as aspergillosis.

[0075] subject The subject to be treated according to the present invention may be any animal or human. The subject is preferably a mammal, such as a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, more preferably a human. The subject may be male or female. The subject may be a patient. The subject may have been diagnosed with a disease or condition (e.g., cancer in need of treatment), may be suspected of having such a disease or condition, or may be at risk of developing such a disease or condition. The subject may be a young person, such as a human child (e.g., a child with pediatric cancer).

[0076] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, have, where appropriate, been expressed in a specific form, or in terms of means for performing a disclosed function, or a method or process for obtaining a disclosed result, and may be utilized to realize the invention in its diverse forms, either separately or in any combination of such features.

[0077] Although the present invention has been described in relation to the exemplary embodiments above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present invention described above are considered to be illustrative and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of the present invention.

[0078] For the avoidance of any doubt, any logical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these logical explanations.

[0079] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification (including the claims which follow), unless the context indicates otherwise, the words "comprise" and "include", as well as variations such as "comprises", "comprising", and "including", are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps.

[0080] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. The term "about" in reference to numerical values ​​is arbitrary and means, for example, ±10%. EXAMPLES

[0081] The following data are provided to demonstrate the efficacy and therapeutic utility of the present invention. These data are intended as examples and are not intended to limit the scope of protection.

[0082] background Hedgehog (Hh) signaling is known to control T cell killing by regulating the formation of immune synapses. For example, de la Roche et al. (2013) report that stimulation of the T cell receptor (TCR) of mouse CD8+ T cells drives Hh activation, which initiates the expression of downstream Hh components, including Gli1. The authors clearly identify the role that Hh signaling plays in centrosome polarization and actin reorganization of CTLs, as well as in controlling immune synapse formation and thus effector function of CTLs.

[0083] It is also known that Hh signaling in mouse CD8+ T cells cannot be induced by administration of extracellular Hh ligand (de la Roche et al. (2013)). Hh induction occurs not through the administration but through the regulation of L-type voltage-gated calcium (Cav) channels downstream of the T cell receptor. For example, Hanna et al. (2021) report that nifedipine-sensitive Cav controls Gli1 induction in naive CD8+ T cells and CD8+ CTLs after TCR stimulation. It has also been reported that Cav1 family channels function independently of classical Hh signaling to mediate specific cell killing by mouse CD8+ CTLs in a Gli1-dependent manner. The authors further show that Cav1 family channels are involved in Gli1 induction in human CTLs. As shown in Figure 15, the GLI1 inhibitor GANT61 reduces tumor cell killing of human CD8+ cytotoxic lymphocytes, which is consistent with the known role of Gli1 in tumor cell killing.

[0084] However, it is widely recognized that calcium signaling processes are highly complex and tightly regulated, and the actions of L-type Cav channel agonists in controlling lymphocyte activation and effector functions are not fully understood.

[0085] Experiment 1: L-type Cav agonists specifically enhance Gli1 transcription levels and potentiate specific tumor cell killing in TCR-stimulated mouse CD8+ CTLs To investigate how L-type Cav channel agonists act in regulating the activation and effector function of CD8+ CTLs, CD8+ T cells were isolated from the spleens and inguinal lymph nodes of Rag2- / - OT-I mice and CTLs were generated. For reference, Rag2- / - mice do not develop mature T and B lymphocytes. OT-I mice carry a transgene encoding a TCR that recognizes a chicken ovalbumin (Ova) peptide fragment. Rag2- / - OT-I mice facilitate the isolation of a naive CD8+ T cell population. Plates were coated using 2.5 μg / mL anti-CD3ε mAb and CD8+ CTLs were stimulated via the TCR for 3 hours in the presence of 5 or 10 μM L-type Cav channel agonist FPL 64176 or carrier control. CD8+ CTLs were preincubated with FPL 64176 or vehicle control for 40–50 min before stimulation. Gli1 expression was assessed using qRT-PCR. As shown in Figures 1A and 1C, administration of FPL 64176 increased Gli1 expression in mouse CD8+ CTLs in a dose-dependent manner.

