Treatment of solid tumors with regimens of targeted radionuclide therapy and genetically engineered immunotherapy [Cross-reference of related applications] This application claims priority to U.S. Provisional Application No. 63 / 508,578, filed on 16 June 2023, which is incorporated herein by reference in its entirety.

Abstract

This specification describes a method for treating solid tumors in a subject, comprising administering a low-dose targeted radiotherapy (TRT) agent to the subject, waiting for 1 to 60 days, and then administering chimeric antigen receptor (CAR) T-cell therapy to the subject.

Description

Technical Field

[0001] The present disclosure relates to a method of treating solid tumors by a combination of targeted radionuclide therapy and CAR T cell therapy.

Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy is a form of genetically engineered T cell therapy that has revolutionized cancer immunotherapy. Structurally, a CAR consists of an extracellular domain containing an antigen-binding domain obtained from the single-chain variable fragment (scFv) of an antibody, a transmembrane domain, an intracellular domain consisting of a T cell signaling domain (CD3-zeta) and a costimulatory domain such as OX40, 4-1BB or CD28. CAR T cells targeting CD19 or B cell maturation antigen (BCMA) have been approved by the FDA for the treatment of relapsed / refractory B cell lymphoma / leukemia and multiple myeloma, respectively, after high complete response rates in clinical trials.

[0003] Despite these successes against hematological malignancies, CAR T cell therapy has not been effective against non-hematological solid tumors for many reasons, including fatigue, lack of infiltration into the immunosuppressive tumor microenvironment (TME), and decreased antigen expression by tumor cells.

[0004] What is needed is a novel treatment regimen including CAR T cell therapy for solid tumors.

Summary of the Invention

[0005] In one aspect, a method of treating a solid tumor in a subject includes administering a low dose of a targeted radiation therapy (TRT) agent to the subject, waiting for 1 to 60 days, and after waiting, administering modified immune cells, such as CAR T cells, CAR NK cells, CAR macrophages, etc. to the subject.

Brief Description of the Drawings

[0006] [Figure 1]Figures 1A–1C show third-generation virus-free CRISPR GD2 CAR T cells. (1A) Schematic diagram of the domains of the third-generation CAR. The extracellular domain includes scFV (VH and VL chains linked by a linker) and a hinge. The intracellular domain consists of two costimulatory domains (CD28 and OX40) and a signaling domain (CD3-ζ). These two domains are connected by a transmembrane domain (CD28 transmembrane domain). (1B) Schematic diagram of the GD2 CAR construct inserted into the human T cell receptor alpha constant gene (TRAC). (1C) Schematic diagram of experimentally used GD2 CAR T cells. These cells express GD2 CAR but lack the T cell receptor due to CRISPR knockout of the human TRAC gene. scFv: single-stranded variable fragment; VH: heavy chain; VL: light chain.

[0007] [Figure 2] Figures 2A–2C show the dose-dependent effects of actinium-225 and lutetium-177 on the viability of GD2 CAR T cells. (2A) Experimental scheme: GD2 CAR T cells were incubated in cell culture medium containing active free 225Ac or 177Lu calculated to irradiate with a radiation dose of 1–6 Gy by day 3. CAR T cells were harvested and their viability was analyzed by flow cytometry using live / dead staining. (2B) Sample gating strategy for flow cytometry. Viable CAR T cells were determined as CD45+ and live / dead-Ghost Dye® red. (2C) Both 225Ac and 177Lu resulted in dose-dependent CAR T cell death. One-way ANOVA, comparative *:0.01; ***:0.0001; ****<0.0001.

[0008] [Figure 3]Figures 3A and 3B show the dose-independent effects of actinium-225 and lutetium-177 on the cytotoxicity of GD2 CAR T cells. (3A) Experimental scheme: After irradiation of CAR T cells with 225Ac or 177Lu, the CAR T cells were harvested and co-cultured with the GD2-expressing human neuroblastoma cell line CHLA-20 for 24 hours. The viability of CHLA-20 cells was analyzed by flow cytometry. (3B) Exposure of GD2 CAR T cells to radiation irradiated by radionuclides enhances their cytotoxicity against CHLA-20 cells, but such enhancement occurs dose-independently and regardless of the type of radionuclide. One-way ANOVA, comparison ***<0.001;****<0.0001;ns: not significant.

[0009] [Figure 4] Figures 4A–4C show that 225Ac and 177Lu enhance the cytotoxicity of GD2 CAR T cells against the GD2-expressing melanoma cell line M21. (4A) Human melanoma cell line M21 expresses GD2. (4B) Experimental scheme: After irradiation of CAR T cells with 225Ac or 177Lu, the CAR T cells were harvested and co-cultured with the GD2-expressing human melanoma cell line M21 for 24 hours. The viability of M21 cells was analyzed by flow cytometry. (4C) Exposure of GD2 CAR T cells to radiation irradiated by radionuclides enhances cytotoxicity against M21 cells in a dose-independent manner, regardless of the type of radionuclide.

[0010] [Figure 5]Figures 5A–5D show that actinium-225 and lutetium-177 do not affect the expression of exhaustion and activation markers on GD2 CAR T cells. (5A) Experimental scheme: GD2 CAR T cells were incubated in cell culture medium containing active free 225Ac or 177Lu calculated to irradiate with a radiation dose of 1–6 Gy by day 3. CAR T cells were harvested and the expression of exhaustion and activation markers was analyzed by flow cytometry. (5B) Almost all irradiated GD2 CAR T cells did not upregulate the expression of the T cell exhaustion marker PD-1. (5C) Irradiation did not significantly affect the activation marker NKG2D. (5D) Similarly, no significant effect was observed on the expression of the T cell activation marker CD69. One-way ANOVA, comparison; ns: not significant.

[0011] [Figure 6] Figure 6 shows that the combination of TRT and GD2 CAR-T cell therapy controls tumor growth in a xenograft model of neuroblastoma.

[0012] [Figure 7]Figures 7A–7E illustrate the potential synergistic effects of CAR-T cell-mediated B7-H3 tumor antigen targeting and LMD killing, as well as with radiation (radiopharmaceutical therapy (RPT) and external beam radiation therapy (EBRT)). (7A) The targetable tumor CAR-T antigen B7-H3 is expressed in >95% of cells across multiple cancer lines (SK-N-AS: neuroblastoma, LMD-BR3: breast cancer LMD). (7B) In vitro killing assays demonstrate specific CAR-T cell killing by LMD BR3 cells compared to untransduced (UTD) T cells (E: T = effector:target cell ratio; *p<0.05, **p<0.01). (7C) B7-H3 expression levels on LMD-BR3 tumor cells increase after radiation (EBRT, 2 or 10 Gy, and 225Ac, 2 Gy over 24 hours). (7D) Single-cell cytokine profiling of LMD-BR3 cells demonstrated an increase in inflammatory and chemoattractive cytokines after radiation (EBRT, 2 or 10 Gy, and 225Ac, 2 Gy over 24 hours). (7E) Radiation significantly increased the killing ability of B7-H3 CAR T cells against LMD-BR3 cells compared to UTD T cells (*p<0.05).

[0013] [Figure 8] Figure 8A shows imaging of luciferase-expressing IV-injected SK-N-AS human neuroblastoma in NRG mice 27 days after treatment with anti-B7-H3 CAR-T cells (5 x 10⁶ cells, IV) and / or 177Lu-NM600 (50 μCi, IV). Only combination therapy eradicates the tumor. (8B) In NRG mice with BR3 LMD, intrathecal injection of B7-H3 CAR-T cells (5 x 10⁶ cells) and low-dose 2 Gy of craniospinal irradiation (CSI) EBRT reduces tumor growth compared to untransduced T cells (UTDs) or CAR-T cells alone. (*: p<0.05; **: p<0.01. Previous studies (not shown) showed that EBRT alone up to 5 Gy did not significantly delay BR3 tumor growth.)

