Nanoparticle formulations for in-situ CAR T cell generation

Abstract

In some embodiments, the Disclosure provides a method for generating CAR T cells in situ. The Disclosure provides lipid nanoparticles that selectively target splenic cells, in particular lymphocytes, such as T cells. The lipid nanoparticles provided herein have an apparent pK of less than 6. a It contains a five-component composition that includes a permanent anionic lipid that imparts [a certain characteristic].

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 534,327 filed on 23 August 2023, the contents of which are incorporated herein by reference.

[0002] Description of research and development funded by the federal government. This invention was implemented with government support, receiving grants R01 CA269787-01 and R01 5R01EB025192-06 from the National Institutes of Health (NIH). The government has specific rights to this invention.

[0003] Sequence listing reference This application includes a sequence listing XML file, which has been filed electronically and is incorporated in its entirety herein by reference. The sequence listing XML file, created on 23 August 2024, is named UTFDP4274WO.xml and has a size of 12,216 bytes. [Background technology]

[0004] I. Field The present invention generally relates to the fields of medicine, biochemistry, and nucleic acid delivery. For example, in certain embodiments, the present invention relates to the production of CAR T cells in situ. In certain embodiments, the present invention relates to compositions formulated for the selective delivery of nucleic acid cargo to spleen cells, for example, lymphocytes.

[0005] II. Description of Related Technologies Chimeric antigen receptor (CAR) T-cell therapy has advanced the field of cancer immunotherapy, from the initial reports (Milone et al., 2009, Carpenito et al., 2009, Huang et al., 2008) to six CAR T-cell therapies approved for clinical use as of April 2023 (FDA Kymriah, 2017, FDA Yescarta, 2017, FDA Tecartus, 2020, FDA ABECMA, 2021, FDA Breyanzi, 2021, FDA CARVYKTI, 2022). Approximately 90% of patients with B-cell malignancies respond to CAR T-cell therapy. However, longitudinal studies have shown that 40-60% of patients experience relapse (Gu et al., 2022). Furthermore, current methods for CAR T cell production are complex (Amini et al., 2022), extremely expensive, and cost up to $500,000 (Hernandez et al., 2018, Choi et al., 2022). Ex vivo CAR T cell production requires the collection of the patient's blood and the isolation of the T cell population in a process called leukocyte apheresis. The T cells are then subjected to ex vivo manipulation via viral transduction to insert the CAR gene, followed by the activation and proliferation of the T cells (Figure 1A). Before treatment, patients must undergo lymphocyte depletion through chemotherapy several days prior to CAR T cell infusion (Liang et al., 2020, Owen et al., 2023). Because this process uses the patient's own T cells, CAR T cells cannot be mass-produced and cannot be immediately used when needed for any patient. In addition, facilities capable of manufacturing this type of cell therapy are limited, and the entire process can take 4-6 weeks. All of these factors combined reduce the opportunities for patients to utilize CAR T-cell therapy. Therefore, there is a need for safe, virus-free methods to generate CAR T cells directly within the patient's body (Xin et al., 2022; Parayath et al., 2021) (Figure 1B). [Overview of the Initiative]

[0006] In some embodiments, the disclosure provides lipid compositions, specifically lipid nanoparticles, that can be used in methods for preparing chimeric antigen receptor (CAR) T cells. In particular, the compositions can be used to target spleen cells, for example, T cells.

[0007] In another aspect, the present disclosure provides a method for preparing chimeric antigen receptor (CAR) T cells in a patient, comprising administering mRNA encapsulated within lipid nanoparticles to the patient, wherein the lipid nanoparticles selectively bind to spleen cells, and the administration results in the formation of CAR T cells in vivo.

[0008] In yet another embodiment, the Disclosure provides a method for preparing chimeric antigen receptor (CAR) T cells in a patient, comprising administering mRNA encapsulated in lipid nanoparticles to the patient, wherein the lipid nanoparticles selectively internalize in spleen cells, and the administration results in the formation of CAR T cells in vivo.

[0009] In some embodiments, the spleen cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells.

[0010] In some embodiments, lipid nanoparticles have a pK of less than 6. a It has pK a It is approximately 1 to approximately 6. In some embodiments, pK a It is approximately 3 to 6.

[0011] In some embodiments, the lipid nanoparticles include ionizable cationic lipids. In some embodiments, the ionizable cationic lipids are dendrimers or dendrons. In some embodiments, the dendrimers or dendrons are of formula: Core - Repeating Unit - Terminal Group (I) [In the formula, the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atoms with repeating units. The core has the formula:

Chemical formula

Chemical formula

Chemical formula

[0012] In some embodiments, the terminal group is of formula: [ka] [In the formula, Y4 is Arcandil (C≦18) and R 10 It is further defined by [being hydrogen].

[0013] In some embodiments, the core is given by the formula: [ka] [In the formula, X2 is N(R5) y And, R5 is hydrogen or alkyl (C≦8) , or substituted alkyl (C≦18) and y is 0, 1, or 2, provided that the sum of y and z is 3. R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substitutional form of either of these groups, b is 1, 2, 3, 4, 5, or 6, and z is further defined as 1, 2, or 3, provided that the sum of z and y is 3.

[0014] In some embodiments, the core is given by the formula: [ka] [In the formula, X3 is -NR6-, where R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) alkoxydiyl (C≦8) , Allenziil (C≦8) , heteroalene zil (C≦8), heterocycloalkanediyl (C≦8) or a substituted form of any of these groups, R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) dialkylamino (C≦12) or a substituted form of either of these groups, or the formula: -N(R f )) f (CH2CH2N(R c )) e R d group, wherein e and f are each independently 1, 2, or 3, provided that the sum of e and f is 3, R c R d and R f are each independently hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and are further defined by c and d are each independently 1, 2, 3, 4, 5, or 6.

[0015] In some embodiments, the core is

Chemical formula

[0016] In some embodiments, the dendrimer is

Chemical formula

[0017] In some embodiments, the lipid nanoparticles further comprise a permanent anionic lipid. In some embodiments, the permanent anionic lipid comprises a phosphate group. In some embodiments, the permanent anionic lipid is​ [ka] [In the formula, R1 and R2 are each independently alkyl (C8-C24) Alkenil (C8-C24) , or a substitutional form of either base, R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) It is either -Y1-R4, where, Y1 is Alkanzil (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy (C≦8-24) or substituted acyloxy (C≦8-24) It is further defined as [being].

[0018] In some embodiments, permanent anionic lipids are [ka] It is further defined as follows.

[0019] In some embodiments, the lipid nanoparticles further contain phospholipids. In some embodiments, the lipid nanoparticles further contain steroids such as cholesterol. In some embodiments, the lipid nanoparticles further contain polymer complex lipids. In some embodiments, the polymer complex lipids are PEGylated lipids. In some embodiments, the polymer complex lipids are of formula: [ka] [In the formula, R 12 and R 13 Each of them is independently alkyl (C≦24) Alkenil (C≦24) , or a substitutional form of either of these groups, R e hydrogen, alkyl (C≦8) , or substituted alkyl(C≦8) and x is further defined by being from 1 to 250.

[0020] In other embodiments, the polymeric complex lipid is dimyristoyl-sn-glycerol or a compound of the formula: [Chemical formula] [wherein, n1 is from 5 to 250, and n2 and n3 are each independently from 2 to 25].

[0021] In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In some embodiments, the mRNA encodes two or more chimeric antigen receptors. In some embodiments, the mRNA further encodes a costimulatory molecule. In some embodiments, the mRNA encodes two or more costimulatory molecules. In some embodiments, the mRNA further encodes a signaling domain. In some embodiments, the mRNA encodes two or more signaling domains. In some embodiments, the mRNA further encodes one or more cytokines. In some embodiments, the mRNA is (i) one or more chimeric antigen receptors, (ii) one or more signaling domains, and (iii) encodes one or more costimulatory molecules.

[0022] In some embodiments, the mRNA is (i) one or more chimeric antigen receptors, (ii) one or more signaling domains, (iii) one or more costimulatory molecules, and (iv) encodes one or more cytokines.

[0023] In some embodiments, the encoded chimeric antigen receptor is an antigen of a tumor marker. In some embodiments, the tumor marker is CD19 or CD20. In some embodiments, the encoded co-stimulatory molecule is CD28 or 41BB. In some embodiments, the encoded signaling domain is CD3ζ.

[0024] In some embodiments, the lipid nanoparticles contain about 1% to about 45% ionizable cationic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 30% ionizable cationic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 1% to about 40% permanent anionic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 5% to about 20% permanent anionic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 1% to about 45% phospholipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 30% phospholipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 70% steroids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 25% to about 60% polymer composite lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain polymer composite lipids at a rate of about 0.01% to about 15% of the lipid nanoparticles as a molar percentage. In some embodiments, the lipid nanoparticles contain polymer composite lipids at a rate of about 0.1% to about 10% of the lipid nanoparticles as a molar percentage.

[0025] In some embodiments, lipid nanoparticles are (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, and (iii) Contains steroids.

[0026] In some embodiments, lipid nanoparticles are (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids, and (iv) Contains polymer complex lipids

[0027] In some embodiments, lipid nanoparticles are (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids, and (iv) Contains phospholipids

[0028] In some embodiments, lipid nanoparticles are (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids, (iv) Phospholipids, and (v) Contains polymer complex lipids

[0029] In some embodiments, the method involves systemically administering lipid nanoparticles to a patient. In some embodiments, systemic administration is by injection. In some embodiments, systemic administration is by intravenous administration.

[0030] In another embodiment, the present disclosure provides a method for treating a disease or disorder in a patient, comprising administering lipid nanoparticles containing mRNA to the patient, wherein the mRNA encodes a chimeric antigen receptor and the lipid nanoparticles selectively bind to spleen cells. In some embodiments, the disease is cancer. In some embodiments, the cancer is a cancer of the lymphoid system. In some embodiments, the cancer of the lymphoid system is lymphoma. In other embodiments, the disease is a cardiovascular disease. In some embodiments, the cardiovascular disease is trauma or heart failure. In other embodiments, the disease or disorder is a fibrous disease.

[0031] In yet another aspect, the present disclosure provides a method of modifying lymphocytes, comprising administering to a patient a lipid nanoparticle comprising mRNA encoding a chimeric antigen receptor, wherein the lipid nanoparticle selectivity binds to lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells.

[0032] In yet still another aspect, the present disclosure (A) a lipid nanoparticle comprising (i) an ionizable cationic lipid, (ii) a permanently anionic lipid, and (iii) one or more additional lipids, and (B) mRNA encoding a chimeric antigen receptor, a composition comprising, wherein the mRNA is encapsulated within the lipid nanoparticle, and the lipid nanoparticle has an apparent pK of less than 6 a to provide a composition.

[0033] In some embodiments, the additional lipid includes steroids such as cholesterol; In some embodiments, the additional lipid includes phospholipids. In some embodiments, the phospholipid is a neutral phospholipid. In some embodiments, the additional lipid includes polymer complex lipids. In some embodiments, the polymer complex lipid is a PEGylated lipid.

[0034] In some embodiments, the lipid nanoparticles contain about 1% to about 45% ionizable cationic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 30% ionizable cationic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 1% to about 40% permanent anionic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 5% to about 20% permanent anionic lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 1% to about 45% phospholipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 30% phospholipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 10% to about 70% steroids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain about 25% to about 60% polymer composite lipids as a mole percentage of the lipid nanoparticles. In some embodiments, the lipid nanoparticles contain polymer composite lipids at a rate of about 0.01% to about 15% of the lipid nanoparticles as a molar percentage. In some embodiments, the lipid nanoparticles contain polymer composite lipids at a rate of about 0.1% to about 10% of the lipid nanoparticles as a molar percentage.

[0035] Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are provided only as illustrative examples, while illustrating specific embodiments of this disclosure, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0036] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure. The present invention may be better understood by referring to one of these drawings in conjunction with a detailed description of the specific embodiments presented herein.