[0086] To determine whether this increase in Gli1 expression correlates with increased cellular effector function, an LDH cytotoxicity assay was performed. LDH (lactate dehydrogenase) is a stable cytoplasmic enzyme that is released into the extracellular environment upon cell membrane damage. The LDH assay evaluates cell viability by measuring LDH enzyme activity in cell culture supernatants. Briefly, CD8+ CTLs from Rag2- / - OT-I mice were co-cultured with EL-4 target cells pulsed with Ova peptide at effector:target ratios shown in Figures 1B and 1D in the presence of 10 μM FPL 64176 or carrier control. As shown, specific cell killing of EL-4 tumor cells over a range of effector-to-target ratios is enhanced by at least 100% by administration of FPL 64176, suggesting that increased expression of Gli1 acts to enhance the effector function of CD8+ CTLs.

[0087] To evaluate whether other clinically approved drugs with potential L-type Ca channel agonist activity, in addition to FPL 64176, could enhance Gli1 transcription, we stimulated CTLs in the presence of FPL 64176, (R)-baclofen and nefiracetam and assessed Gli1 transcription using qRT-PCR. 2+ While (R)-baclofen acts as a potent selective activator of the GABA channel, B Nefiracetam has been described to act as a receptor agonist and facilitate L-type channel currents in some circumstances, and it has been shown to have some agonist activity at L-type voltage-gated calcium channels and to inhibit GABA A It is presumed to act as a receptor agonist.

[0088] Briefly, naive CD8+ T cells were isolated from the spleens and inguinal lymph nodes of C57BL / 6 wild-type mice to generate CTLs. CTLs were restimulated for 6 h on plates precoated with anti-CD3ε (2.5 μg / ml) antibody in the presence of the indicated concentrations of FPL 64176 (Figure 2A); (R)-baclofen (Figure 2B); nefiracetam (Figure 2C) (or the respective carrier controls). Note that CD8+ CTLs were also preincubated for 40–50 min with the appropriate drug or carrier control before restimulation. RNA was extracted from the cells, and Gli1 expression was assessed by qRT-PCR. As shown in Figure 2, only FPL 64176 succeeds in enhancing Gli1 expression after TCR activation. In contrast, administration of (R)-baclofen had little effect on Gli1 expression, and nefiracetam reduced Gli1 expression by approximately 50%. These data demonstrated that a potent and specific agonist of L-type Cav channels can increase Gli1 expression in mouse CTLs.

[0089] Experimental 2 - L-type Cav agonists enhance GLI1 expression and specific tumor cell killing in TCR-stimulated naive CD8+ T cells and CTLs To evaluate the effect of L-type Cav channel agonists in controlling the activation and effector function of human CD8+ T cells, naive primary human CD8+ T cells were purified from the buffy coat layer of blood isolated from healthy donors. The cells were stimulated for 24 hours using ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator beads in the presence of 5 or 10 μM FPL 64176 or carrier control, and GLI1 mRNA expression was evaluated by qRT-PCR. As shown in Figure 3A, administration of FPL 64176 increased GLI1 expression in a dose-dependent manner. Human CD8+ CTLs were then restimulated with plate-bound anti-CD3 for 24 hours between days 15 and 20 after the first TCR stimulation in the presence of 10 μM FPL 64176 or carrier control, and GLI1 mRNA expression was evaluated by qRT-PCR. As shown in Figure 3B, administration of FPL 64176 increased GLI1 expression. The data from Figures 3A and 3B together support the data generated using mouse CD8+ CTLs obtained in experiment 1.

[0090] The effect of L-type Cav agonists on specific tumor cell killing by human CD8+ CTLs was then evaluated by performing a VITAL assay. The VITAL assay is a flow cytometry-based assay that is widely used to evaluate cell killing. It was hypothesized that increased GLI1 expression would lead to enhanced specific tumor cell killing. Briefly, CTLs were co-cultured with CFSE-labeled P815 target cells at the indicated effector-to-target cell ratios in the presence of FPL 64176 or carrier control (Figure 3C and 3D). Note that CD8+ CTLs were also pre-incubated with the appropriate drug or carrier control for 40–50 min prior to the VITAL assay. As shown, administration of FPL 64176 enhanced cell killing by approximately 70%, supporting the hypothesis that increased GLI1 expression downstream of L-type Cav channel activation acts to enhance the effector function of CD8+ T cells. For clarity, this observation confirms the data generated using mouse CD8+ CTLs obtained in experiment 1.