[0014] The above and other features will be recognized and understood by those skilled in the art from the following detailed description, drawings, and attached claims. Detailed Description of the Invention

[0015] As explained in the background technology section, modified immunotherapy, such as CAR T-cell therapy (Figure 1A), has not been effective against non-hematological solid tumors. In targeted radionuclide therapy (TRT), radioactive compounds are selectively delivered to malignant cells using tumor-homing ligands. TRT allows for whole-body irradiation of all sites of disease in patients with metastatic disease and has demonstrated survival benefits in patients with castration-resistant prostate cancer. Furthermore, new data suggest that low-dose radiation delivered by TRT induces a favorable immune response by activating and enhancing the infiltration of endogenous T cells into TMs. TRT may help overcome some of the shortcomings of modified immunotherapy for non-hematological solid tumors. However, before testing this hypothesis in vivo, many questions remain, such as the type of radionuclide, alpha(α) particle emitter vs. beta(β) particle emitter, and the timing and sequence of such combinations, which may affect the viability and function of modified immune cells. Actinium-225, an alpha particle emitter, affects the viability, cytotoxicity, and expression of fatigue and activation markers of third-generation GD2 CAR T cells. 225 Ac), and lutetium-177, which is a beta particle emitter. 177 The effects of Lu) are described herein. These GD2 CAR T cells were generated using a virus-free CRISPR-based approach with a CAR construct knocked into the T cell receptor alpha constant gene (TRAC) locus (Figure 1B), resulting in GD2 CAR T cells lacking the T cell receptor (Figure 1C). The experiments provided herein provide proof of concept of TRT and subsequent modified immunotherapy regimens for the treatment of solid tumors in subjects requiring treatment of solid tumors.

[0016] [TRT] Radiotherapy (RT) is used in both curative and palliative care for over 50% of cancer patients. By inducing potentially lethal DNA damage, RT induces immunogenic tumor cell death characterized by the transfer of calreticulin to the plasma membrane and the release of ATP and HMGB1 proteins into the extracellular environment. This promotes the migration and activation of immune cells in the tumor microenvironment (TME). At sublethal doses, RT also activates the STING / cGAS pathway in tumor cells and stroma, resulting in the upregulation of type I interferon (IFN) response and immune cell adhesion molecules on tumor endothelial cells. Furthermore, RT increases MHC-I expression on tumor cells, potentially promoting tumor recognition by the patient's own tumor-specific T cells. However, in the case of low immunogenic tumors such as pediatric neuroblastoma, the low tumor mutagenesis means there are few tumor-associated neoantigens and limited potential for endogenous T cell recognition.

[0017] Targeted radionuclide therapy (TRT) is a growing category of cancer treatment that selectively irradiates malignant cells with radioactive reticular activity (RT) in vivo using tumor-selective ligands (small molecules or antibodies) labeled with radionuclides. After intravenous injection of the TRT agent, it accumulates in the tumor, and as the radionuclide decays, the RT irradiates the systemic TME with significantly lower toxicity than systemic RT. By activating the STING / cGAS pathway, low-dose TRT stimulates a pro-inflammatory response that enhances endogenous T cell infiltration and activation in the TME of solid tumors.

[0018] Examples of TRT agents include metaiodobenzylguanidine (MIBG), where the iodine atom in the MIBG is a radioactive iodine isotope; radiolabeled tumor targeting antibodies; radioactive isotopes of radium such as Ra-223; and formulas: [ka] Radioactive phospholipid ether metal chelate having Or a salt thereof is included. R1 is (a) a chelating agent that chelates with a metal atom, where the metal atom is an alpha, beta, or Auger-emitting metal isotope having a half-life of more than 6 hours and less than 30 days, or (b) contains a radioactive halogen isotope, a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is -H, -OH, -COOH, -COOX, -OCOX, or -OX, and X is alkyl or arylalkyl; R2 is -N + H3, -N + H2Z, N + HZ2, or -N + Z3, where each Z is independently alkyl or aryl; b is 1 or 2. In some embodiments, R1 is (a) a chelating agent that chelates with a metal atom, and the metal atom is an alpha, beta, or Auger-emitting metal isotope having a half-life of more than 6 hours and less than 30 days. Examples of metal isotopes that can be used include Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, and Th-227.

[0019] In some embodiments, when R1 is (b) a radioactive halogen isotope, the radioactive halogen isotope is 123 I, 124 I, 125 I, 131 I, 211 At, 76 Br, or 77 Br. In some embodiments, a is 1 and m is 0. In some embodiments, n is 18. In some embodiments, R2 is -N + (CH3)3. In some such embodiments, a is 1, m is 0, and n is 18. In some such embodiments, the radioactive halogen isotope is 123 I, 124 I, 125 I, or 131 I (the radioactive halogenated phospholipid ether is 123 I]-NM404,124 I]-NM404, etc. 125 I]-NM404, etc. 131 I]-NM404, etc. 211 At]-NM404, 76 Br]-NM404, or [ 77 (Br]-NM404).

[0020] In one embodiment, (i)m is 0, b is 1, n is an integer between 12 and 30, and R2 is -N + Z3 is where each Z is independently alkyl or aryl, or (ii) m is 1, b is 1, n is an integer between 12 and 30, and R2 is -N + Z3 is where each Z is independently alkyl or aryl; or (iii) m is 0, b is 1, n is 18, and R2 is -N + Z3 is where each Z is independently alkyl or aryl; or (iv)m is 1, b is 1, n is 18, and R2 is -N + Z3, where each Z is independently alkyl or aryl.

[0021] Examples of chelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one of its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or one of its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or one of its derivatives; and 1,4,7-triazacyclononane, 1-glucan. 4,7-Talar acid-4,7-diacetic acid (NODAGA) or one of its derivatives; 1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid (DOTAGA) or one of its derivatives; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or one of its derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) or one of its derivatives; diethylenetriaminepentaacetic acid (DTP A) its diester or one of its derivatives; 2-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A''-DTPA) or one of its derivatives; dephoroxamine (DFO) or one of its derivatives; 1,2-[[6-carboxypyridine-2-yl]methylamino]ethane (H2dedpa) or one of its derivatives; and DADA or one of its derivatives, wherein DADA has the structure: One example is DADA or a derivative thereof, which has the following properties. [ka] In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In other embodiments, a is 0 (aliphatic alkyl chain). In some embodiments, m is 1 (acyl phospholipid series). In some such embodiments, n is an integer from 12 to 20. In some embodiments, Y is -OCOX, -COOX, or -OX. In some embodiments, X is -CH2CH3 or -CH3. In some embodiments, m is 0 (alkyl phospholipid series). In some embodiments, b is 1. In some embodiments, n is 18.

[0022] In some embodiments, R2 is -N + Z is Z3. In some such embodiments, each Z is independently -CH2CH3 or -CH3. In some such embodiments, each Z is -CH3.

[0023] In some embodiments, the chelating agent that chels to metal atoms is, [ka] The filename is JPEG2026522330000004.jpg221170.

[0024] In some embodiments, the chelating agent that chels to metal atoms is, [ka] JPEG2026522330000006.jpg242170JPEG2026522330000007.jpg237170JPEG20265223300 00008.jpg247170JPEG2026522330000009.jpg225170JPEG2026522330000010.jpg171170 That is the case.

[0025] In some embodiments, in the phospholipid ether metal chelate structure, a is 1, b is 1, m is 0, n is 18, and R2 is -N - It is (CH3)3. In some such embodiments, the phospholipid ether metal chelate is 90 This is NM600 chelated to metal atoms such as Y-NM600 (but not limited to this). 64 The following shows NM600 containing Cu. [ka]

[0026] In another embodiment, the TRT agent is metaiodobenzylguanidine (MIBG), and the iodine atom in MIBG is a radioactive iodine isotope.

[0027] In another embodiment, the TRT agent is a radiolabeled tumor-targeting antibody. Examples of radiolabeled tumor-targeting antibodies include: 177 Lu-dilentuximab, yttrium-90 and lutetium-177-labeled rosopatamab, 131 I-rabetsumab, panitumumab conjugated to an α-releaser, 212 Pb, 90 Y-labeled anti-MUC1 antibody, nti-TAG-72 intact antibody radiolabeled with iodine-131, yttrium-90, and lutehume-177, 90 Y-cribatuzumab tetraxetan, 31 Examples include I-labeled mAb 81C6.