[0037] [Figure 1] In situ CAR T cell transfection avoids the cumbersome ex vivo CAR T cell production process. (A) Conventional ex vivo method for CAR T cell preparation. (B) The present disclosure method for in situ CAR T cell production using Spleen SORT LNP for in vivo transfection of T cells. [Figure 2] Spleen SORT LNP is transfected into T cells in vivo via intravenous administration. (A) Structure of the lipid used in the LNP. (B) Formulation details of Spleen SORT LNP and control LNP used in the method of this disclosure. (C) Bioluminescence of organs in C57BL / 6 mice 24 hours after intravenous administration of 0.3 mg / kg firefly luciferase mRNA with 0% 18:1 PA LNP (n=3). (D) Bioluminescence of organs in C57BL / 6 mice 24 hours after intravenous administration of 0.3 mg / kg firefly luciferase mRNA with 10% 18:1 PA LNP (n=3). (E) Quantification of luminescence in the spleen, lung, and liver with 0% 18:1 PA LNP and 10% 18:1 PA LNP. (F) Percentage of TdTomato+ T cells (CD3+) after two 48-hour interval doses of 0.5 mg / kg Cre mRNA IV treatment with Spleen SORT LNP (n=3). (G) Percentage of TdTomato+ CD8 T cells and TdTomato+ CD4 T cells after two 48-hour interval doses of 0.5 mg / kg Cre mRNA IV treatment with Spleen SORT LNP (n=3). (H) Percentage of TdTomato+ B cells, macrophages, monocytes, and neutrophils. [Figure 3] Characterization of the 5A2-SC8 LNP component. (A) Proton nuclear magnetic resonance (1H-NMR) of 5A2-G1, an intermediate of 5A2-SC8. (B) Proton nuclear magnetic resonance (1H-NMR) of 5A2-SC8, the final product. [Figure 4]The addition of 18:1 PA to LNP formulations reduces the overall pKa of the LNPs. LNPs with 0% and 10% 18:1 PA were formulated together with CAR19-41BBz mRNA. Particle size distribution (A), polydispersity index (PDI) (B), zeta potential (C), and overall pKa (D) were measured. [Figure 5] A gating strategy was used to sort CD3+, CD4+, CD8+, and TdTom+ T cells. [Figure 6] Characterization of LNPs used in lymphocyte-rich lymphoma models. (A) Capillary electrophoresis of IVT mRNA from three batches of CAR19-41BBz mRNA and CAR19-28z. Spleen SORT LNP was formulated with CAR19-41BBz mRNA and Cre mRNA. Particle size distribution (B), polydispersity index (PDI) (C), and zeta potential (D) were measured. [Figure 7] Spleen SORT LNP-mediated in-situ CAR T cell generation reduced hepatic tumor volume in a lymphocyte-rich lymphoma model. (A) Protocol and treatment plan followed to create a lymphocyte-rich syngeneic model of B-cell lymphoma in Balb / c mice. After tumor engraftment, Balb / c mice were divided into a saline control group, a Cre mRNA Spleen SORT LNP treatment group, and a CAR19-41BBz mRNA Spleen SORT LNP treatment group. (B) mRNA design used in the treatment model. (C) Bioluminescence tracking of luciferase-expressing lymphoma tumors (n=12). (D) Liver samples taken from tumor-bearing mice 5 weeks after treatment (n=2). (E) Abdominal circumference of mice 3.5 weeks, 4 weeks, and 4.5 weeks after treatment (n=12). One-way ANOVA with multiple comparisons (Dunnett). [Figure 8] Initial survival curve for an invasive lymphocyte-rich B-cell lymphoma model. [Figure 9]Treatment with CAR19-41BBz mRNA Spleen SORT reduces tumor burden. (A) Protocol and treatment plan followed to create a lymphocyte-rich syngeneic model of B-cell lymphoma in Balb / c mice. After tumor engraftment, Balb / c mice were divided into two groups: a saline control group (n=5) and a CAR19-41BBz mRNA Spleen SORT LNP group (n=9). (B) Design of mRNA used in the treatment model. (C) Bioluminescence tracking of luciferase-expressing lymphoma tumors. Abdominal circumference of mice at 4 weeks (D) and 5 weeks (E) after treatment. (F) Survival analysis comparison of the two treatment groups. Unpaired t-test (two-sided). Kaplan-Meier simple survival analysis using Logrank (Mantel-Cox test). [Figure 10] Survival extension of an aggressive lymphocyte-rich lymphoma model after LNP-mediated in situ CAR T cell generation. (A) Protocol and treatment plan followed to create a lymphocyte-rich syngeneic model of B-cell lymphoma in Balb / c mice. After tumor engraftment, Balb / c mice were divided into three groups: a saline control group (n=6), a CAR19-41BBz mRNA Spleen SORT LNP group (n=6), and a CAR19-28z mRNA Spleen SORT LNP group (n=5). (B) Design of mRNA used in the treatment model. (C) BLI images of luciferase-expressing lymphoma tumors. (D) BLI values ​​of tumor-bearing mice over time. (E) BLI values ​​of mice 3 weeks after treatment. (F) Survival analysis comparison of the three treatment groups. Unpaired t-test (two-sided). Kaplan-Meier simple survival analysis using Logrank (Mantel-Cox test). [Figure 11]Lymphocyte tumor infiltration increased after treatment with CAR19-41BBz mRNA Spleen SORT LNP. (A) Tumor tissue excised from tumor-bearing mice in Figure 7 was stained, and CD3+ T cells (red) and DAPI+ nuclei (blue) were quantified and imaged using a confocal microscope. (B) Tumor-infiltrating lymphocytes were quantified from confocal images (n=14). (C) Similarly, tumor tissue excised from tumor-bearing mice in Figure 9 was stained, and CD3+ T cells were quantified and imaged using a confocal microscope. (D) Tumor-infiltrating lymphocytes were quantified from confocal images (n=10). One-way ANOVA with multiple comparisons (Dunnett). Unpaired t-test (two-sided). [Figure 12] Tumor-infiltrating lymphocytes in the saline group. Tumor tissue from the liver of saline-treated mice (Figure 3) was thin-sectioned and stained for CD3+ T cells. Confocal imaging was used to quantify tumor-infiltrating lymphocytes. [Figure 13] Tumor-infiltrating lymphocytes in the mRNA Cre group. Tumor tissue from the liver of mRNA Cre mice (Figure 3) was thin-sectioned and stained for CD3+ T cells. Confocal imaging was used to quantify tumor-infiltrating lymphocytes. [Figure 14] Tumor-infiltrating lymphocytes in the mRNA CAR19-41BBz group. Tumor tissue from the liver of mRNA CAR19-42BBz mice (Figure 3) was thin-sectioned and stained for CD3+ T cells. Confocal imaging was used to quantify tumor-infiltrating lymphocytes. [Figure 15] Tumor-infiltrating lymphocytes in the saline group. Tumor tissue from the liver of saline-treated mice (Figure 4) was thinly sectioned and stained for CD3+ T cells. Confocal imaging was used to quantify tumor-infiltrating lymphocytes. [Modes for carrying out the invention]

[0038] The present disclosure provides a method for generating in situ CAR T cells. The method enables selective LNP mRNA delivery, for example, to extrahepatic targets such as the spleen or lung. The method may be beneficial in that it does not involve antibody-mediated targeting (Cheng et al., 2020). The method involves adding a chemically defined supplemental fifth lipid to the LNP, enabling selective organ targeting (SORT). In some embodiments, the SORT molecule is an anionic lipid. In some embodiments, the SORT LNP of the method exhibits spleen-specific targeting, including delivery to T cells. The method may be useful for mRNA in situ CAR T cell transfection. In some embodiments, the method may be useful for treating cancers such as lymphoma.

[0039] This disclosure provides a method for transfecting T cells after intravenous injection, containing up to 5.8% CD8+ T cells. CAR T cells produced in situ according to the method of this disclosure can reduce tumor burden in invasive models and decrease the number of lesions in the liver. Furthermore, CAR T cells produced in situ according to the method of this disclosure reduced the waist circumference of mice treated with CAR mRNA. CAR T cells produced in situ according to the method of this disclosure extended the survival of mice in a less invasive lymphoma model. Production of CAR T cells in situ according to this method resulted in an increase in tumor-infiltrating lymphocytes in hepatic lesions, which may (without being constrained by theory) explain the observed smaller waist circumference and fewer metastatic lesions in the liver of mice. In some embodiments, the method of this disclosure facilitates in situ CAR T cell therapy for B-cell lymphoma. The method of this disclosure is intended for the use of other CARs to treat equally or more invasive tumors.

[0040] Furthermore, as the design of more potent CARs continues in this field to target hematological malignancies and solid tumors, this method provides a promising way to produce CAR T cells in situ for a variety of applications.

[0041] A.CAR T cells Adoptive immunotherapy has traditionally involved the transfer of autologous antigen-specific T cells generated ex vivo. This specification provides a method for generating antigen-specific T cells in situ by genetic engineering. Novel specificity in T cells has been successfully induced through the introduction of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors consisting of a target moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv) and includes the light chain and variable fragment of a monoclonal antibody linked by a mobile linker. Binding moieties based on receptor or ligand domains have also been successfully utilized. The signaling domains of first-generation CARs are derived from the cytoplasmic region of CD3ζ or the Fc receptor gamma chain. CARs have been successfully used to redirect T cells to antigens expressed on the surface of tumor cells from various malignancies, such as lymphomas and solid tumors.

[0042] i. Chimeric antigen receptor Chimeric antigen receptor (CAR) molecules are recombinant fusion proteins distinguished by both their ability to bind to antigens and their ability to transmit activation signals via an immunoreceptor activation motif (ITAM) located at their cytoplasmic terminal to activate genetically modified immune effector cells for killing, proliferating, and producing cytokines. Receptor constructs that utilize the antigen-binding moiety (e.g., those made from single-chain antibodies (scFv)) offer the additional advantage of being "universal" in that they bind to native antigens on the surface of target cells in an HLA-independent manner.

[0043] Embodiments of CARs described herein include nucleic acids encoding antigen-specific CAR polypeptides comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain including an antigen-binding domain. CARs can recognize epitopes consisting of spaces shared between one or more antigens. Optionally, CARs may include a hinge domain located between the transmembrane domain and the antigen-binding domain. CARs may further include a signal peptide on the cell surface that induces CAR expression. For example, CARs may include a signal peptide from GM-CSF. CARs may be co-expressed with membrane-bound cytokines to improve persistence. For example, CARs may be co-expressed with membrane-bound IL-15.

[0044] Depending on the arrangement of the CAR domain and the specific sequence used in the domain, immune effector cells expressing the CAR may exhibit different activity against target cells. Different CAR sequences may be introduced into immune effector cells to create engineered cells, these engineered cells may be selected for their SRC elevation, and the selected cells may be tested for activity to identify the CAR construct predicted to have the greatest therapeutic efficacy.

[0045] Chimeric antigen receptors can be produced by any means known in the art, but are preferably produced using recombinant DNA technology. Nucleic acid sequences encoding several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence using standard molecular cloning techniques (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.). The resulting coding regions can be inserted into an expression vector and used to transform allogeneic or autologous immune effector cells of a suitable expression host, such as T cells or NK cells.

[0046] Chimeric constructs can be introduced into immune effector cells as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in an orientation suitable for expression. Alternatively, a viral vector (e.g., retroviral vector, adenovirus vector, adeno-associated virus vector, or lentiviral vector) can be used to introduce a chimeric construct into immune effector cells. Vectors suitable for use according to the methods of the present invention do not replicate in immune effector cells. Numerous virus-based vectors are known, such as vectors based on HIV, SV40, EBV, HSV, or BPV, and the viral copy number maintained in the cell is small enough to maintain cell survival.

[0047] ii. Antigen-binding domain The antigen-binding domain may include the complementarity-determining region of a monoclonal antibody, the variable region of a monoclonal antibody, and / or their antigen-binding fragments. The antigen-binding region or domain may include VH and VL chain fragments of a single-stranded variable fragment (scFv) derived from a specific mouse antibody, humanized antibody, or human monoclonal antibody. The fragments may also be any number of different antigen-binding domains of an antigen-specific antibody. The fragments may be antigen-specific scFv encoded by a sequence optimized for human codon usage frequency for expression in human cells. In certain embodiments, the VH and VL domains of the CAR are separated by a linker sequence such as a Whitlow linker.

[0048] A prototype CAR encodes an scFv containing VH and VL domains derived from a single monoclonal antibody (mAb), bound to a transmembrane domain and one or more cytoplasmic signaling domains (e.g., a costimulatory domain and a signaling domain). Thus, the CAR may contain the LCDR1-3 and HCDR1-3 sequences of an antibody that binds to the coronavirus spike protein. However, in a further embodiment, two or more antibodies that bind to the target antigen are identified, and a CAR is constructed containing (1) the HCDR1-3 sequences of a first antibody that binds to the antigen, and (2) the LCDR1-3 sequences of a second antibody that binds to the antigen. Such a CAR containing HCDR and LCDR sequences from two different antigen-binding antibodies may have the advantage of selectively binding to a specific conformation of the antigen (e.g., the pre-fusion conformation of the spike protein).

[0049] Alternatively, CARs may be manipulated using VH and VL chains derived from different mAbs to generate a panel of CAR+ immunoeffector cells. The antigen-binding domain of the CAR may contain any combination of the LCDR1-3 sequences of the first antibody and the HCDR1-3 sequences of the second antibody.