[0091] Experiment 3 – Gli1 expression is important for the cytolytic function of mouse NK cells It was hypothesized that the therapeutic effects of L-type Cav channel agonists may be conserved between CD8+ T cells and NK cells. To examine this hypothesis, NK cells were isolated from the spleens of C57BL / 6 wild-type mice using the MACS negative isolation protocol and expanded in vitro for 7 days in the presence of 500 IU / L IL-2. LDH assays (Figure 4, left panel) and VITAL assays (Figure 4, right panel; RMA tumor target cells are insensitive to killing by NK cells and were used as reference cells in the VITAL assay) were performed to assess specific tumor cell killing. Briefly, NK cells were co-cultured with NK-sensitive RMA / S cells in the presence of 5 μM GANT61 or carrier control. For reference, GANT61 (NSC 136476) inhibits the binding of both Gli1 and Gli2 to DNA. As shown in Figure 4A, NK cells treated with GANT61 were found to have approximately 15% reduced cell killing as measured by LDH assay compared to NK cells treated with carrier control. Similarly, specific tumor cell killing as determined by VITAL assay (Figure 4A, right panel) was significantly reduced in cells treated with GANT61 compared to carrier control.

[0092] To elucidate in more detail the contribution of Gli1 to NK cell killing, NK cells were isolated from WT and Gli1- / - (KO) mice. Cells were co-cultured with RMA / S cells as described above (without GANT61 treatment) and cell killing was assessed using both the LDH assay (left panel) and the VITAL assay (right panel, NK-insensitive RMA cells as control). As shown in Figure 4B, Gli1 KO NK cells were found to have reduced cell killing to the same extent as that by GANT61-treated NK cells (Figure 4A). Combined, these data indicate that Gli1 plays an important role in mediating the cytolytic function of NK cells, supporting the data obtained in experiments 1 and 2.

[0093] Experiment 4 - L-type Cav agonists drive Gli1 transcription and promote specific cell killing by human NK cells To examine whether L-type Cav channel agonists could also enhance GLI1 mRNA levels in the human NK cell line NK-92 (a non-Hodgkin's lymphoma cell line, expressing only Cav1.3), NK-92 cells were stimulated for 4 hours using anti-NKp46 mAb (5 μg / mL) in the presence of either 10 μM FPL 64176 or 100 μM Bay K8644. For reference, Bay K8644 is a structural analogue of nifedipine and acts as an L-type Cav channel agonist. GLI1 expression was assessed using qRT-PCR. As shown in Figure 5A, GLI1 expression was increased in NK-92 cells stimulated using anti-NKp46 mAb in the presence of FPL 64176 compared to cells stimulated in the presence of carrier control. No significant increase was observed in unstimulated cells in the presence of FPL 64176. Similarly, GLI1 mRNA levels were increased (almost 2-fold) in NK-92 cells stimulated with Bay K8644 compared to cells treated with carrier control (Figure 5B). These observations confirm that the GLI1-inducing effect of Cav agonists is not limited to compound FPL 64176, particularly in NK cells.

[0094] To determine whether increased GLI1 expression correlates with increased effector function of NK-92 cells, NK-92 were co-cultured with K562 myeloid leukemia cells in the presence of 10 μM FPL 64176 or carrier control at the effector:target ratios shown in Figure 5C. Cell killing was assessed using an LDH assay as previously described. As shown in Figure 5C, NK-92 cells exhibited enhanced target cell killing upon treatment with FPL 64176 compared to cells treated with carrier control.

[0095] Combined, these data support the data generated with mouse and human CD8+ CTLs from experiments 1 and 2, thereby confirming that the mechanism of action of L-type Cav agonists (including, for example, FPL 64176 and Bay K8644) is conserved within cytotoxic lymphocyte populations. In particular, these data indicate that the effects of L-type Cav channel agonists are conserved at least between mouse and human CD8+ CTLs and NK cells.