[0028] In another embodiment, the TRT agent is a radioactive isotope of radium, such as radium-223 dichloride.

[0029] Radionuclides are radioactive atoms, and historically, TRT patients have been prescribed a given activity (unit curie, Ci), which is the rate of decay or radioactive decay. However, in research and institution-driven therapeutic approaches, the absorbed dose (unit gray: Gy) of the RT administered is determined from the given activity. This is because the absorbed dose, defined as the energy absorbed per unit mass of tissue, mediates the biological effects of the radionuclide.

[0030] In one embodiment, a low-dose TRT agent may be used in the method described herein. Although not bound by theory, since the transplanted tumors are derived from human cancer cell lines, the radiation doses (Gy) described herein for tumors transplanted in mice should be converted to human doses. As is known in the art, the optimal radiation dose may vary from tumor to tumor, but generally, a low-dose TRT agent is a dose lower than the typical dose used in TRT monotherapy, i.e., the dose of TRT expected to kill tumor cells. In one embodiment, the low-dose TRT is administered at a dose of less than 10%, specifically less than 6%, and more specifically less than 5%, of the dose used to kill tumor cells (which may be as high as 36 Gy).

[0031] In one embodiment, a low-dose TRT agent provides a radiation dose of 1 to 6 Gy, particularly 1 to 3 Gy, to a human patient, including children or adults.

[0032] [Modified immune cells including CAR T cells] Adoptive cell therapy using genetically modified immune cells such as CAR T cells is emerging as a novel immunotherapy approach. T cells typically interact with targets on their endogenous T cell receptors (TCRs), enabling a highly specific response to peptides presented in relation to human leukocyte antigens (HLA). Immune cells like T cells can be engineered to bind to novel antigens and targets by inserting new receptors with desired specificity. Genetically modified immune cells contain an extracellular domain that includes an antigen-recognition domain linked to a first intracellular domain via a first transmembrane domain. Typically, in the case of CARs, the antigen-recognition domain is a monoclonal antibody-derived single-strand variable fragment (scFv) that can target antigens such as specific tumor-associated antigens.

[0033] In some embodiments, the immune cells are T cells, natural killer (NK) cells, innate lymphoid cells, cytokine-induced killer (CIK) cells, hematopoietic progenitor cells, peripheral blood (PB)-derived immune cells, bone marrow-derived immune cells, macrophages, or umbilical cord blood (UCB)-derived immune cells. In some embodiments, the immune cells are immune cells derived from embryonic or induced pluripotent stem cells (iPSCs). In some embodiments, the immune cells are modified autologous cells isolated from patients requiring cancer treatment, or modified cells derived from allogeneic healthy donors intended to treat patients with cancer. The aforementioned immune cells may be genetically engineered to express CARs.

[0034] Immune cells can be isolated from subjects, particularly mammalian subjects such as human subjects and companion animals. Immune cells can be obtained from subjects of interest, such as those suspected of having a specific disease or condition, those suspected of being predisposed to a specific disease or condition, or those receiving treatment for a specific disease or condition. Immune cells can be concentrated / purified from any tissue in which they exist, including but not limited to blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. The tissues / organs from which immune cells are concentrated, isolated, and / or purified can be isolated from both living and non-living subjects, where non-living subjects are organ donors. Isolated immune cells can be used directly or stored for a period of time, such as by freezing.

[0035] A population of immune cells can be obtained from a subject in need of treatment or suffering from a disease associated with reduced immune cell activity. Therefore, the cells are autologous to the subject in need of treatment. Alternatively, a population of immune cells can be obtained from a donor, such as an allogeneic healthy donor. The immune cell population can be recovered from PB, umbilical cord blood, bone marrow, spleen, or any other organ / tissue where immune cells are present in the subject or donor. Immune cells can be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood. The immune cell population may originate from iPSCs and / or any other stem cells known in the art. In some embodiments, the iPSCs and / or stem cells used to induce a population of immune cells can be obtained from a subject in need of treatment or suffering from a disease associated with reduced immune cell activity, and therefore these iPSCs and / or stem cells are autologous to the subject in need of treatment. Alternatively, iPSCs and / or stem cells can be obtained from a healthy donor and therefore may be allogeneic to the subject in need of treatment.

[0036] When a population of immune cells is obtained from a donor different from the target, the donor is preferably allogeneic, provided that the obtained cells are target-compatible in the sense that they can be introduced into the target. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. Allogeneic cells can be treated to reduce their immunogenicity in order to make them compatible with the target.

[0037] For example, immune cells and human T cells can be edited so that CARs are randomly inserted into the genome using viral vectors (retroviruses, lentiviruses, AAVs, etc.) or into targeted regions of the T cell genome using non-viral approaches (electroporation, mRNA, lipid nanoparticles, etc.) combined with CRISPR / Cas9, TALENs, or zinc finger nucleases. CARs can be inserted into the endogenous T cell receptor alpha subunit constant gene (TRAC) or the endogenous T cell receptor beta subunit constant gene (TRBC).

[0038] In one embodiment, the CAR comprises an antigen-specific extracellular domain (e.g., a single-stranded variable fragment [scFV] capable of binding to surface-expressed antigens of malignant tumors) bound to an intracellular domain (e.g., CD28, ICOS, CD27, 4-1BB, OX40, CD40L, CD3-ζ, or a combination thereof) by a transmembrane domain (e.g., derived from CD4, CD8, CD28, CH2CH3, NKG2D, IgG, or CD3-ζ transmembrane domain).

[0039] The antigen-specific extracellular domain can also contain a spacer that links the Vh and VL chains of the scFV, which could be the hinge region of IgG1 and is sufficient for most scFv-based constructs.

[0040] The antigen-specific extracellular domain of a CAR recognizes and specifically binds to an antigen, typically a surface-expressed antigen of a malignant tumor. The antigen-specific extracellular domain specifically binds to the antigen if it binds with an affinity interaction (KD) of, for example, about 0.1 pM to about 10 μM, more specifically about 0.1 pM to about 1 μM, and more specifically about 0.1 pM to about 100 nM. Methods for determining the affinity of the interaction are known in the art. Suitable antigen-specific extracellular domains for use in CARs can be any antigen-binding polypeptide, and one or more scFvs or other antibody-based recognition domains (cAb VHH (camel antibody variable domain) or its humanized version, IgNAR VH (shark antibody variable domain) and its humanized version, sdAb VH (single-domain antibody variable domain) and "camelized" antibody variable domains) are suitable for use. In some examples, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs, can be used similarly to cytokine receptor ligands.

[0041] This disclosure provides chimeric antigen receptors (CARs) that bind to an antigen of interest. In certain embodiments, the CARs bind to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, oncoproteins. Antigens may be expressed as peptides or as part of an intact protein. The intact protein or any part thereof may be native or mutagenic.Non-limiting examples of tumor antigens that are CAR targets include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein Protein 40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), adult AChR subunit, folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit IL-13Rα2, κ-light chain, kinase insertion domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), claudin-18.2, FAP, CA19, B7-H3, calreticulin, ERBB2, MAGEA3, p53, MARTI, GP100, proteinase 3 (PR1), Examples include rosinase, survivorbin, hTERT, EphA2, NKG2D ligand, carcinometris antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, and combinations thereof.

[0042] The intracellular domain transmits T cell activation signals. The intracellular domain can increase CAR T cell cytokine production and promote T cell replication. The intracellular domain reduces CAR T cell exhaustion in patients, increases T cell antitumor activity, and enhances CAR T cell survival. Exemplary intracellular domains include costimulatory domains derived from CD27, CD28, CD137 or 4-1BB, CD154 or CD40L, CD244 or 2B4, CD278 or ICOS, CD134 or OX40, CD3-ζ and combinations thereof, as well as signaling domains (also called cytotoxic domains) derived from CD16, DAP10, DAP12, CD28, ICOS, CD27, OX40, CD40L, CD3-ζ and combinations thereof. The costimulatory domains are derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and / or persistence in vivo.