[0050] iii. Hinged Domain CAR polypeptides may contain a hinge domain located between the antigen-binding domain and the transmembrane domain. In some cases, the hinge domain may be included within the CAR polypeptide to provide sufficient distance between the antigen-binding domain and the cell surface, or to mitigate the possibility of steric hindrance that could adversely affect antigen binding or effector function of CAR-modified immunoeffector cells. The hinge domain may contain sequences that bind to Fc receptors, such as FcγR2a or FcγR1a. For example, the hinge sequence may contain an Fc domain from a human immunoglobulin that binds to an Fc receptor (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE).

[0051] The CAR hinge domain may originate from the constant region or portion of human immunoglobulin (Ig) containing the Ig hinge, or from the transmembrane domain and CD8a-hinge region of human CD8α. The CAR hinge domain may contain the hinge-CH2-CH3 region of antibody isotype IgG4. The hinge domain (and / or CAR) may not contain the CH2 and CH3 sequences of wild-type human IgG4. Point mutations may be introduced into the antibody heavy chain CH2 domain to reduce glycosylation and nonspecific Fc gamma receptor binding in CAR-modified immunoeffector cells.

[0052] The CAR hinge domain may contain an Ig Fc domain that has at least one mutation compared to the wild-type Ig Fc domain, which reduces Fc-receptor binding. For example, the CAR hinge domain may contain an IgG4-Fc domain that has at least one mutation compared to the wild-type IgG4-Fc domain, which reduces Fc-receptor binding. The CAR hinge domain may contain an IgG4-Fc domain that has a mutation (such as an amino acid deletion or substitution) at the position corresponding to L235 and / or N297 compared to the wild-type IgG4-Fc sequence. For example, the CAR hinge domain may contain an IgG4-Fc domain that has an L235E and / or N297Q mutation compared to the wild-type IgG4-Fc sequence. The CAR hinge domain may contain an IgG4-Fc domain that has an amino acid substitution at the L235 position for a hydrophilic amino acid such as R, H, K, D, E, S, T, N, or Q, or for an amino acid that has similar properties to "E," such as D. The CAR hinge domain may contain an IgG4-Fc domain having an amino acid substitution at position N297 for an amino acid that has similar properties to "Q," such as S or T.

[0053] The hinge domains may contain sequences that are approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the IgG4 hinge domain, CD8a hinge domain, or CD28 hinge domain, or manipulated hinge domains.

[0054] iv. Transmembrane domain or co-stimulatory molecule Antigen-specific extracellular domains and intracellular signaling domains may be linked by transmembrane domains or co-stimulatory molecules. Polypeptide sequences that can be used as part of a transmembrane domain include, but are not limited to, human CD4 transmembrane domains, human CD28 transmembrane domains, transmembrane human CD3z domains, cysteine-mutated human CD3z domains, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16, CD8, and erythropoietin receptors. For example, a transmembrane domain may include a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequences described in U.S. Patent Publication 2014 / 0274909 (e.g., CD8 and / or CD28 transmembrane domains) or U.S. Patent No. 8,906,682 (e.g., CD8α transmembrane domains), which are incorporated herein by reference. The transmembrane domain may originate from the alpha, beta, or zeta chains of the T cell receptor, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., including at least one of their transmembrane domains). In certain specific embodiments, the transmembrane domain may be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CD8a or CD28 transmembrane domain.

[0055] v. Signaling Domain The signaling domain or intracellular signaling domain of a CAR is involved in the activation of at least one of the normal effector functions of immune cells engineered to express the CAR. The term "effector function" refers to a specific function of a differentiated cell. For example, the effector function of a T cell may be helper activity such as cytolytic activity or cytokine secretion. Effector functions in naive, memory, or memory-type T cells include antigen-dependent proliferation. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transmits effector function signals and induces cells to perform specific functions. Intracellular signaling domains may originate from the intracellular signaling domains of innate receptors. Examples of such innate receptors include any of the zeta chains or homologs of T cell receptors (e.g., eta, delta, gamma, or epsilon), MB1 chains, B29, Fc RIII, Fc RI, and combinations of signaling molecules such as CD3z and CD28, CD27, 4-1BB / CD137, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, CD40, OX40 / CD134, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling moieties of other members of the protein-activating family can also be used.

[0056] While the entire intracellular signaling domain can be used, it is often not necessary to use the entire intracellular polypeptide. To the extent that a cleaved portion of the intracellular signaling domain has a use, such a cleaved portion can be used in place of the complete chain, as long as it transmits the effector functional signal. Thus, the term “intracellular signaling domain” means that it includes a cleaved portion of the intracellular signaling domain sufficient to transmit the effector functional signal upon CAR binding to a target. One or more cytoplasmic domains may be used so that so-called third-generation CARs have at least two or three signaling domains fused together for additive or synergistic effects; for example, CD28 and 4-1BB can be combined within the CAR construct. In certain specific embodiments, the intracellular signaling domain contains a sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a domain containing a CD3z intracellular domain, a CD28 intracellular domain, a CD137 intracellular domain, or a CD28 intracellular domain fused to a 4-1BB intracellular domain.

[0057] B. Anionic lipids In some embodiments, the disclosure provides one or more lipids having one or more hydrophobic components and permanent anionic groups. One of the anionic groups that can be used in permanent anionic lipids is a phosphate group. The phosphate group may be a negatively charged compound that is deprotonated at a pH below 8, 9, 10, 11, 12, 13, or 14. Another anionic group that can be used in permanent anionic lipids is a carboxylic acid group. The carboxylic acid group may be a negatively charged compound that is deprotonated at a pH below 3, 4, 5, 6, or 7. The hydrophobic component is one or more C6-C 24 The alkyl or alkenyl group may be present. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0058] In some embodiments, the permanent anionic lipid is given by formula: [ka] [In the formula, R1 and R2 are each independently alkyl (C8-C24) Alkenil (C8-C24) , or a substitutional form of either base, R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) It is either -Y1-R4, where, Y1 is Alkanzil (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy (C≦8-24) or substituted acyloxy (C≦8-24) It has the structure of [being].

[0059] C. Ionizable lipids In some embodiments of this disclosure, compositions are provided that contain a compound comprising a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers exhibiting regular dendritic branching formed by the sequential or generational addition of branched layers toward or from the core, and characterized by a core, at least one internal branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994). In other embodiments, the term “dendrimer” as used herein is intended to encompass, without limitation, molecular structures having an internal core, an internal layer (or “generation”) which is a repeating unit regularly bonded to this initiating core, and an external surface which is a terminal group bonded to the outermost generation. A “dendron” is a species of dendrimer, having branches that extend directly from a core or a focal which can be bonded to a core, or that extend through a bonding portion to form a larger dendrimer. In some embodiments, the dendrimer structure has repeating groups radiating from a central core, doubling with each repeating unit per branch. In some embodiments, the dendrimers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like substances. These terms may be used to describe the compounds described herein that exhibit a dendron-like appearance (e.g., molecules radiating from a single focal point).

[0060] Dendrimers are polymers, but they may be preferred over conventional polymers due to their controllable structure, single molecular weight, numerous and controllable surface functional groups, and conventionally, the adoption of a spherical conformation after reaching a certain generation. Dendrimers can be prepared by sequentially reacting each repeating unit to produce monodisperse, dendritic, and / or generational polymeric structures. Each dendrimer consists of a central core molecule and dendritic wedges bonded to one or more functional sites on the central core. Depending on the assembly monomer used during preparation, a variety of functional groups such as anionic, cationic, hydrophilic, or lipophilic groups can be arranged on the surface layer of the dendrimer. In some embodiments, ionizable cationic lipids are expressed by formula: Core-repeating unit-end group (DI) [In the formula, the core is linked to repeating units by removing one or more hydrogen atoms from the core and replacing the atoms in repeating units.] The core is, formula: [ka] It has, During the ceremony, X1 is an amino or alkylamino (C≦12) , dialkylamino (C≦12) heterocycloalkyl (C≦12) heteroaryl (C≦12) , or their replacement forms, R1 is amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) , or a substitutional form of either of these groups, a is 1, 2, 3, 4, 5, or 6, or The core is, formula: [ka] It has, During the ceremony, X2 is N(R5) y And, R5 is hydrogen, alkyl (C≦18) , or substituted alkyl (C≦18) and y is 0, 1, or 2, provided that the sum of y and z is 3. R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substitutional form of either of these groups, b is 1, 2, 3, 4, 5, or 6, and z is 1, 2, or 3, provided that the sum of z and y is 3, or The core is, formula: [ka] It has, During the ceremony, X3 is -NR6-, where R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) alkoxydiyl (C≦8) , Allenziil (C≦8) , heteroalene zil (C≦8) heterocycloalkanediyl (C≦8) , or a substitutional form of any of these groups, R3 and R4 are independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) , or a substitution form of either of these groups, or formula: -N(R f ) f (CH2CH2N(R c )) e R d , [ka] It is the basis of, During the ceremony, e and f are independently 1, 2, or 3, provided that the sum of e and f is 3. R c , R d , and R f These are, independently, hydrogen and alkyl. (C≦6) , or substituted alkyl (C≦6) And, c and d are independently 1, 2, 3, 4, 5, or 6, or The core is alkylamine (C≦18) , dialkylamine (C≦36) heterocycloalkanes (C≦12) , or a substitutional form of any of these groups, The repeating units include a degradable diacyl and a linker. Degradable diacyl group, formula: [ka] It has, During the ceremony, A1 and A2 can be independently -O-, -S-, or -NR. a -and here, R a hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) And, Y3 is Arcanziel (C≦12) , Alkenzil (C≦12) , Allenziil (C≦12) , or a substitution form of any of these bases, or formula: [ka] It is the basis of, During the ceremony, X3 and X4 are Arkanzil (C≦12) , Alkenzil (C≦12) , Allenziil (C≦12) , or a substitutional form of any of these groups, Y5 is a covalent bond, alkanediyl (C≦12), Alkenzil (C≦12) , Allenziil (C≦12) , or a substitutional form of any of these groups, R9 is alkyl (C≦8) or substituted alkyl (C≦8) And, The linker group is, formula: [ka] It has, During the ceremony, Y1 is Alkanzil (C≦12) , Alkenzil (C≦12) , Allenziil (C≦12) , or a substitutional form of any of these groups, If the repeating unit contains a linker group, the linker group contains independent degradable diacyl groups bonded to both the nitrogen and sulfur atoms of the linker group (when n is greater than 1), the first group of the repeating unit is a degradable diacyl group, and for each linker group, the next repeating unit contains two degradable diacyl groups bonded to the nitrogen atom of the linker group, where n is the number of linker groups present in the repeating unit. The terminal group is, formula: [ka] It has, During the ceremony, Y4 is Arcandil (C≦18) is or alkandiil (C≦18) Alkanediyls in which one or more of the hydrogen atoms on top are replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≦18) And, R 10 is hydrogen, carboxy, hydroxy, or Ariel (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) N-heterocycloalkyl (C≦12) ,-C(O)N(R 11)-Arkanzil (C≦6) -heterocycloalkyl (C≦12) -C(O)-alkylamino (C≦12) -C(O)-dialkylamino (C≦12) -C(O)-N-heterocycloalkyl (C≦12) And here, R 11 hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) And, The last degradable diacyl in the chain is attached to the terminal group, n is a dendrimer or dendron, further defined by [0, 1, 2, 3, 4, 5, or 6] or a pharmaceutically acceptable salt thereof.

[0061] In some embodiments, the terminal group is of formula: [ka] [In the formula, Y4 is Arcandil (C≦18) and R 10 It is further defined by [being hydrogen].

[0062] In some embodiments, A1 and A2 are independently -O- or -NR a - is

[0063] In some embodiments of a dendrimer or dendron of formula (DI), the core is of formula: [ka] [In the formula, X2 is N(R5) y And, R5 is hydrogen or alkyl (C≦8) , or substituted alkyl (C≦18) and y is 0, 1, or 2, provided that the sum of y and z is 3. R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substitutional form of either of these groups, b is 1, 2, 3, 4, 5, or 6, and z is further defined as 1, 2, or 3, provided that the sum of z and y is 3.

[0064] In some embodiments of a dendrimer or dendron of formula (DI), the core is of formula: [ka] [In the formula, X3 is -NR6-, where R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) alkoxydiyl (C≦8) , Allenziil (C≦8) , heteroalene zil (C≦8) heterocycloalkanediyl (C≦8) , or a substitutional form of any of these groups, R3 and R4 are independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) , or a substitution form of either of these groups, or formula: -N(R f ) f (CH2CH2N(R c )) e R d , [ka] It is the basis of, During the ceremony, e and f are independently 1, 2, or 3, provided that the sum of e and f is 3. R c , R d , and R fThese are, independently, hydrogen and alkyl. (C≦6) , or substituted alkyl (C≦6) And, c and d are further defined independently by [1, 2, 3, 4, 5, or 6].