[0096] Experiment 5 - Mutations driving a gain-of-function Hh phenotype act to enhance specific cell killing by NK cells Gorlin syndrome patients are known to have one of several possible germline mutations and deletions that cause loss of function of PTCH1 in the protein patch homolog 1 (PTCH1) gene. PTCH1 is a negative regulator of the pathway. Thus, PTCH1 loss-of-function mutations enhance classical Hh signaling and enhance GLI1. To evaluate the effect of PTCH1 mutations, NK cells were isolated from Gorlin syndrome patients and healthy control individuals and cultured for 2 days with IL-2. On day 2, the levels of GLI1 mRNA were determined by qRT-PCR (Figure 6A). As shown, NK cells from Gorlin patients had higher GLI1 mRNA levels. Next, NK cells from Gorlin syndrome patients and healthy control individuals were co-cultured with K562 target cells at the effector-to-target ratio shown in Figure 6B. As shown, it was observed that NK cells isolated from patients with Gorlin syndrome acted as more potent killers than NK cells isolated from healthy control individuals. Without wishing to be bound by any particular theory, the inventors believe that approaches aimed at inhibiting, reducing or downregulating PTCH1 function (and concomitantly increasing GLI1 signaling) in immune cells will recapitulate the enhanced cell killing observed in NK cells isolated from patients with Gorlin's syndrome. Thus, immune cells engineered to reduce PTCH1 function and / or expression, or gene silencing approaches such as CRISPR or siRNA-based silencing of PTCH1 in immune cells, may provide novel immune cell-based therapeutic avenues for the treatment of various cancers.

[0097] To determine whether the increased specific cell killing seen in Gorlin patients could be achieved by engineering cells to express gain-of-function mutations that increase Gli1 signaling, NK-92 cells were engineered to stably express the I756T GOF mutation in Cav1.4. Empty vector (EV) and Cav1.4 (WT) were used as controls. The loss-of-function mutation S229P was used as a negative control. K562 tumor cell killing was assessed after 24 hours by LDH killing assay. As shown in Figure 7, the I756T mutant increased cell killing. Thus, gain-of-function effects in the non-classical Hedgehog pathway can be achieved by genetic engineering of Cav channels. These mutations do not affect cell survival and proliferation (data not shown).

[0098] Experiment 6 - Transcription of Gli1 mediates specific cell killing by mouse cytotoxic CD4+ T cells Experiments 1, 2, 3, and 4 identify Gli1 transcription as a mediator of specific cell killing of CD8+ CTLs and NK cells in humans and mice. To examine whether Gli1 also plays a role in driving effector function of CD4+ T cells, cells were isolated from the spleens of OT-II mice and CTLs were generated. Cells were treated with GANT61 at a concentration of 5 μM or carrier control 24 hours prior to co-culture with B cells (as target cells) at the effector:target ratio shown in FIG. 8. As shown, CD4+ CTLs treated with GANT61 exhibited a 50% reduction in specific cell killing compared to CD4+ CTLs treated with carrier control. This observation suggests that the function of Gli1 in promoting cell effector function is conserved between CD8+ and CD4+ CTLs, as well as NK cells, which supports the results of experiments 1, 2, 3, and 4.

[0099] Experiment 7 - Cytokine production and maintenance of cytolytic effectors are unaffected by Gli1 inhibition in all cytotoxic lymphocytes The data presented in Examples 1-6 show a novel L-type Cav channel-mediated regulation of killing, which is mediated by Gli1 and TCR-induced. To further investigate this regulation, the effect of GANT61 inhibition on granzyme B and IFNγ secretion was examined. Cytotoxic CD4+ cells were generated from OT-II mice and treated with 5 μM GANT61 or carrier control for 24 hours on day 7, followed by flow cytometric analysis of granzyme B (GzmB) and IFNγ levels on day 8. NK cells were isolated from C57BL / 6 mice and treated with 10 μM GANT61 or carrier control (EtOH) overnight (approximately 18 hours) before analysis of intracellular granzyme B (GzmB) and IFNγ expression by flow cytometry. In both cytotoxic CD4+ cells (Figure 9) and NK cells (Figure 10), GzmB content and IFNγ secretion were not affected by Gli inhibition.