[0043] Typically, the antigen-specific extracellular domain is linked to the intracellular domain of the CAR by a transmembrane domain, such as a transmembrane domain derived from CD4, CD8, CD28, CH2CH3 or NKG2D, IgG or CD3-ζ transmembrane domain. The transmembrane domain traverses the cell membrane, anchoring the CAR to the T cell surface and connecting the extracellular domain to the intracellular signaling domain, thus influencing the expression of the CAR on the T cell surface.

[0044] CARs may also further include one or more spacers. Spacers or hinges connect (i) the antigen-specific extracellular domain to the transmembrane domain, (ii) the transmembrane domain to the costimulatory domain, (i) the costimulatory domain to the intracellular domain, and / or (iv) the transmembrane domain to the intracellular domain. For example, including a spacer domain (e.g., IgG1, IgG2, IgG4, CD28, CD8) between the antigen-specific extracellular domain and the transmembrane domain may affect the flexibility of the antigen-binding domain and thereby affect the CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art.

[0045] In certain embodiments, the CAR comprises a tumor antigen-binding domain targeting ganglioside G2 (GD2), which is bound to an intracellular domain containing a costimulatory domain derived from CD27, CD28, CD137, CD154, CD244, CD278, or a combination thereof, and a cytotoxic domain derived from CD3ζ, DAP10, DAP12, CD16, or a combination thereof.

[0046] CARs can be inserted into the genome of unmodified T cells using viral or nonviral methods. Viral vectors include retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses. The generation of CAR T cells using viral methods for CAR insertion is well known in the art. Methods for producing nonviral CAR T cell products are described in U.S. Patent Application Publication 2020 / 0000851, International Publication 2021 / 173925, and International Publication 2023 / 023635, which are incorporated herein by reference for their disclosures for producing nonviral CAR T cell products.

[0047] Unmodified T cells include autologous T cells collected from patients, such as cancer patients, by peripheral blood collection or leukocyte apheresis. Unmodified T cells may also include T cells from allogeneic healthy donors or induced pluripotent stem cells that can be used to produce pluripotent T cells for administration to patients. T cells are generally modified ex vivo, i.e., outside the patient, and then modified T cells, such as CAR T cells, are returned to the patient by intravenous, subcutaneous, intratumoral, intraperitoneal, or intraventricular injection, etc.

[0048] An exemplary nonviral method described in International Publication No. 2021 / 173925 involves introducing a nonviral double-stranded HDR template containing Cas9 RNP and CAR into unmodified T cells by electroporation to provide genome-edited T cells containing CAR.

[0049] Genome editing of T cells can be performed using either the CRISPR system or the Cas9 ribonucleoprotein. CRISPR refers to a clustered and regularly arranged short palindromic sequence repeat type II system used by bacteria and archaea for adaptive defense. This system allows bacteria and archaea to detect and silence foreign nucleic acids, such as viruses or plasmids, in a sequence-specific manner. In the type II system, guide RNA interacts with Cas9, directing the nuclease activity of Cas9 to a target DNA sequence complementary to that present in the guide RNA. The guide RNA base pairs with the complementary sequence in the target DNA. The Cas9 nuclease activity then causes a double-strand break in the target DNA.

[0050] [Dosage regimen] In one embodiment, a method for treating a solid tumor in a subject includes administering a low-dose targeted radiotherapy (TRT) agent to the subject, waiting for 1 to 60 days, and then administering modified immune cell (e.g., chimeric antigen receptor (CAR) T cell) therapy to the subject after the waiting period.

[0051] In one embodiment, modified immune cells (e.g., CAR T cells) for use in a method of treating solid tumors in a subject, wherein the method comprises administering a low dose of a TRT agent to the subject, waiting for 1 to 60 days, and administering the modified immune cell therapy to the subject after the waiting period, are included herein.

[0052] In another embodiment, modified immune cells (e.g., CAR T cells) for use in a method of treating solid tumors in a subject, wherein the method comprises administering the modified immune cells to a subject, the subject having been administered a low dose of a TRT agent 1 to 60 days prior to the administration of the modified immune cells (e.g., CAR T cells).

[0053] In another embodiment, the use of modified immune cells (e.g., CAR T cells) in the manufacture of a pharmaceutical for a method of treating solid tumors in a subject, the method comprising administering a low dose of a TRT agent to the subject, waiting for 1 to 60 days, and administering modified immune cells to the subject after the waiting period.

[0054] The waiting period between therapies is based on the half-life of the TRT agent and the initial radiation dose. Since TRT is expected to reduce the viability of CAR T cells, the waiting period is a crucial part of this method. Examples of waiting periods include 1-60 days, 1-30 days, 2-30 days, 2-20 days, 2-12 days, 3-10 days, 3-9 days, or 3-6 days.

[0055] In one embodiment, the subject is a mammal, specifically a human or a dog.

[0056] Non-limiting examples of cancers presented as malignant solid tumors that can be treated using the disclosed methods include melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, renal cell carcinoma, non-small cell lung cancer, head and neck cancer, bladder cancer, hepatocellular carcinoma, spinal chordoma, cholangiocarcinoma, liver cancer, subcutaneous cancer, squamous cell carcinoma of the skin or head and neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma, Wilms' tumor, medulloblastoma, ependymoma, pineoblastoma, peripheral neuroectodermal tumor, or germ cell tumor.

[0057] This disclosure includes the compounds described herein (including intermediates) in any pharmaceutically acceptable form, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, etc. The term “compound” should be understood to include any or all of such forms, whether expressly stated or not (sometimes “salt” may be expressly stated).

[0058] "Pharmacologically acceptable," as used herein, means that the compound, composition, or carrier is suitable for administration to a subject to achieve the treatment described herein without adverse side effects that are excessively harmful in light of the need for the treatment.

[0059] When used herein, the term “effective dose” refers to the amount of compound or dosage that elicits a biological or medical response in a subject, tissue, or cell as sought by a researcher, veterinarian, physician, or other clinician.

[0060] The term "pharmaceutically acceptable carrier" includes all kinds of dry powders, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retarders, etc. A pharmaceutically acceptable carrier is a material useful for the purpose of administering compounds in the method of the present invention, preferably non-toxic, and may be a solid, liquid, or gaseous material, otherwise inert, pharmaceutically acceptable, and compatible with the compounds described herein. Examples of such carriers include, but are not limited to, various lactoses, oils such as mannitol and corn oil, buffers such as PBS, saline solutions, amides such as polyethylene glycol, glycerin, polypropylene glycol, dimethyl sulfoxide and dimethylacetamide, proteins such as albumin, and detergents such as Tween 80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.

[0061] When used herein, the terms “administer” or “administer” refer to providing the compounds or pharmaceutical compositions of the present invention to subjects who are suffering from or at risk of suffering from the disease or condition to be treated or prevented.

[0062] In pharmacology, the route of administration is the path by which a drug is taken into the body. Routes of administration can generally be classified according to the site where the substance is applied. Common examples include oral administration and intravenous administration. Routes can also be classified based on where the target of action lies. Action can be local (subject to a specific location), enteral (an action on the entire system, but delivered through the gastrointestinal tract), or parenteral (a systemic action, but delivered via a route other than the GI tube) via inhalation into the lungs. One form of local administration is intratumoral (IT), where the drug is injected directly or adjacent to a known tumor site.

[0063] Local administration emphasizes a local effect, where the substance is applied directly to the site where its action is desired. However, sometimes the term local may be defined as application to a local area or surface of the body without necessarily involving a targeted effect, thus making the classification a variation of the classification based on the site of application. In enteral administration, the desired effect is systemic (non-local), and the substance is delivered via the gastrointestinal tract. In parenteral administration, the desired effect is systemic, and the substance is delivered via a route other than the gastrointestinal tract.