[0065] In some embodiments of the dendrimer or dendron of formula (I), the terminal group is of formula: [ka] Represented by, During the ceremony, Y4 is Arcandil (C≦18) and R 10 It is hydrogen.

[0066] In some embodiments of the dendrimer or dendron of formula (DI), the core is [ka] It is further defined as follows.

[0067] In some embodiments of the dendrimer or dendron of formula (DI), the degradable diacyl is [ka] It is further defined as follows.

[0068] In some embodiments of the dendrimer or dendron of formula (DI), the linker is [ka] Further defined as (D-VI), where Y1 is alkanediyl (C≦8) or substitute alkanediyl (C≦8) That is the case.

[0069] 1. In some embodiments of the dendrimer or dendron of formula (DI), the dendrimer or dendron is [ka] Selected from the group consisting of TIFF2026529161000037.tif192159, TIFF2026529161000038.tif206159, TIFF2026529161000039.tif79159 and their pharmaceutically acceptable salts.

[0070] B. Dendrimer or dendron of formula (X) A. In some embodiments of lipid compositions, ionizable cationic lipids are of the formula [ka] It is a dendrimer or dendron of the formula. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron of the formula. [ka] It is a dendrimer or dendron.

[0071] B. In some embodiments of the lipid composition, the ionizable cationic lipid has the structural formula: [ka] A dendrimer or dendron of generation (g) having or a pharmaceutically acceptable salt thereof, in the formula, (a) The core has the structural formula (X Core ): [ka] Includes, During the ceremony, Q is independent in each occurrence of covalent, -O-, -S-, and -NR. 2 -, or -CR 3a R 3b -and, R 2 In each occurrence, R 1g or -L2 -NR 1e R 1f And, R 3a and R 3b Each instance independently consists of an alkyl group that is substituted with hydrogen or optionally substituted (e.g., C1-C6, e.g., C1-C3), R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (If present) each occurrence is independently substituted by branching, hydrogen, or optional (e.g., C1-C 12 ) is a connection point to alkyl, L 0 , L 1 , and L 2 Each instance is independently selected from covalent bonds, alkylenes, heteroalkylenes, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyls, and arylenes, or Alternatively, L 1 A part of R 1c and R 1d Together with one of the following (e.g., C4-C6), it forms a heterocycloalkyl (e.g., containing one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur), and x 1 is 0, 1, 2, 3, 4, 5, or 6, (b) Each of the multiple (N) branches is independent of the structural formula (X Branch ): [ka] Including, in the formula, * This indicates the connection point of the branch to the core. g is 1, 2, 3, or 4. Z=2 (g-1) And, When g=1, G=0, or when g≠1,

number

[0072] X Core In some embodiments, Q is independently covalent, -O-, -S-, and -NR in each occurrence. 2 -, or -CR 3a R 3b X Core In some embodiments of X, Q is a covalent bond, independently in each occurrence. Core In some embodiments of X, Q is -O- independently in each occurrence. Core In some embodiments of X, Q is independently -S- in each occurrence. Core In some embodiments, Q is independently -NR in each occurrence. 2 And R 2 In each occurrence, R 1g or -L 2 -NR 1e R 1f X Core In some embodiments, Q is independently -CR in each occurrence. 3a R 3b R 3a And R 3a and R 3bEach instance is independently of the others, and is an alkyl group (e.g., C1-C6, e.g., C1-C3) substituted with hydrogen or of any choice.

[0073] X Core In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (If present) each instance independently represents a connection point to a branched, hydrogenated, or optionally substituted alkyl group. Core In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (If present) each occurrence is independently a branching point, a connection point to hydrogen. Core In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (If present) each instance is independently and branched, optionally substituted alkyl (e.g., C1-C) 12 It is a connection point to ).

[0074] X Core In some embodiments, L 0 , L 1 , and L 2 In each instance, independently, the elements are selected from covalent bonds, alkylenes, heteroalkylenes, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyls, and arylenes, or, alternatively, L 1 A part of R 1c and R 1dTogether with one of them, it forms a heterocycloalkyl (for example, C4-C6, containing one or two nitrogen atoms and optionally an additional heteroatom selected from oxygen and sulfur). Core In some embodiments, L 0 , L 1 , and L 2 Each of these can be a covalent bond, independently of the others in their respective appearances. Core In some embodiments, L 0 , L 1 , and L 2 Each of these can be hydrogen independently in its respective appearance. Core In some embodiments, L 0 , L 1 , and L 2 Each of these occurrences is independent of the alkylene (e.g., C1-C 12 For example, this could be C1-C6 or C1-C3). Core In some embodiments, L 0 , L 1 , and L 2 Each of these occurrences is independent of the heteroalkylene (e.g., C1-C) 12 For example, this could be C1-C8 or C1-C6). Core In some embodiments, L 0 , L 1 , and L 2 Each of these can independently be a heteroalkylene (e.g., C2-C8 alkylene oxide, e.g., oligo(ethylene oxide)) in each of its occurrences. Core In some embodiments, L 0 , L 1 , and L 2 Each of these can independently be [alkylene]-[heterocycloalkyl]-[alkylene][(e.g., C1-C6)alkylene]-[(e.g., C4-C6)heterocycloalkyl]-[(e.g., C1-C6)alkylene] in each instance. Core In some embodiments, L 0 , L 1 , and L2 Each instance can independently be [alkylene]-(arylene)-[alkylene][(e.g., C1-C6)alkylene]-(arylene)-[(e.g., C1-C6)alkylene]. Core In some embodiments, L 0 , L 1 , and L 2 Each instance can independently be [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6)alkylene]-phenylene-[(e.g., C1-C6)alkylene]). Core In some embodiments, L 0 , L 1 , and L 2 Each of these can independently be a heterocycloalkyl (e.g., C4-C6 heterocycloalkyl) in its respective appearance. Core In some embodiments, L 0 , L 1 , and L 2 Each of these can independently be an arrine (e.g., phenylene) in its respective appearance. Core In some embodiments, L 1 A part of R 1c and R 1d It forms a heterocycloalkyl group with one of the two. Core In some embodiments, L 1 A part of R 1c and R 1d Together with these atoms, a heterocycloalkyl group (e.g., a C4-C6 heterocycloalkyl group) is formed, which may contain one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur.

[0075] X Core In some embodiments, L 0 , L 1 , and L 2 In each instance, they are independently covalent bonds, C1-C6 alkylenes (e.g., C1-C3 alkylenes), and C2-C 12(For example, C2-C8) alkylene oxide (for example, oligo(ethylene oxide), for example, -(CH2CH2O) 1-4 -(CH2CH2)-), [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene](for example, [ka] and [(C1-C4)alkylene]-phenylene-[(C1-C4)alkylene](for example, [ka] Selected from: X Core In some embodiments, L 0 , L 1 , and L 2 In each instance, C1-C6 alkylene (e.g., C1-C3 alkylene) and -(C1-C3 alkylene-O) appear independently. 1-4 Selected from -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-, and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-. Core In some embodiments, L 0 , L 1 , and L 2 In each instance, independently, L is a C1-C6 alkylene (e.g., a C1-C3 alkylene). In some embodiments, L 0 , L 1 , and L 2 Each of these occurrences is independent of the others, C2-C 12 (For example, C2-C8) alkylene oxide (for example, -(C1-C3 alkylene-O) 1-4 -(C1-C3 alkylene)) X Core In some embodiments, L 0 , L 1 , and L 2Each of these is independently selected in its respective appearance from [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-phenylene-(C1-C3alkylene)-) and [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-).

[0076] X Core In some embodiments, x 1 X is 0, 1, 2, 3, 4, 5, or 6. Core In some embodiments, x 1 X is 0. Core In some embodiments, x 1 X is 1. Core In some embodiments, x 1 The answer is 2. Core In some embodiments, x 1 X is 0. Core In some embodiments, x 1 X is 1. Core In some embodiments, x 1 The answer is 2. Core In some embodiments, x 1 The answer is 3. Core In some embodiments, x 1 It is 4. Core In some embodiments, x 1 It is 5. Core In some embodiments, x 1 The answer is 6.

[0077] 2.X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] Includes. X Core In some embodiments, the core has the structural formula: [ka] The formula includes, where Q' is -NR2- or -CR3aR3b-, and q1 and q2 are independently 1 or 2. Core In some embodiments, the core has the structural formula: [ka] X Core In some embodiments, the core is a structural formula [ka] The formula includes, where ring A is an optionally substituted aryl or an optionally substituted (e.g., C3-C12, e.g., C3-C5) heteroaryl. Core In some embodiments, the core is a structural formula [ka] Includes.

[0078] X Core In some embodiments, the core comprises the structural formula shown in Table A and a pharmaceutically acceptable salt thereof, wherein, * This indicates the bonding point of the core to one of the multiple branches. In some embodiments, the core examples in Table A are not limited to the stereoisomers (i.e., enantiomers, diastereomers) described. [Table 1] TIFF2026529161000062.tif199159TIFF2026529161000063.tif201159TIFF2026529161000064.tif211159TIFF2026529161000065.tif170159

[0079] X Core In some embodiments, the core is [ka] A structural formula selected from the group consisting of TIFF2026529161000067.tif47159, and pharmaceutically acceptable salts thereof, wherein in the formula, * indicates a connection point of the core to one of the multiple branches or to H. In some embodiments, in the formula, * This indicates the connection point of the core to one of several branches.

[0080] X Core In some embodiments, the core is structure [ka] It has, in the formula, * This indicates a connection point of the core to one of several branches or to H. In some embodiments, at least two branches are connected to the core. In some embodiments, at least three branches are connected to the core. In some embodiments, at least four branches are connected to the core.

[0081] X Core In some embodiments, the core is structure [ka] It has, in the formula, * This indicates a connection point of the core to one of the multiple branches or to H. In some embodiments, at least four branches are connected to the core. In some embodiments, at least five branches are connected to the core. In some embodiments, at least six branches are connected to the core.

[0082] In some embodiments, the multiple (N) branches include at least three branches, at least four branches, and at least five branches.

[0083] X Branch In some embodiments of X, g is 1, 2, 3, or 4. Branch In some embodiments, g is 1. Branch In some embodiments of X, g is 2. Branch In some embodiments of X, g is 3. Branch In some embodiments, g is 4.

[0084] In some embodiments of XBranch, Z=2(g-1), and when g=1, G=0. BranchIn some embodiments, Z = 2(g-1), and when g ≠ 1,

number

[0085] X Branch In some embodiments, g=1, G=0, Z=1, and each branch of the multiple branches contains a structural formula. Structural formula [ka] Includes.

[0086] X Branch In some embodiments, g=2, G=1, Z=2, and each branch of the multiple branches is a structural formula [ka] Includes.

[0087] X Branch In some embodiments, g=3, G=3, Z=4, and each branch of the multiple branches is a structural formula [ka] Includes.

[0088] X Branch In some embodiments, g=4, G=7, Z=8, and each branch of the multiple branches is a structural formula [ka] Includes.

[0089] In some embodiments, the dendrimer or dendron described herein for generation (g)=1 has the following structure: [ka] It holds.

[0090] In some embodiments, the dendrimer or dendron described herein for generation (g)=1 has the following structure: [ka] It holds.

[0091] Table B shows examples of formulations of dendrimers or dendrons of generations 1-4 described herein. The number of diacyl groups, linker groups, and terminal groups can be calculated based on g. [Table 2]

[0092] In some embodiments, the diacyl group is independently of the structural formula. [ka] Includes, * This indicates the bond point at the proximal end of the diacyl group, ** This indicates the bonding site at the distal end of the diacyl group.

[0093] X Branch In some embodiments of the diacyl group, Y 3 In each instance, independently, the substituts are an alkylene, an alkenylene, or an allenylene, which are optionally substituted. Branch In some embodiments of the diacyl group, Y 3 In each occurrence, the alkylene (e.g., C1-C) is independently substituted by choice. 12 ) is X Branch In some embodiments of the diacyl group, Y 3 In each occurrence, the alkenylenes (e.g., C1-C) are independently substituted by choice. 12 ) is. X Branch In some embodiments of the diacyl group, Y 3 In each occurrence, the arerinenes (e.g., C1-C) are independently substituted by choice. 12 )

[0094] X Branch In some embodiments of the diacyl group, A 1 and A 2 Each instance independently represents -O-, -S-, or -NR. 4 - is X Branch In some embodiments of the diacyl group, A 1 and A 2 In each instance, independently, it is -O-. Branch In some embodiments of the diacyl group, A 1 and A 2 Each instance is independently -S-. Branch In some embodiments of the diacyl group, A 1 and A 2 Each instance is independently of -NR 4 - and R 4 X is an alkyl group (e.g., C1-C6) substituted with hydrogen or optionally. Branch In some embodiments of the diacyl group, m 1 and m 2 In each occurrence, independently, X is 1, 2, or 3. Branch In some embodiments of the diacyl group, m 1 and m 2 X is 1 in each instance, independently of the others. Branch In some embodiments of the diacyl group, m 1 and m 2 In each instance, independently, X is 2. Branch In some embodiments of the diacyl group, m 1 and m 2 In each instance, independently, it is 3. Branch In some embodiments of the diacyl group, R 3c , R 3d , R 3e , and R 3f Each instance independently consists of an alkyl group substituted with hydrogen or of any choice.Branch In some embodiments of the diacyl group, R 3c , R 3d , R 3e , and R 3f Each of these instances is independently hydrogen. Branch In some embodiments of the diacyl group, R 3c , R 3d , R 3e , and R 3f Each instance is independently and optionally substituted with an alkyl group (e.g., C1-C8).