[0100] Figures 9 and 10 show that the pathway we uncovered behaves differently from the reported role of Ca2+ channels in cytokine production and maintenance of cytolytic effectors. Interestingly, we have previously shown that the effect in cytotoxic CD8+ T cells is the same (De la Roche, 2013), and these results indicate that this mechanism is shared among all cytotoxic lymphocytes.

[0101] Experiment 8 - L-type Cav agonist treatment inhibits CD8 + It does not impair T cell viability or proliferation, does not affect memory phenotype, and does not inhibit extracellular Ca 2+ Enhances CTL killing via To ensure that the use of L-type Cav agonists does not adversely affect immune cells, we assessed general cellular function. +T cells were stimulated with plate-bound anti-CD3 / CD28 antibodies in the presence of 10 μM FPL64176 or carrier control for 24 h, followed by flow cytometry analysis (Figure 11A-D). L-type Cav agonists significantly increased CD8 T cell proliferation, as shown by analysis of viable cell counts (A), analysis of cells positive for the apoptotic marker Apotracker™ Green (B), and analysis of cells positive for the cell viability dye DAPI (C). + There was no effect on T cell viability or proliferation. Central and effector memory were also shown to be unaffected (D), and analysis of T cell subsets based on CD44 and CD62L expression showed no significant differences between agonist and control: CD44 - CD62L + (Naive), CD44 + CD62L + (Central Memory), and CD44 + CD62L - (effector memory). CTLs maintained viability and proliferation as determined by flow cytometry analysis after restimulation with plate-bound anti-CD3 antibody for 24 h on day 10 in the presence of 10 μM FPL64176 or carrier control (E–G). 2+ To determine whether the Gli1 pathway requires extracellular calcium, the calcium chelator BAPTA was added to the LDH cytotoxicity assay. BAPTA is a calcium chelator that can chelate any extracellular Ca2+ binding sites if the Gli1 pathway requires extracellular calcium. 2+ As can be seen in Figure ​ Figure1H,1H, in the presence of BAPTA, killing by immune cells was dramatically reduced. Thus, Gli1-mediated killing is mediated by extracellular Ca 2+ It depends on the existence of

[0102] Experimental 9 - L-type Cav agonists enhance the killing capacity of human gamma delta T cells Experiments 1-6 demonstrated that GLI1 transcription is involved in the expression of CD8 + A mediator of specific cell killing by CTL and NK cells, and in mice, CD4 +It is shown that GLI1 also plays a role in driving T cell effector function. To investigate whether GLI1 also plays a role in driving gamma delta T cell effector function, Vy9Vd2 cells were prepared from PBMCs of healthy human donors. Killing assays were performed between days 16 and 20. Figure 12A shows that gamma delta T cells pretreated with the GLI1 inhibitor GANT61 have a decreased ability to kill K562 target cells. Gamma delta T cells were also pretreated with 10 μM FPL 64176 or ETOH (vehicle control) before co-culture with K562 target cells at the indicated effector-to-target ratios and subjected to flow cytometric cytotoxicity assays. Figure 12B shows that gamma delta T cells treated with FPL 64176 have an increased killing activity compared to the control. Thus, Gli1 also controls the killing capacity of gamma-delta T cells, and the killing activity of these cells can be enhanced by L-type Cav agonists.