[0064] Examples of parenteral administration include intravenous (intravenous), e.g., many drugs and total parenteral nutrition; intra-arterial (in arteries), e.g., vasodilators in the treatment of vasospasm and thrombolytics for the treatment of embolism; intraosseous (in bone marrow), intramuscular, intracerebral (in brain parenchyma), intraventricular (in the ventricular system), intrathecal (injection into the spinal canal); and subcutaneous (under the skin). Of these, intraosseous injection is effectively an indirect form of intravenous access, as the bone marrow is directly drained into the venous system. Intraosseous injection may occasionally be used for drugs and fluids in emergency medicine and pediatrics when intravenous access is difficult.

[0065] The present invention can be further illustrated by the following non-limiting examples. [Examples]

[0066] material and method Cell lines: GD2-expressing CHLA-20 cells (human neuroblastoma cells) and M21 cells (human melanoma cells) were donated by Dr. Mario Otto and Dr. Paul Sondel, respectively, and grown in Dulbecco's Modified Eagle Medium (DMEM) containing high glucose (Gibco) supplemented with 10% fetal bovine serum (Avantor) and 1% penicillin-streptomycin (Gibco). These cells were maintained at 37°C in 5% CO2. Cell line authentication was performed by cell morphology using genome short tandem repeat analysis (Idexx BioAnalytics) according to ATCC guidelines. Mycoplasma testing was performed periodically to eliminate contamination using the Mycoplasma Detection Kit MycoStrip® (InvivoGen).

[0067] GD2 CAR T cells: Virus-free CRISPR GD2 CAR T cells were prepared as previously described in Mueller et al. 2022 J Immunother Cancer, 2022;10:e004446.doi:10.1136 / jitc-2021-004446, cultured in ImmunoCult™-XF T cell Expansion Medium supplemented with 500 U / mL IL-2 (Peprotech), and maintained at 37°C in 5% CO2.

[0068] In vitro dosimetry: All in vitro dosimetry studies were performed in 6-well cell culture plates. 177 Lu or 225 Serial dilutions of various Ac activity levels were prepared in cell culture medium and added to each well. A thermoluminescent dosimeter (TLD) was placed beneath each well. For each radionuclide, the TLD was collected after one half-life and analyzed by the University of Wisconsin-Madison Radiation Calibration Laboratory (calibration certificate number 1664.01). As previously reported in the art, a standard curve was obtained to determine the amount of radiation needed to irradiate a given dose. 177 Lu or 225Ac was used to determine its activity. To confirm the validity of this method, the mean absorbed dose to cells was calculated using the Geant4 Monte Carlo toolkit and the RAPID extension. A model of a flat-bottomed 6-well plate was developed in Geant4 using manufacturing specifications where each well had a diameter of 36 mm and a height of 10.7 mm. Cell volume can be defined as the thin water equivalent layer at the bottom of the well.

[0069] In vitro GD2 CAR T cell irradiation: GD2 CAR T cells were treated with 3 mL of ImmunoCult™-XF T cell Expansion Medium. 177 Lu or 225 The subjects were exposed to various doses of radiation from Ac over a three-day period. 177 The radiation doses irradiated by Lu were 1, 2, and 6 Gy, 225 Ac was irradiated with 1 and 2 Gy.

[0070] In vitro GD2 CAR T cell survival rate: After irradiation, the survival rate of GD2 CAR T cells was determined by flow cytometry.

[0071] In vitro co-culture of GD2 CAR T cells and tumor cells: GD2 CAR T cells were irradiated, harvested, washed three times with PBS, and co-cultured with CHLA-20 cells for 24 hours in an effector-to-target (E:T) ratio of 10:1.

[0072] Flow cytometry: Cells were harvested, washed with PBS, and resuspended in a single-cell solution in PBS as previously reported in the art. Live / dead staining with Fc blocking (Biolegend, 422302) and Ghost Dye® Red 780 (Tonbo Biosciences, 13-0865-T100) was performed at 4°C for 10 minutes. Fluorophore conjugate antibodies containing anti-CD45-APC (Biolegend, 304012), anti-GD2-PE-Dazzle584 (Biolegend, 357320), anti-PD-1-PE (Biolegend, 329906), anti-CD69-BV510 (Biolegend, 310936), and anti-NKG2D-BV605 (Biolegend, 320832) were incubated at 4°C for 20 minutes and washed with 2% FBS in PBS. Samples were analyzed using an Attune® NxT Flow Cytometer (ThermoFisher), and the collected data was analyzed using FlowJo software.

[0073] Statistical Analysis: All statistical analyses were performed using Prism 9 (GraphPad Software). For comparisons between multiple groups, a two-way ANOVA with Tukey's multiple comparison test was used.

[0074] [Example 1: 177 Lu and 225 Radiation irradiated by Ac leads to dose-dependent GD2 CAR T cell death. The inventors hypothesize that combining in vivo TRT with CAR T cell therapy exposes CAR T cells to radiation, which is detrimental to their survival rate. Therefore, to evaluate the effects of radiation irradiated by TRT on CAR T cells, GD2 CAR T cells were used. 177 Lu or 225 The cells were exposed to various doses of radiation irradiated by Ac. A 6-well plate containing GD2 CAR T cells was irradiated with a specific activity of 1, 2, or 6 Gy by day 3. 177 LU or 225The cells were incubated in a medium containing Ac. After irradiation, GD2 CAR T cells were harvested, and their viability was evaluated by flow cytometry (Figure 2A) using viability / death staining (sample gating strategy; see Figure 2B). Dose-dependent GD2 CAR T cell death was observed for both radionuclides. However, 177 Lu 225 It appears to have lower cytotoxicity compared to Ac. 225 1 Gy of radiation administered by Ac reduced the survival rate of GD2 CAR T cells by almost one-third compared to unirradiated cells, but 2 Gy... 225 In Ac, such survival rates 7 This decreased to 2.34% (Figure 2C). 177 After irradiation with 1 Gy of Lu, the survival rate of GD2 CAR T cells decreased to 25%–19.6% compared to unirradiated cells, and this survival rate decreased to 16.1% and 12.2% after 2 Gy and 6 Gy, respectively (Figure 2C). [Example 2: 177 Lu and 225 Radiation irradiated by Ac enhances the cytotoxic activity of GD2 CAR T cells against the GD2-expressing neuroblastoma cell line CHLA-20.

[0075] In addition to evaluating the effect of radiation irradiated with radionuclides on the viability of GD2 CAR T cells, we also determined the effect of such radiation on the effector function of GD2 CAR T cells. For this purpose, GD2 CAR T cells were co-cultured with the GD2-expressing neuroblastoma cell line CHLA-20 for 24 hours after irradiation. The toxicity of irradiated GD2 CAR T cells was determined by measuring the viability of CHLA-20 cells by flow cytometry (Figure 3A). Viable CHLA-20 cells were identified as CD45-negative and viable / dead staining (Ghost Dye® Red)-negative (CD45- / Ghost Dye® Red-). As expected, unirradiated GD2 CAR T cells showed potent cytotoxic activity against CHLA-20 cells. The viability of CHLA-20 cells decreased to 6.9% after 24 hours of co-culture with GD2 CAR T cells, compared to 52.2% in the absence of GD2 CAR T cells (Figure 3B). Irradiation of GD2 CAR T cells prior to co-culture with CHLA-20 cells enhanced the cytotoxic activity of the CAR T cells, resulting in near-complete eradication of CHLA-20 cells (Figure 3B). However, the enhanced cytotoxicity of GD2 CAR T cells was independent of the type of particles released (α vs. β particles) and the radiation dose administered (Figure 3B). Radiation-induced enhancement of the cytotoxic activity of GD2 CAR T cells was also observed against the human melanoma cell line M21, which also expresses GD2 (Figures 4A-C).

[0076] [Example 3: 177 Lu and 225 Radiation irradiated by Ac does not affect the expression of fatigue and activation markers on GD2 CAR T cells. against GD2 CAR T cells 225 Ac or 177 To further characterize the effects of radiation irradiated by Lu and to understand the underlying mechanisms of radiation-induced enhancement of GD2 CAR T cells, the expression of the T cell exhaustion marker PD-1, the activation marker CD9, and the activation receptor NKG2D was evaluated by flow cytometry. As mentioned above, various radiation doses were used.225 Ac or 177 After irradiating GD2 CAR T cells with Lu, the GD2 CAR T cells were harvested and the expression of cell surface markers was determined by flow cytometry (Figure 5A). Compared to unirradiated GD2 CAR T cells, 225 1 Gy of irradiation with Ac resulted in increased PD-1 expression, 177 Radiation administered by Lu (1, 2, and 6 Gy) showed a tendency towards decreased PD-1 expression. However, the changes in PD-1 expression after irradiation were not statistically significant (Figure 5B).