[0095] In some embodiments of the diacyl group, A 1 is -O- or -NH-. In some embodiments of the diacyl group, A 1 It is -O-. In some embodiments of the diacyl group, A 2 is -O- or -NH-. In some embodiments of the diacyl group, A 2 It is -O-. In some embodiments of the diacyl group, Y 3 C1-C 12 (For example, C1-C6, for example, C1-C3) These are alkylenes.

[0096] In some embodiments of the diacyl group, the diacyl group independently appears in the structural formula. [ka] Includes, and optionally R 3c , R 3d , R 3e , and R 3f Each of these elements is independently either hydrogen or a C1-C3 alkyl group in its respective appearance.

[0097] In some embodiments, the linker group is independently of the structural formula [ka] Includes, ** This indicates the linker bonding site to the proximal diacyl group.*** This indicates the linker's bonding site to the distal diacyl group.

[0098] X Branch In some embodiments of the linker group (if present), Y1 is, independently in each occurrence, an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. Branch In some embodiments of the linker group (if present), Y1 is independently, in each occurrence, optionally substituted alkylene (e.g., C1-C 12 ) is. X Branch In some embodiments of the linker group (if present), Y1 is independently, in each occurrence, optionally substituted with an alkenylene (e.g., C1-C 12 ) is. X Branch In some embodiments of the linker group (if present), Y1 is independently, in each occurrence, optionally substituted with an allerylene (e.g., C1-C 12 )

[0099] X Branch In some embodiments of the terminal groups, each terminal group is independently selected from optionally substituted alkylthiols and optionally substituted alkenylthiols. Branch In some embodiments of the terminal groups, each terminal group is optionally substituted with an alkylthiol (e.g., C1-C 18 For example, C4-C 18 ) is. X Branch In some embodiments of the terminal groups, each terminal group is optionally substituted with an alkenylthiol (e.g., C1-C 18 For example, C4-C 18 )

[0100] X Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 Alkenylthiol or C1-C 18It is an alkylthiol, and the alkyl or alkenyl portion is optionally a halogen, C6-C 12 Ariel, C1-C 12 Alkylamino, C4-C6N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 Alkylamino), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6N-heterocycloalkyl), -C(O)-(C1-C 12 The molecule is substituted with one or more substituents independently selected from alkylamino and -C(O)-(C4-C6N-heterocycloalkyl), and any of the C4-C6N-heterocycloalkyl moieties of the preceding substituents may optionally be substituted with a C1-C3 alkyl or C1-C3 hydroxyalkyl molecule.

[0101] X Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 (For example, C4-C 18 ) Alkenylthiol or C1-C 18 (For example, C4-C 18 ) an alkylthiol, where the alkyl or alkenyl portion is optionally a halogen, C6-C 12 Aryl (e.g., phenyl), C1-C 12 (For example, C1-C8) alkylamino (for example, C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino) [ka] C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl) [ka] N-piperidinyl [ka] N-azepanyl [ka] -OH, -C(O)OH, -C(O)N(C1-C3alkyl)-(C1-C6 alkylene)-(C1-C 12 Alkylamino (e.g., monoalkylamino or dialkylamino) (e.g., [ka] ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6N-heterocycloalkyl)(for example, [ka] -C(O)-(C1-C 12 Alkylaminos (e.g., monoalkylaminos or dialkylaminos), and -C(O)-(C4-C6N-heterocycloalkyls) (e.g., [ka] Each of the preceding substituents is independently substituted with one or more substituents, and any C4-C6N-heterocycloalkyl moiety of the preceding substituent is optionally substituted with a C1-C3 alkyl or C1-C3 hydroxyalkyl molecule. Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 (For example, C4-C 18 ) is an alkylthiol, and the alkyl portion is optionally substituted with one substituent -OH. Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 (For example, C4-C 18 ) is an alkylthiol, and the alkyl portion is optionally C1-C 12 (For example, C1-C8) alkylamino (for example, C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino) [ka] (etc.)) and C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl [ka] N-piperidinyl [ka] N-azepanyl [ka] It is replaced with one substituent selected from X. Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 (For example, C4-C 18 ) Alkenylthiol or C1-C 18 (For example, C4-C 18 ) It is an alkylthiol. Branch In some embodiments of the terminal groups, each terminal group is independently C1-C 18 (For example, C4-C 18 It is an alkylthiol.

[0102] X Branch In some embodiments of the terminal groups, each terminal group independently has the structure shown in Table C. In some embodiments, the dendrimers or dendrones described herein may include the terminal groups or pharmaceutically acceptable salts, or pharmaceutically acceptable salts of those selected in Table C. In some embodiments, the examples of terminal groups in Table C are not limited to the stereoisomers (i.e., enantiomers, diastereomers) described herein. [Table 3] TIFF2026529161000093.tif214159TIFF2026529161000094.tif36159

[0103] In some embodiments, the dendrimer or dendron of formula (X) is selected from those listed in Table D and their pharmaceutically acceptable salts.

Table 4

[0104] C. The physical properties of dendrimers can be modified by altering the functional groups and / or chemical properties of the core, repeating units, and surface or terminal groups. Some modifiable properties include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment ability. Dendrimers are often described by the generation or number of repeating units in the branching. A dendrimer consisting only of the core molecule is called generation 0, and each subsequent repeating unit along all branches is generation 1, generation 2, etc., continuing to the terminal or surface groups. In some embodiments, a half-generation may arise only from the first condensation reaction with an amine, rather than a second condensation reaction with a thiol.

[0105] The preparation of dendrimers requires a degree of synthetic control, achieved through a series of stepwise reactions involving the construction of the dendrimer by each continuum. Dendrimer synthesis can be convergent or divergent. In divergent dendrimer synthesis, the molecule is constructed stepwise from core to periphery, involving the bonding of one generation to the previous generation and then the alteration of the functional group for the reaction of the next step. The transformation of the functional group is necessary to prevent uncontrolled polymerization. Such polymerization results in highly branched molecules that are not monodisperse, otherwise known as hyperbranched polymers. Due to steric effects, if the repeating units of the dendrimer continue to react, steric overcrowding can prevent complete reaction in certain generations, resulting in spherical or globular molecules until the monodispersity of the molecule is destroyed. Therefore, in some embodiments, G1-G10 generation dendrimers are specifically intended. In some embodiments, the dendrimer includes one, two, three, four, five, six, seven, eight, nine, or ten repeating units, or any range that can be derived therefrom. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (11, 12, 13, 14, 15, 20, or 25, etc.) can be increased by reducing the spatial arrangement units of the branched polymer.

[0106] Furthermore, dendrimers possess two main chemical environments: one created by specific surface groups in the terminal generation, and another within the dendritic structure, shielded from the bulk medium and surface groups due to its higher-order structure. Due to these distinct chemical environments, dendrimers have been found to have numerous diverse applications, including therapeutic uses.

[0107] In some embodiments, dendrimers that can be used in the composition are constructed using the suggestive reactivity of acrylate and methacrylate groups with amines and thiols. The dendrimers may include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines, and methacrylate groups with mercapto groups. Furthermore, the repeating units of the dendrimer may contain groups that are biodegradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diether groups, ester groups, amide groups, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine having multiple distinct dendritic branches, each of which may contain one or more repeating units. The dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the terminal group is a lipophilic group such as a long-chain alkyl or alkenyl group. In other embodiments, the terminal group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the terminal group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In yet another embodiment, the terminal group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0108] In some embodiments, the composition may further include a molar ratio of ionizable lipids to total lipid composition of about 15 to about 60. In some embodiments, the molar ratio is from about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 to about 60, or any range that can be derived therein. In some embodiments, the molar ratio is about 30 to about 45.

[0109] The cationic ionizable lipids of this disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated as optically active or racemic forms. Therefore, unless a specific stereochemistry or isomer is specifically indicated, all chiral, diastereomer, racemic, epimeric, and geometric isomers of the chemical formula are intended. Cationic ionizable lipids may appear as racemics and racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers. In some embodiments, single diastereomers are obtained. The chiral centers of the cationic ionizable lipids of this disclosure may be in either an S or R configuration. Furthermore, it is intended that one or more of the cationic ionizable lipids may exist as structural isomers. In some embodiments, compounds may have the same formula but different bonding characteristics to the core nitrogen atom. While we do not wish to be bound by any theory, it is conceivable that such cationic ionizable lipids exist because the starting monomer first reacts with a primary amine and then statistically reacts with any secondary amine present. Therefore, in structural isomers, a completely reacted primary amine may be shown, followed by a mixture of the reacted secondary amines.

[0110] In the chemical formulas used to represent cationic ionizable lipids in this disclosure, typically only one of several different possible tautomers is shown. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol group. Similarly, many types of imine groups exist in equilibrium with the enamine group. Regardless of which tautomers are shown for a given formula, and regardless of which is more frequently observed, all tautomers of a given chemical formula are intended.

[0111] The cationic ionizable lipids of this disclosure may also have advantages over compounds known in the prior art, whether used in the indications described herein or elsewhere, such as being more potent, less toxic, longer-acting, more potent, producing fewer side effects, being more readily absorbed, and / or having a better pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance), and / or possessing other useful pharmacological, physical, or chemical properties compared to compounds known in the prior art.

[0112] Furthermore, the atoms constituting the cationic ionizable lipids of this disclosure are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms with the same atomic number but different mass numbers. As a general example, and without limitation, isotopes of hydrogen include tritium and deutherium, and isotopes of carbon include, 13 C and 14 C is included.

[0113] It should be recognized that, as long as the salt as a whole is pharmacologically acceptable, the specific anions or cations that form part of any salt form of the cationic ionizable lipids provided herein are not important. Further examples of pharmaceutically acceptable salts and methods of their preparation and use are shown in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0114] D. Additional lipids in lipid nanoparticles In some embodiments of this disclosure, a composition is produced by mixing a composition containing one or more lipids with a cationic ionizable lipid. In some embodiments, the polymer is mixed with one, two, three, four, or five different types of lipids. It is intended that the cationic ionizable lipid can be mixed with a single type of multiple different lipids. In some embodiments, the cationic ionizable lipid composition includes at least a steroid or steroid derivative, a PEG lipid, and a phospholipid.

[0115] Veterinary and Veterinary Fractions In some aspects of this disclosure, a composition is produced by mixing a cationic ionizable lipid with one or more steroids or steroid derivatives. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” refers to a class of compounds having a 4-ring, 17-carbon cyclic structure, which may further include one or more substitutions such as alkyl groups, alkoxy groups, hydroxyl groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms. In one embodiment, the cyclic structure of the steroid includes three condensed cyclohexyl rings and a condensed cyclopentyl ring, as shown in the following formula: [ka]

[0116] In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative has the formula, [ka] It is a sterol that is further defined as follows.

[0117] In some embodiments of this disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is given by formula: [ka] It is further defined by...

[0118] As described above, cholestane derivatives include one or more non-alkyl substitutions of the cyclic system described above. In some embodiments, cholestane or cholestane derivatives are cholestene or cholestene derivatives or sterol or sterol derivatives. In other embodiments, cholestane or cholestane derivatives are both cholestere and sterol or derivatives thereof.

[0119] In some embodiments, the composition may further include a steroid-to-total lipid composition molar ratio of about 10 to about 60. In some embodiments, the molar ratio is from about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 to about 60, or any range that can be derived therein. In some embodiments, the molar ratio is about 25 to about 50, for example, 30.