[0103] Experiment 10 - Treatment of tumor-specific CD8 T cells with the Cav channel agonist FPL 64176 followed by adoptive transfer into tumor-bearing mice increases tumor cell death in vivo As mentioned above, the present invention can be utilized for adoptive transfer. To examine the applicability of Cav channel agonist treatment in adoptive transfer treatment regimens, cytotoxic OTI T cells were adoptively transferred via tail vein injection into MC38-Ova tumor-bearing Rag2 KO mice after in vitro restimulation in the presence of FPL 64176 or carrier control. 24 hours after adoptive transfer, tumors were excised and analyzed for cleaved caspase 3 by immunohistochemistry. Figures 13A and B show that treating tumor-specific T cells with Cav channel agonist prior to adoptive transfer increases tumor cell killing compared to adoptive transfer with T cells that were not treated with Cav channel agonist. This data validates the feasibility of the present invention for use in adoptive transfer. FPL treatment does not appear to affect effector and central memory phenotypes in tumors, spleens and draining lymph nodes in vivo (data not shown).

[0104] Experiment 11 - L-type voltage-gated Ca 2+ Ca channel agonist FPL 64176 2+ Effect on flux To investigate the mechanism by which L-type Cav agonists exert their effects, calcium flux experiments were performed on mouse CD8 + The experiment was carried out with cytotoxic T lymphocytes. 2+ Treatment with the channel agonist FPL 64176 reduced Ca 2+ Promotes flux initiation and inhibits murine CD8 + CaV-mediated Ca in cytotoxic T lymphocytes 2+ These results show that it specifically increases the flux.

[0105] summary Cytotoxic lymphocytes, particularly CD8+ T cells and NK cells, are important in the elimination of infected and malignant cells. The unprecedented success of immunotherapy utilizing the killing potential of cytotoxic lymphocytes has prompted research into ways to enhance their killing potential. The advantages of the present invention will become apparent from a comparison of the results disclosed herein with other approaches.

[0106] One example described in the art is the deletion of the transcription factor Bach2 in CD8+ T cells described in Barton (2022). Bach2 is highly expressed in naive T cells and prevents terminal differentiation into effector cells by restricting TCR-driven gene expression and by increasing memory cell generation. Furthermore, Bach2 is important for the differentiation of stem-like CD8+ T cells, which are essential for tumor control in vivo and for responding to checkpoint inhibition.

[0107] Barton (2022) shows that genetic knockout of Bach2 in CD8+ T cells enhances in vitro killing by murine CTLs by 50-75% in most populations (Figure 1 in Barton 2022). In contrast, the present invention increases killing by murine CTLs by 100% (Example 1 and Figure 1). Furthermore, Barton (2022) shows that the killing phenotype is enhanced by Bach2 - / - Changes in the differentiation phenotype of CD8+ T cells, differences in the proportion of CD8 T cells in the populations tested, and Bach2 - / - This was due in part to enhanced T-cell proliferation. When CTLs were derived from similar starting populations, CTL killing was enhanced by only 15-50% and was due to an increase in cytolytic granules containing high levels of lytic proteins.

[0108] The present invention provides better overall killing and does not affect the overall levels of proliferation, differentiation, and solubility proteins, which are predicted to be terminally differentiated in vivo by Barton (2022)'s Bach2 - / - In contrast to T cells, the present invention specifically targets the killing mechanism.

[0109] The present invention utilizes a novel non-classical Hedgehog signaling pathway downstream of the TCR that is independent of NFAT, AP-1, and NFkB signaling and is associated with calcium flux (Hanna, 2021). Importantly, NFAT has been shown to be a key driver of T cell exhaustion (Martinez, 2015). As such, the present invention is not expected to induce an exhaustion phenotype.

[0110] Data from experiments 1, 2, 3, and 4 show that L-type Cav agonists act to enhance Gli1 expression in mouse and human CD8+ CTLs and NK cells; and to enhance specific cell killing of multiple tumor cell types. Data from experiment 7 show that this effect extends to CD4+ CTLs, and experiment 9 shows that this effect extends to gamma-delta T cells. These are the first experimental data to link L-type Cav channel agonists (e.g., FPL 64176 and Bay K8644) to hedgehog activation and Gli1 transcription, and to utilize L-type Cav channel agonists to enhance cytotoxic effector function of CD8+ CTLs, NK cells, and CD4+ CTLs. Experiment 10 shows that this approach can be used for adoptive transfer.

[0111] In summary, the experimental data obtained support the efficacy and therapeutic utility of the present invention and indicate that immune cells expressing L-type Cav agonists and / or engineered L-type Cav channels are likely to be useful tools for use in the treatment of disease.