[0077] Although not statistically significant, an upward adjustment trend was observed in the expression of CD69 and NK.G2D after irradiation with both radionuclides (Figures 5C and 5D).

[0078] [Discussion of Examples 1-3] Due to the increasing therapeutic role of TRT in the clinical management of cancer and its immunostimulatory effects at low doses, the combination of TRT and CAR T-cell therapy constitutes a therapeutic approach for selected patients with metastatic solid tumors. Therefore, the in vitro effects and functions of TRT on CAR T-cell survival are determined to identify their maximum permissible radiation dose (irradiated by radionuclides) and radionuclides (α vs β emitters) that can enhance the effector function of CAR T-cells, while minimizing adverse effects on CAR T-cell survival.

[0079] 177 Lu and 225 The dose-dependent death of GD2 CAR T cells observed in response to radiation irradiated by Ac is expected because the radiation is cytotoxic to lymphocytes, particularly CD8+ T cells, which are the precursors of GD2 CAR T cells. Such dose-dependent death is present with radiation irradiated by any radionuclide, but not with alpha-emitting radionuclides. 225 The biological effects of a similar dose of radiation (1 Gy) irradiated by Ac are as follows: 177The biological effects are higher with α-emitters than with the same dose irradiated by Lu. While not constrained by theory, this difference in biological effects is attributed to the LET difference between these two radionuclides. Despite having a shorter range in tissue (0.05–0.08 mm) compared to β-emitters (1–5 mm), α-emitters induce 10–20 double-strand DNA breaks (DSBs) per 10 μm, resulting in potent cytotoxicity, due to their higher LET (50–230 keV / μm), whereas β-emitters induce more single-strand DNA breaks that are readily repaired. It has been suggested in the art that α-particle emitters induce multiple very close-proximity DSBs, leading to depletion of p53-binding protein 1 (53BP1), a DNA damage response protein necessary for double-strand DNA breaks. Such depletion results in insufficient amounts of 53BP1 to adequately repair all DSBs, further highlighting the biological efficacy of high-LET α-particle emitters.

[0080] In this study, 1 Gy 225 Ac-induced in vitro CAR T cell death is 177 This is only 2.2 times Lu (Figure 2C), which is lower than what has been observed in prior art. This discrepancy may be due to the different in vitro models (GD2 CAR T cells) used in this study, the methods used to access therapeutic efficacy, and the fact that our in vitro dosimetry allowed for a comparison of similar doses of radiation irradiated by radionuclides rather than active ones.

[0081] The radiation delivered by external beam radiation therapy (EBRT) upregulates the expression of PD-1 on CD8+ T cells, which can be detrimental to CAR T cell function, but surprisingly, this upregulation 177 Lu or 225This was not observed in Ac. While not bound by theory, this may be due to the inherent design of this third-generation GD2 CAR, which has a CRISPR knock-in of the CAR construct to the TRAC locus, resulting in CAR T cells lacking the T cell receptor, which could represent another therapeutic benefit of combining TRT with CAR T cell therapy instead of EBRT.

[0082] Radiation delivered by EBRT enhances the cytotoxic activity of CAR T cells, including those against antigen-negative malignant cells. Prior to this study, such enhancement had not been demonstrated with TRT. The presence of TRT in the environment may have selected more radiation-resistant CAR T cell subclones with potent cytolytic activity.

[0083] Based on in vitro studies, the low doses of radiation (1 or 2 Gy) tested herein, irradiated by α-emitters, may not be ideal for potential therapeutic combinations with CAR T cells, making β-emitters preferred candidates. Instead, 1 Gy irradiated by β-particle emitters may be ideal, as all other radiation doses evaluated (2 Gy or 6 Gy) enhanced the cytotoxic function of CAR T cells to a similar degree, while inducing a higher rate of GD2 CAR T cell death. These findings suggest that, regarding the ordering between TRT and CAR T cell combinations, CAR T cell infusion should be performed after TRT administration to minimize CAR T cell exposure to radiation. In vitro studies have shown that β-emitters... 177While 1 Gy of irradiation by Lu may be ideal, higher test doses of 2 Gy or 6 Gy may be considered if there is an appropriate delay between TRT irradiation and CAR T cell injection. This delay allows for the radioactive decay of radionuclides so that, when CAR T cells are injected, the residual activity of the radionuclides in the TME is irradiated with a dose of less than 1 Gy. Although not bound by theory, it is speculated that using doses exceeding 1 Gy may be an attractive option, as it would allow for the utilization of the tumor-killing effect of TRT on malignant cells, and subsequently reduce the tumor volume to which CAR T cells must be effective.

[0084] This study provides answers to key questions such as the optimal dose of radiation, the type of radionuclide, and the timing and order of combinations, enabling the rational design of in vivo studies evaluating the combination of CAR T cell therapy and TRT.

[0085] [Example 4: In vivo combination of TRT and GD2 CAR T cells] Ten million LUC-GFP CHLA-20 human neuroblastoma cells were subcutaneously injected into the right flank of 6-8 week old male or female NRG mice. Tumor transplantation was confirmed 5 days after cell injection by bioluminescence using an in vivo imaging system (IVIS). Tumor volume was monitored and was approximately 75 mm². 3 Once the volume reached a certain level, the mice were randomly divided into different treatment groups: 1) control (no treatment; n=4); 2) radiation only (1.75 Gy; n=5). 177 Lu-NM600 was administered intravenously 6 days after tumor injection; 3) GD2 CAR T cells (10 million cells; n=5) were administered intravenously 15 days after tumor injection; 4) A combination therapy group (n=8) received intravenous radiation (1.75 Gy) 6 days after tumor injection, followed by intravenous administration of GD2 CAR T cells (10 million cells) 9 days later (15 days after tumor injection). Tumor volume was 1500 mm². 3 The patient was monitored until euthanasia was performed if the condition reached a certain level or ulcer formation was observed.

[0086] CAR T cells have limited success against solid tumors. In patients with metastatic disease, it is impossible to irradiate all disease sites with EBRT. While TRT can irradiate all disease sites, it is likely to be ineffective as a monotherapy at low doses. By administering CAR T cells after TRT, the killing of metastatic solid tumors can be significantly increased without affecting the survival or proliferation of CAR T cells.

[0087] [Example 5: B7-H3 in combination with radiotherapy] + Cancer CAR-T cell targeting] Cancer metastasis and spread into the cerebrospinal fluid (CSF), known as leptomeningeal disease (LMD), is a major clinical challenge given that current treatments are both ineffective and excessively toxic. LMD occurs in 5–10% of adult patients with metastatic solid tumors and has a serious prognosis. Without treatment, survival in these patients is typically measured in weeks. Patients with LMD may experience severe headaches as well as loss of vision, hearing, speech, and facial motor and sensory impairment. Some pediatric patients with cancers such as medulloblastoma are at a sufficiently high risk of CSF spread and require preemptive treatment. Currently, EBRT targeting the entire brain and spine (craniospinal irradiation, CSI) is the only proven effective treatment available for most patients at high risk of LMD or cancer CSF dissemination. CSI EBRT slows progression, alleviates symptoms, and extends survival in these patients, although LMD often recurs. To reduce the risk of cardiac, pulmonary, esophageal, and oral side effects, patients with LMDs are ideally treated with high-precision proton beam irradiation; however, access to proton beams is limited, particularly in rural and low-income patient populations. Even when patients achieve permanent control or prevention of LMDs, they often experience serious late-stage toxicity from CSI EBRT, even with proton beam irradiation. These toxicities include memory impairment and weakening of the spinal bones. In children, CSI EBRT can also result in stunted growth and potentially severe cognitive impairment. These toxicities arise because CSI EBRT treats not only the CSF space where the LMDs are located, but also the entire brain, spinal cord, and vertebral bodies. This radiation to normal tissue results in long-term, persistent toxicity that impairs the quality of life of survivors. Therefore, there is a great need for new therapeutic approaches that can effectively treat LMDs with greater specificity and efficacy to eliminate cancer cells while minimizing the impact on normal tissue surrounding the CSF.