[0120] Polymer complex lipids In some embodiments of this disclosure, a polymer is mixed with one or more polymer-compound lipids, such as PEGylated lipids (or PEG lipids), to produce a dendrimer composition. In some embodiments, this disclosure includes using any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride, also containing a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups, or C6-C24 fatty acid groups, attached to a linker group having a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG ceramide conjugate, PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropane-3-amine, PEG-modified diacylglycerol and dialkylglycerol. In some embodiments, PEG-modified diastearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG modification is measured by the molecular weight of the PEG component in the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weights of PEG-modified lipids range from approximately 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to approximately 15,000. Some non-limiting examples of lipids that can be used in the present invention are taught by U.S. Patent No. 5,820,873, WO2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0121] In another embodiment, PEG lipids are given by formula: [ka] [In the formula, R 12 and R 13 Each of them is independently alkyl (C≦24) Alkenil (C≦24) , or a substitutional form of either of these groups, R e hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) And x is 1 to 250]. In some embodiments, R e is alkyl (C≦8) For example, methyl. 12 and R 13 Each of them is independently alkyl (C≦4-20) In some embodiments, x is 5 to 250. In one embodiment, x is 5 to 125 or x is 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0122] In another embodiment, PEG lipids are given by formula: [ka] [wherein the formula n1 is an integer between 1 and 100, and n2 and n3 are integers independently selected from 1 to 29]. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range that can be derived from there. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0123] In some embodiments, the composition may further include molar ratios of PEG lipids with ionizable total lipid composition ranging from about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12 to about 12.5, or any range that can be derived therefrom. In some embodiments, the molar ratio is about 1 to about 6.

[0124] Phospholipids In some embodiments of this disclosure, a polymer is mixed with one or more phospholipids to produce a composition. In particular, in some embodiments, the phospholipid is a neutral phospholipid. In some embodiments, it is any lipid including a phosphate group. In some embodiments, the phospholipid has a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally an organic small molecule. In some embodiments, the organic small molecule is an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.

[0125] In some embodiments, the composition may further include a phospholipid to total lipid composition molar ratio of about 5 to about 50. In some embodiments, the molar ratio is from about 5, 10, 15, 20, 25, 30, 35, 40, 45 to about 50, or any range that can be derived therein. In some embodiments, the molar ratio is from about 20 to about 40.

[0126] E. Nucleic acids In some aspects of this disclosure, the composition comprises one or more nucleic acids. In some embodiments, the composition comprises one or more nucleic acids present in a weight ratio of about 5:1 to about 1:100 relative to ionizable lipids. In some embodiments, the nucleic acid to dendrimer weight ratio is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any range that can be derived therefrom. Furthermore, it should be clear that this disclosure is not limited to the specific nucleic acids disclosed herein. The present invention is not limited to any particular source, sequence, or type of nucleic acid, but those skilled in the art can easily identify relevant homologs in nucleic acids from various other sources of nucleic acids, for example, from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cows, pigs, horses, sheep, cats, and other species).

[0127] In some embodiments, the mRNA includes approximately 250 to 15,000 nucleotides, approximately 500 to 5,000 nucleotides, approximately 800 to 2,500 nucleotides, or approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000 to approximately 15,000 nucleotides, or any range that can be derived within that range.

[0128] In some embodiments, the composition includes a molar ratio of lipid component to nucleic acid component of about 1,000:1 to about 5,000:1, about 2,000:1 to about 4,000:1, or about 1,500:1, from about 1,000:1, 1,500:1, 2,000:1, 2,500:1, 3,000:1, 3,500:1, 4,000:1, 4,500:1, or any range that can be derived therefrom. In some embodiments, the compositions include N:P ratios ranging from approximately 1:1 to approximately 20:1, approximately 2:1 to approximately 10:1, approximately 4:1 to approximately 8:1, or from approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 to approximately 20:1, or any range of N:P ratios that can be derived within that range.

[0129] Modified nucleic acid bases In some embodiments, the nucleic acids of the Disclosure comprise one or more modified nucleosides containing a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may possess desirable properties, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid, compared to oligonucleotides comprising only nucleosides containing a native sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar substitute. Such a sugar substitute may comprise one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0130] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-crosslinked sugar substituents, including but not limited to substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position may be allyl, amino, azide, thio, O-allyl, or O--C1-C 10 Alkyl, O--C1-C 10Substitutive alkyl groups are selected from OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C1-C 10 It is alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar contains more than one non-crosslinked sugar substituent, e.g., TF-5'-methyl sugar moiety (for additional 5',2'-bis-substituted sugar moieties and nucleosides, see, for example, PCT international application WO2008 / 101157).

[0131] A nucleoside containing a 2'-substituted sugar moiety is referred to as a 2'-substituted nucleoside. In some embodiments, the 2'-substituted nucleoside is a halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O, S, or N(R) m )-alkyl; O, S, or N(R m )-alkenyl; O, S or N(R m )-Alkynyl;O-Alkyrenyl-O-alkyl,Alkynyl,Alkalyl,Aralkyl,O-Alkalyl,O-Aralkyl,O(CH2)2SCH3,O(CH2)2--O--N(R m )(R n ), or O--CH2--C(=O)--N(R m )(R n The formula includes a 2'-substituted selected from ), where each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10 These are alkyl groups. These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl groups.

[0132] In some embodiments, the 2'-substituted nucleoside is F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O--CH2--C(=O)--N(R m )(R n The formula includes a 2'-substituted selected from ), where each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10 It is alkyl.

[0133] In some embodiments, the 2'-substituted nucleoside includes a sugar moiety comprising a 2'-substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0134] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituted substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0135] Certain modified sugar moieties contain a bridging sugar substituent, which forms a second ring to produce a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety contains a bridging between the 4' and 2' furanose ring atoms. An example of such a 4' to 2' sugar substituent is --[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O--or,--C(R a R b)--O--N(R)--;4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2'(LNA);4'-(CH2)--S-2';4'-(CH2)2--O-2'(ENA);4'-CH(CH3)--O-2'(cEt) and 4'-CH(CH2OCH3)--O-2', and their analogues (see, for example, U.S. Patent No. 7,399,845);4'-C(CH3)(CH3)--O-2' and its analogues ( See, for example, WO2009 / 006478); 4'-CH2--N(OCH3)-2' and its analogues (see, for example, WO2008 / 150729); 4'-CH2--O--N(CH3)-2' (see, for example, US2004 / 0171570 published 2 September 2004); 4'-CH2--O--N(R)-2', and 4'-CH2--N(R)--O-2'-[wherein each R is independently H, a protecting group, or C1-C 12 [It is alkyl]; 4'-CH2--N(R)--O-2' [wherein R is H, C1-C 12 Examples include, but are not limited to, alkyl groups or protecting groups (see U.S. Patent No. 7,427,672); 4'-CH2--C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2--C(=CH2)-2' and its analogues (see PCT International Application No. WO2008 / 154401).

[0136] In some embodiments, such a bridging from 4' to 2' is independently --[C(R a )(R b )] n --, --C(R a )=C(R b )--,--C(R a )=N--, ​​--C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a )2--,--S(=O) x --, and --N(R a)-- comprises 1 to 4 linking groups independently selected from, where, x is 0, 1, or 2. n is 1, 2, 3, or 4. Each R a and R b These are independently H, protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl substitution C2-C 12 Alkinyl, C5-C 20 Aryl, substitution C5-C 20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), and Each of J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl substitution C2-C 12 Alkinyl, C5-C 20 Aryl, substitution C5-C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl group or a protecting group.

[0137] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Examples of bicyclic nucleosides include (A) α-L-methyleneoxy(4'-CH2--O-2')BNA, (B) β-D-methyleneoxy(4'-CH2--O-2')BNA (also called locked nucleic acid or LNA), (C) ethyleneoxy(4'-(CH2)2--O-2')BNA, (D) aminooxy(4'-CH2--O--N(R)-2')BNA, and (E) oxyamino(4'-CH2--N( (R)--O-2')BNA, (F)Methyl(methyleneoxy)(4'-CH(CH3)--O-2')BNA (also called restricted ethyl or cEt), (G)Methylene-thio(4'-CH2--S-2')BNA, (H)Methylene-amino(4'-CH2-N(R)-2')BNA, (I)Methyl carbon ring(4'-CH2--CH(CH3)-2')BNA, (J)Propylene carbon ring(4'-(CH2) 3~ Examples include, but are not limited to, 2')BNA and (K)methoxy(ethyleneoxy)(4'-CH(CH2OMe)-O-2')BNA (also known as restricted MOE or cMOE).

[0138] Further bicyclic sugar moieties are described, for example, in Singh et al., Chem. et al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222,Singh et al.,J.Org.Chem.,1998,63,10035-10039,Srivastava et al. al., J.Am.Chem.Soc., 129(26)8362-8379(Jul.4,2007), Elayadi et al. al.,Curr.Opinion Invens.Drugs,2001,2,5561, Braasch et al.,Chem.Biol.,2001,8,1-7,Orum et al.,Curr.Opinion Mol.Ther., 2001, 3,239-243, U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845, WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, and WO2007 / 134181, U.S. Patent Publication Nos. US2004 / 0171570 and US2007 / 028783 Patent No. 1, and US2008 / 0039618, U.S. Patent Applications No. 12 / 129,154, No. 60 / 989,574, No. 61 / 026,995, No. 61 / 026,998, No. 61 / 056,564, No. 61 / 086,231, No. 61 / 097,787, and No. 61 / 099,844, as well as PCT International Applications PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922 are known in the art.

[0139] In some embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by the isomer configuration. For example, a nucleoside containing a 4'-2' methylene-oxy bridge can be in either an α-L or β-D configuration. To date, α-L-methyleneoxy(4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that have exhibited antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0140] In some embodiments, the substituted sugar moiety includes one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents (e.g., 5'-substituted sugar and 4'-2'-crosslinked sugar, PCT International Application WO2007 / 134181 (where LNA is substituted with, for example, a 5'-methyl group or a 5'-vinyl group)).

[0141] In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of a naturally occurring sugar is substituted with, for example, a sulfur atom, a carbon atom, or a nitrogen atom. In some such embodiments, such modified sugar moiety also includes bridging and / or non-bridging substituents as described above. For example, certain sugar substitutes include substitution at the 4'-sulfur atom and the 2' position (see, e.g., U.S. Patent Application Publication US2005 / 0130923) and / or substitution at the 5' position. As an additional example, carbocyclic bicyclic nucleosides with 4'-2' bridging have been reported (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org.Chem., 2006, 71, 7731-7740).

[0142] In some embodiments, the sugar substitute includes a ring having more than five atoms. For example, in some embodiments, the sugar substitute includes a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoroHNA (F-HNA).

[0143] In some embodiments, a modified THP nucleoside of formula VII is provided, where q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is not H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In some embodiments, a THP nucleoside of formula VII is provided, where one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

[0144] Many other bicyclo and tricyclo sugar substitute ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0145] Combinations of modifications, such as 2'-F-5'-methyl-substituted nucleosides (see PCT International Application WO2008 / 101157 for other disclosed 5',2'-bis-substituted nucleosides) and substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see U.S. Patent Publication US2005 / 0130923), or otherwise, 5'-substitution of bicyclic nucleic acids (see PCT International Application WO2007 / 134181, in which a 4'-CH2--O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). Their synthesis and preparation have also been reported, along with oligomerization and biochemical studies of carbocyclic and bicyclic nucleosides (see, for example, Srivastava et al., 2007).

[0146] In some embodiments, the present invention provides oligonucleotides comprising modified nucleosides. These modified nucleotides may include modified sugars, modified nucleic acid bases, and / or modified bonds. Specific modifications are selected so that the resulting oligonucleotide has desirable characteristics. In some embodiments, the oligonucleotide comprises one or more RNA-like nucleosides. In some embodiments, the oligonucleotide comprises one or more DNA-like nucleotides.

[0147] In some embodiments, the nucleoside of the present invention comprises one or more unmodified nucleic acid bases. In certain embodiments, the nucleoside of the present invention comprises one or more modified nucleic acid bases.

[0148] In some embodiments, the modified nucleic acid base is selected from universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases, as defined herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl, and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, and cytosine. Tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. Further modified nucleic acid bases include tricyclic pyrimidines, e.g., phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), G-clamps, e.g., substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazine-2(3H)-one), carbazolecytidine ( 2These include H-pyrimido[4,5-b]indole-2-one) and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified nucleic acid bases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, those disclosed in Englisch et al., 1991, and those disclosed in Sanghvi, YS, 1993.

[0149] Representative U.S. patents teaching the preparation of certain modified nucleic acid bases and other modified nucleic acid bases include, without limitation, U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, and 5,5 Examples include Nos. 02,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0150] In some embodiments, the present invention provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides may be linked together using any internucleoside linkage. Two main classes of internucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside links include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative phosphorus-free internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiesters (--O--C(O)--S--), thionocarbamates (--O--C(O)(NH)--S--); siloxanes (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Compared to natural phosphodiester links, modified links can typically be used to increase the nuclease resistance of oligonucleotides, as this alters the nuclease resistance. In some embodiments, internucleoside links with chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral links include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free nucleoside bonds are well known to those skilled in the art.