[0112] References Several publications are cited above in order to more fully describe and disclose the invention and the state of the art to which it pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety. Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature (2019) 576:149-157 Badou et al., Emerging roles of L-type voltage-gated and other calcium channels in T lymphocytes. Front Immunol 4, 243 (2013). Barton, P.R. et al. Super-killer CTLs are generated by single gene deletion of Bach2. Eur J Immunol 52, 1776-1788 (2022) de la Roche et al., Hedgehog Signaling Controls T Cell Killing at the Immunological Synapse. Science (2013) Hamara-Wahanui et al., A CACNA1Fmutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation. PNAS (2005) 102(21) Hanna et al., Non-canonical Hedgehog signaling through L-type voltage gated Ca 2+ channels controls CD8 + T cell killing. BioRχiv (2021) Martinez, G.J. et al. The transcription factor NFAT promotes exhaustion of activated CD8(+) T cells. Immunity 42, 265-278 (2015). Kotturi and Jefferies, Molecular characterization of L-type calcium channel splice variants expressed in human T lymphocytes. Molecular Immunology (2005) Striessnig, et al. Voltage-Gated Ca 2+-Channel α1-Subunit de novo Missense Mutations: Gain or Loss of Function - Implications for Potential Therapies. Frontiers in Synaptic Neuroscience (2021) 13(634760) Williams et al., Functional impact of a congenital stationary night blindeness type 2 mutation depends on subiunit composition of Cav1.4 Ca(2+) channels. J Biol. Chem. (2020) 295:17215-17226

[0113] For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

1. An engineered immune cell, the engineered immune cell comprising a gain-of-function mutation in a Ca channel and / or overexpressing an L-type voltage-gated calcium (Ca) channel.

2. 2. The engineered immune cell of claim 1, comprising a gain-of-function mutation in CACNA1F.

3. 3. The engineered immune cell of claim 1 or claim 2, which overexpresses Cav1.4 compared to a wild-type immune cell of the same type.

4. The engineered immune cell of any one of claims 1 to 3, wherein the CACNA1F mutation comprises I745T.

5. The engineered immune cell of any one of claims 1 to 4, selected from the group consisting of T cells, CAR-T cells, NK cells and CAR-NK cells.

6. 1. An engineered immune cell for use in medicine, the engineered immune cell comprising a gain-of-function mutation in an L-type Cav channel and / or overexpressing an L-type Cav channel.

7. 1. An engineered immune cell for use in a method of treating cancer in a subject, the engineered immune cell comprising a gain-of-function mutation in an L-type Cav channel and / or overexpressing an L-type Cav channel.

8. 8. The engineered immune cell for use according to claim 7, wherein the cancer is selected from melanoma, lung cancer, colorectal cancer, breast cancer, lymphoma, leukemia, multiple myeloma, liver cancer, gastric cancer, central nervous system (CNS) tumors, pediatric glioma, ovarian cancer, cervical cancer, colon cancer, pancreatic cancer, prostate cancer, and gastric cancer.

9. 9. The engineered immune cell for use according to any one of claims 6 to 8, comprising a gain-of-function mutation in CACNA1F.

10. 10. The engineered immune cell for use according to any one of claims 6 to 9, which overexpresses Cav1.4 compared to a wild-type immune cell of the same type.

11. 11. The engineered immune cell for use according to any one of claims 6 to 10, wherein the CACNA1F mutation comprises I745T.

12. The engineered immune cell for use according to any one of claims 6 to 11, selected from the group consisting of T cells, CAR-T cells, NK cells and CAR-NK cells.

13. An L-type Cav channel agonist for use in a method of stimulating the cell killing activity of immune cells in a subject having a proliferative disorder.

14. A pharmaceutical composition comprising immune cells treated with or admixed with an L-type Cav channel agonist.

15. 15. The pharmaceutical composition of claim 14 for use in a method of stimulating cell killing activity against a target cell population.