[0088] The methods described herein may provide effective and less toxic treatments for patients at high risk of LMD or CSF dissemination of cancer. The hypothesis is that intrathecal irradiation of novel radiopharmaceutical therapies (RPTs) allows for more effective irradiation of LMDs while reducing toxicity by minimizing radiation exposure to normal tissue compared to CSI EBRT. Specifically, combining intrathecal administration of RPTs with CAR-T cell therapy would overcome the limitations of intrathecal administration of RPTs alone. Although CAR-T cells are used as standard in the treatment of hematological malignancies, their effectiveness against large solid tumors has not yet been proven due to their limited invasion, activation, and persistence in the established tumor microenvironment (TME). However, recent studies have shown promising therapeutic potential of intrathecally injected CAR-T cells against central nervous system tumors. Radiation can debulk and immunomodulate solid tumor TMEs, enhancing T cell invasion and activation, including in the CNS. The optimal dose of RPT in combination with immunotherapy is very low and can differ significantly from the optimal dose of the same drug used as monotherapy. While RPT has a potential synergistic effect in promoting solid tumor cell destruction by CAR-T cells, this is only observed at very low doses of RPT. A combination of intrathecal RPT and CAR-T cells for evaluating the curative potential of this combination for LMD is described herein.

[0089] EBRT can enhance CAR T cell infiltration and expansion in the TME of brain tumors in mouse models, resulting in a potent and persistent antitumor response partially mediated by CAR T cells. When tumor cells are treated with RPT, increased CAR-T cell killing activity can be achieved in vitro without inducing exhaustion markers. It is hypothesized that RPT exhibits synergistic therapeutic efficacy in combination with CAR-T cells by immunomodulating the TME at the tumor site, inducing inflammation in the TME, and increasing the susceptibility of tumor cells to CAR-T cell killing. The small absolute volume of disease in LMD settings can be particularly well targeted by such combinations. Furthermore, intrathecal CAR-T cells may have fewer inhibitory components of the host immune system in the CSF compared to blood, as treatment via this pathway has yielded some of the most promising clinical results to date for solid tumors. Intrathecally injected CAR-T cells recently showed an antitumor response in 3 out of 4 patients with median glioma, resulting in an increase in immunostimulatory cytokines in the CSF. Importantly, these clinical studies demonstrate the safety of intrathecal administration of CAR-T cells with limited and manageable toxicity.

[0090] B7-H3 tumor-specific antigen expression in cancer and CAR-T cell targeting. The B7-H3 (CD276) antigen is an immune checkpoint overexpressed on the cell surface of tumor cells in pediatric brain cancer, neuroblastoma, melanoma, sarcoma, and various other cancers, while B7-H3 is rarely or never expressed in normal tissues. B7-H3 is a pan-oncological antigen for targeting multiple forms of cancer with a single CAR-T cell product. Nearly homogeneous cell surface expression of B7-H3 on human BR3 LMD breast cancer cell lines and human SK-N-AS neuroblastoma cell lines was observed by flow cytometry (Figure 7A).

[0091] B7-H3 +Targeting of cancer cells with CAR-T cells. CAR-T cells targeting human B7-H3 demonstrate preclinical efficacy against microscopic disease in neuroblastoma, medulloblastoma, and other xenograft tumor models in immunodeficient mice. B7-H3 CAR-T cells were designed as a second-generation CD8H / Tm-CD28-CD3ζ(28ζ)CAR format containing CD8a H / Tm sequences and CD28 and CD3ζ signaling domains. These B7-H3 CAR-T cells induced rapid and robust killing of LMD BR3 cells in vitro compared to minimal or no killing from untransduced (UTD) control T cells (Figure 7B).

[0092] Radiation promotes the antitumor activity of anti-B7-H3 CAR-T cells. The inflammatory effects of radiation, including EBRT and RPT, promote the upregulation of tumor cell surface expression of various immune checkpoint receptors. To evaluate whether radiation can affect B7-H3 tumor cell expression, 4 Gy or 12 Gy of EBRT or 225 BR3 LMD cells were administered 12 Gy from Ac in vitro, and flow cytometry was performed 24 hours later. Radiation increased B7-H3 expression on the surface of tumor cells in this human breast cancer LMD model, and this effect was maximized by the RPT agent in this preliminary analysis (Figure 7C). These findings are important because the efficacy of B7-H3 CAR-T cells may depend on a high density of target antigens on the surface of tumor cells. The induction of B7-H3 tumor cell surface expression may be a mechanism for synergistic action between RPT and anti-B7-H3 CAR-T cells.

[0093] Since radiation is known to induce inflammatory cytokine production in tumor cells, EBRT or 225 We studied cytokine secretion from BR3 LMD cells after Ac radiation. Using multi-cytokine single-cell proteome analysis with the Isoplexis system, preliminary studies showed that radiation increased the production of T cell-stimulating cytokines and inflammatory cytokines by BR3 LMD cells, which is 225The effect was most pronounced after Ac radiation (Figure 7D). This suggests that RPT may enhance CAR-T cell invasion, activation, and persistence in this tumor model. Pre-treatment of LMD BR3 cells in vitro with 2 Gy of EBRT 24 hours prior to the addition of anti-B7-H3 CAR-T cells resulted in enhanced tumor cell killing, as evidenced by efficacy at a lower effector:target cell (E:T) ratio when irradiated compared to control tumor cells (Figure 7E). This suggests that low-dose radiation may enhance the sensitivity of these cells to CAR-T cell killing. In summary, these preliminary findings support the possibility of a therapeutic synergy between RPT and CAR-T cells.

[0094] In vivo collaborative therapeutic efficacy of CAR-T cells and radiation. Luciferase-expressing SK-N-AS neuroblastoma cells were intravenously injected into NRG mice to induce systemic metastasis (primarily in the liver, not leptomania (LMD) in this study). Once tumor engraftment was confirmed, 50 μCi was administered. 177 Lu-NM600 injection (irradiating an estimated tumor dose of 2 Gy in this model) was performed. Thirteen days later, these mice were intravenously injected with anti-B7-H3 CAR-T cells. Evidence of a synergistic therapeutic effect between low-dose RPT and CAR-T cells was observed 27 days after the start of treatment, and all mice receiving combination therapy appeared disease-free at this point based on bioluminescent luciferase imaging (Figure 8A). In another study, LMD tumors were initiated by carotid injection of luciferase-expressing BR3 cells in NRG mice. On day 6, bioluminescence was performed to confirm LMD growth, and the mice were randomly divided into treatment groups: control untransduced T cells (UTD), anti-B7-H3 CAR-T cells, or a combination of anti-B7-H3 CAR-T cells and 2 Gy of CSI EBRT. EBRT was administered 6 days post-engraftment (Xstrahl CIX3 cabinet irradiator) to CAR-T cells or UTC cells (5 × 10⁶). 6The cells were intrathecally injected 24 hours later. Previous experiments (not shown) have demonstrated no change in LMD growth with CSI EBRT up to 5 Gy. In this study, the results suggest that mice receiving the combination of anti-B7-H3 CAR-T cells and low-dose CSI EBRT show reduced LMD load compared to controls with UTC or CAR-T cells alone (Figure 8B).

[0095] These results are expected to demonstrate the dose- and time-dependent effects of RPT on B7-H3 expression and immune-related markers in TME. Effective therapeutic regimens will be identified for each isotope irradiated by NM600 in combination with CAR T cells. The optimal dose of RPT for this combination is predicted to be at a low dose level, not MTD, as the radioactive effect on CAR T cells may be detrimental to efficacy. While not bound by theory, the higher biological efficacy of alpha particle emitters, in contrast to monotherapy, may be counterproductive in combination with CAR-T cells. 177 The combination with Lu-NM600 may be the most effective in promoting CAR-T cell activity.