[0151] The oligonucleotides described herein contain one or more chiral centers and thus give rise to enantiomers, diastereomers, and other stereoisomer configurations that can be defined in terms of absolute stereochemistry as (R) or (S), α or β as in the case of sugar anomers, or (D) or (L) as in the case of amino acids. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0152] Neutral nucleoside interbonding includes, but is not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2--N(CH3)--O-5'), amide-3 (3'-CH2--C(=O)--N(H)-5'), amide-4 (3'-CH2--N(H)--C(=O)-5'), formacetal (3'-O--CH2--O-5'), and thioformacetal (3'-S--CH2--O-5'). Further neutral nucleoside bonds include nonionic bonds containing siloxanes (dialkylsiloxanes), carboxylic acid esters, carboxamides, sulfides, sulfonic acid esters, and amides (see, for example, *Carbohydrate Modifications in Antisense Research*; YSSanghvi and PDCook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral nucleoside bonds include nonionic bonds containing mixed component moieties of N, O, S, and CH2.

[0153] Additional modifications may also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. For example, one of the additional modifications of the ligand-bound oligonucleotide of the present invention involves chemically linking one or more additional non-ligand moieties or non-ligand complexes to the oligonucleotide, which enhances the activity, intracellular distribution, or intracellular uptake of the oligonucleotide. Such parts include lipid portions, for example, cholesterol portions (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), thioethers (e.g., hexyl-5-tritylthiol (Manoharan et al., 1992, Manoharan et al., 1993)), thiocholesterol (Oberhauser et al., 1992), aliphatic chains (e.g., dodecanediol residues or undecyl residues (Saison-Behmoaras et al., 1991, Kabanov et al., 1990, Svinarchuk et al., 1993)), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995, Shea et al.) Examples include, but are not limited to, polyamines or polyethylene glycol chains (Manoharan et al., 1995), adamantane acetate (Manoharan et al., 1995), palmityl moiety (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996).

[0154] Representative U.S. patents teaching the preparation of such oligonucleotide complexes are U.S. Patents Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,580,731, 5,591,584, and 5,10, respectively, which are incorporated herein by reference. No. 9,124, No. 5,118,802, No. 5,138,045, No. 5,414,077, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587 ,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,737, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,5 No. 82, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,02 2, 5,254,469, 5,258,506, 5,262,536, 5,272,250, 5,292,873, 5,317,098, 5,371,241, 5,391,723, Examples include, but are not limited to, Nos. 5,416,203, 5,451,463, 5,510,475, 5,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,941.

[0155] F. Cancer and hyperproliferative disorders Hyperproliferative disorders can be associated with any disease that causes cells to begin regenerating uncontrollably, the most typical example being cancer. One of the key elements of cancer is the disruption of the normal cell cycle, and therefore, substances that inhibit cell growth are important therapeutic agents for treating these diseases. In some embodiments, target genes or target transcripts that guide polynucleotides can complex together may be found in human cells, e.g., cancer cells. In some embodiments, the compounds of this disclosure may interfere with gene expression in human cells, e.g., cancer cells. Methods of this disclosure aim to interfere with gene expression in either healthy cells or cancerous cells, or both. In this disclosure, the cell membrane disruptive compounds described herein may be used to cause cell number reduction and therefore may be used to treat a variety of cancer strains. In some embodiments, it is anticipated that the compounds and compositions described herein may be used to treat virtually any malignant tumor.

[0156] Cancer cells that can be treated with the compounds or compositions of this disclosure include, but are not limited to, cells from the skin, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, pancreas, testes, tongue, cervix, or uterus. Furthermore, cancer can be, but is not limited to, the following histological types: neoplasm (malignant); carcinoma; cancer (undifferentiated); giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; piloma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma (malignant); cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; cord-like adenocarcinoma; adenomatous polyp-containing adenocarcinoma; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; carcinoid tumor (malignant); bronchioloalveolar adenocarcinoma adenocarcinoma); papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; Sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; Mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease (breast); acinar cell carcinoma of the pancreas; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma (malignant); ovarian stromal tumor (malignant); follicular cell tumor (malignant); granulosa cell tumor (malignant); androblastoma (malignant); Sertoli cell carcinoma; Leydig cell tumor (malignant); lipid cell tumor (malignant); Paraganglioma (malignant); Extramammary paraganglioma (malignant); Pheochromocytoma; Glomangiosarcoma; Malignant melanoma; Apigmented melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus (malignant); Sarcoma; Fibrosarcoma; Fibrous histiocytoma (malignant); Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor (malignant); Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor (malignant); Brenner tumor (malignant); Phyllodes tumor (malignant); Synovial sarcoma; Mesothelioma (malignant); Undifferentiated germ cell tumor; Fetal carcinoma; Teratoma (malignant); Ovarian goiter (malignant); Choriocarcinoma; Mesonephroma (malignant); Angiosarcoma; Hemangioendothelioma (malignant); Kaposi's sarcoma;Perivascular cell tumor (malignant); lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma; chondroblastoma (malignant); mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor (malignant); ameloblastoma; ameloblastoma (malignant); ameloblastoma; pineal gland tumor (malignant); chordoma; glioma (malignant); ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectodermal; cerebellar sarcoma; gangliblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma (malignant); nerve Visarcoma; schwannoma (malignant); granuloma (malignant); malignant lymphoma; Hodgkin's disease; lateral granuloma; malignant lymphoma (small lymphocytic); malignant lymphoma (large cell, diffuse); malignant lymphoma (follicular); mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcomatic leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and hairy cell leukemia. In certain aspects, tumors may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia.

[0157] G. Treatment method Disclosed herein are methods for treating subjects having or suspected to have a disease or disorder, such as a genetic disease or disorder or a disease or disorder associated with mutations in one or more genes, the methods comprising administering a lipid nanoparticle composition that can be used to target and / or modify T cells to the subject. The subject may be a mammal. The subject may be a non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat and other species). The subject may be a human. The subject may be determined to have mutations in its genes. In some embodiments, administration includes systemic (e.g., intravenous) administration. In some embodiments, the subject is selected from the group consisting of mouse, rat, monkey and human. In some embodiments, the subject is human. This disclosure provides methods for using the composition in conjunction with other therapeutic treatments such as surgery, chemotherapy, radiotherapy or immunotherapy.

[0158] chemotherapy A wide variety of chemotherapeutic agents can be used according to embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within cells, for example, whether and at what stage of the cell cycle they affect. Alternatively, agents may be characterized based on their ability to directly crosslink with DNA by affecting nucleic acid synthesis, their ability to intercalate into DNA, or their ability to induce chromosomal and mitotic abnormalities.

[0159] Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonic acids, e.g., busulfan, improsulfan, and picosulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; ethyleneimines and methylamelamamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine); acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (synthetic Including analogs KW-2189 and CB1-TM1); Eloiterobin; Pancratistatin; Sarcodictiin; Spongestatin; Nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, and uracil mustard; Nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; Antibiotics, e.g., engine antibiotics (e.g., calicheamicin, in particular calicheamicin gamma I and calicheamicin omega I1); Dinemicin (including Dinemicin A); Bisphosphonates, e.g., clodronate; Esperamycin;Furthermore, neocardinostatin chromophore and related pigment proteins enediin antibiotic chromophore, acrasinomycin, actinomycin, orthoralnycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin) Including epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalar nisin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolbicin; metabolic antagonists, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, pteropterin, and trimethrexate; pre Pyrimidine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents, e.g., mitotane and trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofamide glycoside ;aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamin; demecoltin; diazicon; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; mytansinoids, e.g., mytansin and anthamitosin; mitogwazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine;PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, beracrine A, loridine A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinate complexes, e.g., cisplatin, oxaliplatin and carboplatin; vinblastine Examples include: platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, precomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans platinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0160] Radiation therapy Other widely used factors that cause DNA damage include gamma rays, commonly known as X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors, such as microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and UV irradiation are also considered. All of these factors are most likely to have broad-ranging damaging effects on DNA, DNA precursor cells, DNA replication and repair, and chromosome construction and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens for long-term exposure (3–4 weeks) to a single dose of 2,000–6,000 roentgens. The dose range for radioisotopes varies considerably, depending on the half-life of the isotope, the intensity and type of radiation being irradiated, and uptake by tumor cells.

[0161] immunotherapy Those skilled in the art will understand that immunotherapy may be used in combination with or in conjunction with the methods of the embodiments. In relation to cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Immune effectors may be antibodies specific to certain markers on the surface of tumor cells, for example. Antibodies alone may function as effectors of a treatment, or they may mobilize other cells to act in actual cell killing. Antibodies may also be bound to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and function solely as targeted agents. Alternatively, effectors may be lymphocytes carrying surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0162] In one embodiment of immunotherapy, tumor cells must possess certain markers that are suitable for targeting, i.e., markers that are not present in most other cells. Many tumor markers exist, and any one of these is suitable for targeting in connection with embodiments of the present invention. Common tumor markers include B cell maturation antigen, CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, GPRC5D, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative embodiment of immunotherapy is to combine anticancer action with immunostimulatory action. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0163] Examples of immunotherapies currently in trial or use include: immunoadjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169, Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, e.g., interferon α, β, ε, ω, and κ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward et al., 1998); and gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward et al., 1998). This includes Villaseca, 1998, U.S. Patent Nos. 5,830,880 and 5,846,945; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998, U.S. Patent No. 5,824,311). It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0164] In some embodiments, the combinations described herein include agents that reduce tumor immunosuppression, such as chemokine (CXC motif) receptor 2 (CXCR2) inhibitors. In some embodiments, the CXCR2 inhibitor is danirixin (CAS registry number: 954126-98-8). Danirixin is also known as GSK1325756 or 1-(4-chloro-2-hydroxy-3-piperidine-3-ylsulfonylphenyl)-3-(3-fluoro-2-methylphenyl)urea. Danirixin is disclosed, for example, in Miller et al. Eur J Drug Metab Pharmacokinet (2014) 39:173-181 and Miller et al. BMC Pharmacology and Toxicology (2015), 16:18. In some embodiments, the CXCR2 inhibitor is reparixin (CAS registry number: 266359-83-5). Reparixin is also known as repertaxin or (2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide. Reparixin is a non-competitive allosteric inhibitor of CXCR1 / 2. Reparixin is disclosed, for example, in Zarbock et al. British Journal of Pharmacology (2008), 1-8. In some embodiments, the CXCR2 inhibitor is navalixin. Navalixin is also known as MK-7123, SCH527123, PS291822, or 2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methylfuran-2-yl)propyl]amino]-3,4-dioxocyclobuten-1-yl]amino]benzamide, and navalixin is disclosed, for example, in Ning et al. Mol Cancer Ther. 2012;11(6):1353-64. In some embodiments, the CXCR2 inhibitor is also known as AZD5069, N-[2-[[(2,3-difluorophenyl)methyl]thio]-6-{[(1R,2S)-2,3-dihydroxy-1-methylpropyl]oxy}-4-pyrimidinyl]-1-azetidine sulfonamide.In some embodiments, the CXCR2 inhibitor is an anti-CXCR2 antibody, such as those disclosed in WO2020 / 028479.

[0165] In some embodiments, the combinations described herein include agents that activate dendritic cells, such as TLR agonists. “TLR agonist” as defined herein is any molecule that activates Toll-like receptors, as described in Bauer et al., 2001, Proc. Natl. Acad. Sci. USA 98:9237-9242. TLR agonists may be small molecules, recombinant proteins, antibodies or antibody fragments, nucleic acids, or proteins. In certain embodiments, TLR agonists may be recombinants, native ligands, immunostimulatory nucleotide sequences, small molecules, purified bacterial extracts, or inactivated bacterial preparations.

[0166] Several agonists of microbial TLRs, such as lipopolysaccharides, peptidoglycans, flagellin, and lipoteichoic acid, have been reported (Aderem et al., 2000, Nature 406:782-787; Akira et al., 2001, Nat.Immunol.2:675-680). Some of these ligands can activate different dendritic cell subsets expressing distinct patterns of TLRs (Kadowaki et al., 2001, J.Exp.Med.194:863-869). Therefore, TLR agonists may be any preparation of microbial material possessing TLR agonist properties. Certain types of untranslated DNA have been shown to stimulate immune responses by activating TLRs. In particular, immunostimulatory oligonucleotides containing CpG motifs are widely disclosed and have been reported to activate lymphocytes (see U.S. Patent No. 6,194,388). As used herein, the “CpG motif” is defined as an unmethylated cytosine-guanine (CpG) dinucleotide. Immunostimulatory oligonucleotides containing a CpG motif can also be used as TLR agonists according to the methods of the present invention. Immunostimulatory nucleotide sequences may be stabilized by structural modifications such as phosphorothioate modifications or encapsulated in cationic liposomes to improve in vivo pharmacokinetics and tumor targeting.