16. The L-type Cav channel agonist for use according to claim 13, or the pharmaceutical composition according to claim 14 or 15, wherein the immune cells are selected from the group consisting of T cells, CAR-T cells, NK cells and CAR-NK cells.

17. 1. An in vitro method for enhancing cell killing activity of an immune cell, comprising contacting an L-type Cav channel agonist with an immune cell, wherein the L-type Cav channel agonist is capable of stimulating L-type Cav channel activity in a Gli1 reporter assay.

18. 18. The method of claim 17, wherein the L-type Cav channel agonist is selected from the group consisting of FPL 64176 and Bay K8644.

19. 1. An in vitro method of engineering immune cells to increase cell killing activity, comprising genetically engineering immune cells to enhance or stimulate expression of L-type voltage-gated calcium (Cav) channel activity in a Gli1 reporter assay.

20. 20. The method of claim 19, wherein the manipulation comprises mutating Cav1.4 to introduce a gain-of-function mutation.

21. 21. The method of claim 20, wherein the Cav1.4 mutation comprises I745T.

22. 22. The method of any one of claims 19 to 21, wherein the manipulation comprises overexpressing Cav1.

4.

23. 23. The method of any one of claims 19 to 22, wherein the manipulation comprises nuclease or CRISPR-based editing of the CACNA1F gene.

24. The method of any one of claims 17 to 23, wherein the immune cell is selected from the group consisting of a T cell, a CAR-T cell, an NK cell, and a CAR-NK cell.

25. 1. A method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune cells, wherein the immune cells comprise a gain-of-function mutation in an L-type Cav channel and / or overexpress an L-type Cav channel.

26. The method of claim 25, wherein the immune cell is selected from the group consisting of a T cell, a CAR-T cell, an NK cell, and a CAR-NK cell.

27. 1. A method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an L-type Cav channel agonist.

28. 1. A method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of immune cells treated with an L-type Cav channel agonist.

29. 29. The method of claim 27 or 28, wherein the L-type Cav channel agonist is selected from the group consisting of FPL 64176 and Bay K8644.

30. 1. An engineered immune cell for use in a method of treating a chronic infection in a subject, the engineered immune cell comprising a gain-of-function mutation in an L-type Cav channel and / or overexpressing an L-type Cav channel.

31. 1. An engineered immune cell for use in a method of treating an autoimmune disease in a subject, the engineered immune cell comprising a gain-of-function mutation in an L-type Cav channel and / or overexpressing an L-type Cav channel.

32. An in vitro method for determining the suitability of cytotoxicity of immune cells derived from a mammalian subject, the method comprising the step of measuring the expression level of GLI1 in immune cells derived from the mammalian subject.

33. 33. The method of claim 32, wherein the immune cell is an engineered immune cell.

34. 33. The method of claim 32, comprising administering a test agent to the immune cells.

35. 35. The method of claim 34, wherein the test agent is an L-type Cav channel agonist.

36. 36. The method of any one of claims 32 to 35, wherein the immune cell is a CAR T cell, a CAR NK cell, a T cell or an NK cell.

37. An engineered immune cell for use in a method of treating a proliferative disease in a subject, the engineered immune cell comprising a loss-of-function mutation in PTCH1 and / or wherein PTCH1 is silenced or downregulated and the PTCH1 gene product is antagonized.

38. 38. The engineered immune cell for use according to claim 37, wherein the loss-of-function mutation in PTCH1 is a mutation found to be present in human patients with Gorlin's syndrome.

39. 39. The engineered immune cell for use according to claim 37 or claim 38, selected from the group consisting of T cells, CAR-T cells, NK cells, and CAR-NK cells.

40. 40. A method of treating a proliferative disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune cells, wherein the immune cells are as defined in any one of claims 37 to 39.

41. 41. The engineered immune cell for use according to any one of claims 37 to 39 or the method according to claim 40, wherein the proliferative disease comprises cancer, optionally wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, breast cancer, lymphoma, leukemia, multiple myeloma, liver cancer, gastric cancer, central nervous system (CNS) tumors, pediatric glioma, ovarian cancer, cervical cancer, colon cancer, pancreatic cancer, prostate cancer, and gastric cancer.

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