[0096] The terms “a,” “an,” and “the,” and similar references (particularly in the context of the following claims) should be interpreted as encompassing both singular and plural, unless otherwise indicated herein or unless clearly inconsistent with the context. Terms such as “first,” “second,” etc., when used herein, do not mean to indicate a particular order, but merely to indicate multiple, e.g., layers, for convenience. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-restrictive terms (i.e., “including but not limited to”) unless otherwise specified herein. Enumerations of value ranges are intended merely as a convenient way to refer individually to each distinct value that falls within that range, unless otherwise indicated herein, and each distinct value is incorporated herein as if it were individually listed herein. The endpoints of all ranges are within the range and can be combined independently. All methods described herein can be performed in appropriate order, unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any and all examples or illustrative language (e.g., "etc.") is merely intended to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. No language in this specification should be construed as indicating an unclaimed element essential to the practice of the invention as used herein.

[0097] While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various modifications can be made and elements can be replaced with equivalents without departing from the spirit of the invention. Furthermore, many modifications may be made to adapt the teachings of the invention to specific situations or materials without departing from the essential spirit of the invention. Accordingly, the present invention is not limited to the specific embodiments disclosed as the best mode intended to carry out the invention, but is intended to include all embodiments that fall within the spirit of the appended claims. Any combination of the above elements in all possible variations is incorporated into the invention unless otherwise indicated herein or is clearly inconsistent with the context.

Claims

1. A method for treating solid tumors in a subject, Administering a low-dose targeted radiotherapy (TRT) agent to the subject, Waiting for 1 to 60 days, A method comprising administering a modified immunotherapy to the subject after a waiting period.

2. The method according to claim 1, wherein the modified immune cells are T cells, natural killer (NK) cells, innate lymphoid cells, cytokine-induced killer (CIK) cells, hematopoietic progenitor cells, peripheral blood (PB)-derived immune cells, bone marrow-derived immune cells, macrophages, or umbilical cord blood (UCB)-derived immune cells.

3. The method according to claim 2, wherein the modified immune system expresses a chimeric antigen receptor (CAR).

4. The method according to claim 1, wherein the waiting period is 1 to 30 days.

5. The method according to claim 1, wherein the low dose of the TRT agent is a radiation dose of 1 to 6 Gy.

6. The modified immunotherapy according to claim 1, wherein the modified immunotherapy comprises modified autologous cells isolated from a patient requiring cancer treatment, or modified cells derived from an allogeneic healthy donor.

7. CAR targets include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP -40), epidermal cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), adult AChR subunit, folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleuk IL-13Rα2 receptor subunit, K-light chain, kinase insertion domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), claudin-18.2, FAP, CA19, B7-H3, calreticulin, ERBB2, MAGEA3, p5 3. MARTI, GP100, Proteinase 3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligand, Oncotesticular antigen NY-ESO-1, Carcinoembryonic antigen (h5T4), Prostate stem cell antigen (PSCA), Prostate-specific membrane antigen (PSMA), ROR1, Tumor-associated glycoprotein 72 (TAG-72), Vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NK. The method according to claim 3, comprising a tumor antigen selected from CS1, EGF1R, EGFR, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, and combinations thereof.

8. The aforementioned CAR is, A tumor antigen-binding domain that targets ganglioside G2 (GD2), Binds to transmembrane domains derived from CD8, CD28, CH2CH3, or NKG2D, The method according to claim 3, comprising a tumor antigen-binding domain bound to an intracellular domain containing a costimulatory domain derived from CD27, CD28, CD137, CD154, CD244, CD278, or a combination thereof, and a cytotoxic domain derived from CD3ζ, DAP10, DAP12, CD16, or a combination thereof.

9. The method according to claim 1, wherein the solid tumor is melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, renal cell carcinoma, non-small cell lung cancer, head and neck cancer, bladder cancer, hepatocellular carcinoma, spinal chordoma, bile duct cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma of the skin or head and neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma, Wilms' tumor, medulloblastoma, ependymoma, pineoblastoma, peripheral neuroectoderm tumor, or germ cell tumor.

10. The method according to claim 1, wherein the TRT agent is metaiodobenzylguanidine (MIBG), wherein the iodine atom in the MIBG is a radioactive iodine isotope; a radiolabeled tumor targeting antibody; a radioactive isotope of radium; or a radiophospholipid ether metal chelate.

11. The aforementioned radioactive phospholipid ether metal chelate is, 【Chemistry 1】 Formula 1 Radioactive phospholipid ether metal chelate having (In the formula, R １ (a) A chelating agent that chelates to a metal atom, wherein the metal atom is an alpha, beta, or Auger-emitting metal isotope having a half-life of more than 6 hours and less than 30 days, or (b) a radioactive halogen isotope. a is either 0 or 1; n is an integer between 12 and 30; m is either 0 or 1; Y is -H, -OH, -COOH, -COOX, -OCOX, or -OX, and X is an alkyl or arylalkyl group; R ２ is -N ＋ H ３ -, -N ＋ H ２ Z, -N ＋ HZ ２ or -N ＋ Z ３ where each Z is independently alkyl or aryl, b is either 1 or 2. The method according to claim 10, comprising:

12. In equation 1(i), m is 0, b is 1, n is an integer from 12 to 30, and R ２ Ha-N ＋ Z ３ Here, each Z is independently alkyl or aryl, or (ii) m is 1, b is 1, n is an integer from 12 to 30, R ２ Ha-N ＋ Z ３ Here, each Z is independently alkyl or aryl; or (iii)m is 0, b is 1, n is 18, R ２ Ha-N ＋ Z ３ Here, each Z is independently alkyl or aryl; or (iv)m is 1, b is 1, n is 18, R ２ Ha-N ＋ Z ３ The method according to claim 11, wherein each Z is independently alkyl or aryl.

13. (1) The metal isotope is selected from the group consisting of Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212 and Th-227; or (2) Radioactive halogen isotopes are １２３ I, １２４ I, １２５ I, １３１ I, ２１１ At, ７７ Br and ７６ The method according to claim 11, selected from the group consisting of Br.

14. The chelating agents are 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) and its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives; 1,4,7-triazacyclononane, 1-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives; 1,4,7,10-tetraaziledodecane, 1-glutaric acid-4,7,10-triacetic acid (DOTAGA) and its derivatives; 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives; 1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11,-diacetic acid (CB-TE2A) and its derivatives; Diethylenetriaminepentaacetic acid (DTPA), its diesters and their derivatives; 2-Cyclohexyldiethylenetriaminepentaacetic acid (CHX-A''-DTPA) and its derivatives; Defoloxamine (DFO) and its derivatives; 1,2-[[6-carboxypyridine-2-yl]methylamino]ethane (H2dedpa) and its derivatives; and DADA and its derivatives, with structure: 【Chemistry 2】 The method according to claim 11, comprising a selection from the group consisting of DADA and its derivatives.

15. The chelating agent that chels onto the metal atom is 【Transformation 3】 【change】 The method according to claim 11, selected from the group consisting of the following.

16. The aforementioned radioactive phospholipid ether metal chelate is 【Chemistry 4】 【change】 【change】 【change】 【change】 【change】 The method according to claim 11, wherein the formula is selected from the group consisting of the following.

17. The method according to claim 16, wherein the radioactive phospholipid ether metal chelate is NM600 which chelates to the metal atom.

18. The aforementioned radioactive phospholipid ether metal chelate is ９０ The method according to claim 17, wherein the product is Y-NM600.

19. The method according to claim 1, wherein the modified immune cells are CAR T cells containing the B7-H3 tumor antigen, and the TRT agent is NM600.

20. The method according to claim 19, wherein the subject has leptomeningeal disease (LMD).

21. The method according to claim 1, wherein the modified immune cells are CAR T cells that recognize the ganglioside GD2 tumor antigen, and the TRT agent is NM600.

22. The method according to claim 21, wherein the subject has neuroblastoma or another cancer that expresses GD2.

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