[0167] In some embodiments, immunotherapy may be immune checkpoint inhibitors. This disclosure may also provide compositions that inhibit immune checkpoints. Immune checkpoints enhance or degrade signals (e.g., costimulatory molecules). Immune checkpoint proteins that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), HLA-DRB1, ICOS (also known as CD278), HLA-DQA1, HLA-E, and indoleamine 2,3-dioxygenator. Examples include ze1 (IDO1), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, OX40 (also known as CD134), programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T cell immune receptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig inhibitor of T cell activation (VISTA, also known as C10orf54). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0168] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant ligands or receptors, or antibodies such as human antibodies (e.g., International Patent Publication WO2015 / 016718, Pardoll, Nat Rev Cancer, 12(4):252-264, 2012, both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogues thereof may be used, in particular chimeric, humanized, or human-type antibodies. As will be apparent to those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0169] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain embodiments, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In yet another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In certain embodiments, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, including those described in U.S. Patent Application Publication Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369 (all of which are incorporated herein by reference).

[0170] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding moiety of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0171] Another immune checkpoint protein that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is analogous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found on regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 results in increased expression of CTLA-4, which is an inhibitory receptor for the B7 molecule.

[0172] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) for use in this method can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, PCT Publication Nos. WO01 / 14424, WO98 / 42752, WO00 / 37504 (tremelimumab; CP675,206, formerly also known as tisilimmab); U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA, 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology, 22(145):Abstract No.2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res, 58:5301-5304 may be used in the methods disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application No. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0173] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or their antigen-binding fragments and variants (see, for example, WO01 / 14424). In other embodiments, the antibody comprises the CDR or VR of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with the above antibody and / or binds to the same epitope on CTLA-4 as the above antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules for modulating CTLA-4 include, for example, CTLA-4 ligands and receptors described in U.S. Patent Nos. 5844905, 5885796 and International Patent Application Nos. WO1995001994 and WO1998042752 (all incorporated herein by reference), and immunoadhesins described in, for example, U.S. Patent No. 8329867 (incorporated herein by reference).

[0174] Another immune checkpoint protein that may be targeted in the methods provided herein is lymphocyte-activating gene 3 (LAG-3), also known as CD223. The complete protein sequence of human LAG-3 has Genbank accession number NP-002277. LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an "off" switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 activates both effector T cells and inhibitory regulatory T cells. In some embodiments, the immune checkpoint inhibitor is an anti-LAG-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-LAG-3 antibodies (or VH and / or VL domains derived therefrom) for use in the present methods can be produced using methods well known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art may be used. Exemplary anti-LAG-3 antibodies are relatrimab (also known as BMS-986016) or its antigen-binding fragments and variants (see, e.g., WO2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, e.g., WO2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in WO2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in WO2017 / 220569.

[0175] Another immune checkpoint protein that may be targeted in the methods provided herein is the V-domain Ig inhibitor of T cell activation (VISTA), also known as C10orf54. The complete protein sequence of human VISTA has Genbank accession number NP_071436. VISTA is found on leukocytes and inhibits T cell effector function. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-VISTA antibodies (or VH and / or VL domains derived therefrom) for use in these methods can be produced using methods well known in the art. Alternatively, anti-VISTA antibodies recognized in the art may be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as ombachilimab) (see, e.g., WO2015 / 097536, WO2016 / 207717, WO2017 / 137830, WO2017 / 175058). VISTA can also be inhibited by the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, e.g., WO2015 / 033299, WO2015 / 033301).

[0176] Another immune checkpoint protein that may be targeted in the methods provided herein is indoleamine 2,3-dioxygenase (IDO). The complete protein sequence of human IDO has Genbank accession number NP_002155. In some embodiments, the immune checkpoint inhibitor is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and napoximod (GDC-0919).

[0177] Another immune checkpoint protein that may be targeted in the methods provided herein is CD38. The complete protein sequence of human CD38 has Genbank accession number NP_001766. In some embodiments, the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-CD38 antibodies (or VH and / or VL domains derived therefrom) for use in these methods can be produced using methods well known in the art. Alternatively, an anti-CD38 antibody recognized in the art may be used. An exemplary anti-CD38 antibody is daratumumab (see, for example, U.S. Patent No. 7,829,673).

[0178] Another immune checkpoint protein that may be targeted in the methods provided herein is ICOS, also known as CD278. The complete protein sequence of human ICOS has Genbank accession number NP_036224. In some embodiments, the immune checkpoint inhibitor is an anti-ICOS antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Anti-human ICOS antibodies (or VH and / or VL domains derived therefrom) suitable for use in these methods can be produced using methods well known in the art. Alternatively, anti-ICOS antibodies recognized in the art can be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, e.g., WO2016 / 154177, WO2018 / 187191) and GSK3359609 (see, e.g., WO2016 / 059602).

[0179] Another immune checkpoint protein that may be targeted in the methods provided herein is the T cell immune receptor (TIGIT) having Ig and ITIM domains. The complete protein sequence of human TIGIT has Genbank accession number NP_776160. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-TIGIT antibodies (or VH and / or VL domains derived therefrom) for use in these methods can be produced using methods well known in the art. Alternatively, anti-TIGIT antibodies recognized in the art can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, for example, WO2017 / 030823 and WO2016 / 028656).

[0180] Another immune checkpoint protein that may be targeted in the methods provided herein is OX40, also known as CD134. The complete protein sequence of human OX40 has Genbank accession number NP_003318. In some embodiments, the immune checkpoint inhibitor is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Suitable anti-human-OX40 antibodies (or VH and / or VL domains derived therefrom) for use in these methods can be produced using methods well known in the art. Alternatively, an anti-OX40 antibody recognized in the art may be used. An exemplary anti-OX40 antibody is PF-04518600 (see, for example, WO2017 / 130076). ATOR-1015 is a bispecific antibody that targets CTLA4 and OX40 (see, for example, WO2017 / 182672, WO2018 / 091740, WO2018 / 202649, and WO2018 / 002339).

[0181] Another immune checkpoint protein that may be targeted in the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), also known as TNFRSF18 and AITR. The complete protein sequence of human GITR has Genbank accession number NP_004186. In some embodiments, the immune checkpoint inhibitor is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Anti-human-GITR antibodies (or VH and / or VL domains derived therefrom) suitable for use in these methods can be produced using methods well known in the art. Alternatively, anti-GITR antibodies recognized in the art can be used. An exemplary anti-GITR antibody is TRX518 (see, for example, WO2006 / 105021).

[0182] In one embodiment, the present application provides a combination therapy for the treatment of cancer, which comprises adoptive T cell therapy and a checkpoint inhibitor. In one embodiment, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another embodiment, autologous and / or allogeneic T cells are directed toward a tumor antigen.

[0183] D.Surgery Approximately 60% of people with cancer undergo some type of surgery, including prophylactic, diagnostic or staging, curative, and palliative surgeries. Curative surgery includes excision, which involves the physical removal, resection, and / or destruction of all or part of the cancerous tissue, and may be used in conjunction with other treatments, such as the treatments of the embodiments of this invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative treatments. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0184] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment may be achieved by perfusion, direct injection, or local application of additional anticancer treatment to the affected area. Such treatment may be repeated, for example, every day, every two days, every three days, every four days, every five days, every six days, or every seven days, or every week, every two weeks, every three weeks, every four weeks, and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months. These therapeutic agents may also be available in different dosages.

[0185] Other drugs To improve the therapeutic efficacy of the treatment, other agents may be used in combination with certain embodiments of the present invention. These additional agents include agents that affect the upregulation of cell surface receptors and GAP binding, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing substances, or other biological substances. Increasing intercellular signaling by increasing the number of GAP bindings enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with certain embodiments of the present invention to improve the anti-hyperproliferative efficacy of the therapeutic agent. Cell adhesion inhibitors are intended to improve the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are adhesion plaque kinase (FAK) inhibitors and lovastatin. To further improve the therapeutic effect, it is intended that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain embodiments of the present invention. The composition may perform one or more of these functions and then be combined with another agent that enhances the activity of the composition.

[0186] H. Kit This disclosure also provides kits. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit comprises the compositions described above or in the claims.

[0187] The kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container that can contain the components, and preferably, can be suitably dispensed. If the kit contains two or more components, the kit generally also includes a second, third, or other additional container that can separately contain the additional components. However, various combinations of components may be contained in a single container. In some embodiments, all of the lipid nanoparticle components are combined in a single container. In other embodiments, some or all of the lipid nanoparticle components are provided in separate containers.

[0188] The kit of the present invention also typically includes packaging for housing various containers strictly limited for commercial sale. Such packaging may include corrugated cardboard or injection-molded or blow-molded plastic packaging in which the desired container is held. The kit may also include instructions for using the kit components. The instructions may include possible modifications.

[0189] I. Chemical definition When used in relation to chemical groups, "hydrogen" means -H, "hydroxy" means -OH, "oxo" means =O, "carbonyl" means -C(=O)-, "carboxy" means -C(=O)OH (also written as -COOH or -CO2H), "halo" independently means -F, -Cl, -Br or -I, "amino" means -NH2, "hydroxyamino" means -NHOH, "nitro" means -NO2, "imino" means =NH, "cyano" means -CN, and "isocyan" "To" means -N=C=O, "azide" means -N3, "phosphate" means -OP(O)(OH)2 or its deprotonated form in a monovalent context, and -OP(O)(OH)O- or its deprotonated form in a divalent context, "mercapto" means -SH, "thio" means =S, "sulfonyl" means -S(O)2-, "hydroxysulfonyl" means -S(O)2OH, "sulfonamide" means -S(O)2NH2, and "sulfinyl" means -S(O)-.

[0190] In relation to chemical formulas, the symbol "-" represents a single bond, "=" represents a double bond, and "≡" represents a triple bond.

number

number

[0191] The base "R" is, for example, in the ring system, given by formula: [ka] In cases where it is depicted as a "floating base", As long as a stable structure is formed, R can replace any hydrogen atom bonded to any of the ring atoms, including the hydrogen atoms depicted, implied, or explicitly defined. The group "R" on the fused ring system, for example, by formula: [ka] In cases where it is depicted as a "floating base", Unless otherwise specified, R can replace any hydrogen atom bonded to any of the ring atoms of either fused ring. Replaceable hydrogens include those depicted (e.g., the hydrogen bonded to nitrogen in the above formula), implied hydrogens (e.g., the hydrogen in the above formula that is not shown but is understood to exist), explicitly defined hydrogens, and optional hydrogens whose presence depends on the identity of the ring atoms (e.g., the hydrogen bonded to X when group X is equal to -CH-). In the examples shown, R may be present in either a five-membered ring or a six-membered ring in the fused ring system. In the above formula, the subscript "y" immediately following the parenthesized group "R" represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, and is limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0192] For chemical groups and compound groups, the number of carbon atoms in a group or group is as follows: "Cn" defines the exact number (n) of carbon atoms in the group / group. "C≦n" defines the maximum number (n) of carbon atoms that can exist in the group / group, such that the minimum number is as small as possible for that group / group. For example, the group "alkenyl" (C≦8) " or group "Alken (C≦8) It is understood that the minimum number of carbon atoms in "alkoxy" is 2. (C≦10) This is compared with "Cn-n'". "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Therefore, "alkyl" (C2-10) " specifies an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may be before or after the chemical group or group they modify, and may or may not be enclosed in parentheses, and this does not imply any change in meaning. Thus, "C5 olefin", "C5-olefin", "olefin" (C5) " and "olefinC5 The terms "..." and "..." are all synonymous.

[0193] The term “saturated” as used to modify a compound or chemical group means a compound or chemical group that does not have carbon-carbon double bonds or carbon-carbon triple bonds, except as described below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substitutional forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. If such bonds are present, carbon-carbon double bonds that may arise as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance will dissolve in the solution.

[0194] When the term "aliphatic" is used without the modifier "substituted," it means that a compound or chemical group that is so modified is acyclic or cyclic but is a non-aromatic hydrocarbon compound or group. In aliphatic compounds / groups, carbon atoms can be linked together in a straight chain, a branched chain, or a non-aromatic ring (alicyclic). Aliphatic compounds / groups can be saturated, i.e., linked by a single carbon-carbon bond (alkane / alkyl), or unsaturated, having one or more carbon-carbon double bonds (alkene / alkenyl), or having one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0195] When the term "aromatic" is used to modify the atoms of a compound or chemical group, it means that the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by the interaction of bonds forming the ring.

[0196] The term "alkyl," when used without the modifier "substituted," refers to a monovalent saturated aliphatic group having a carbon atom as a bond site, a linear or branched acyclic structure, and lacking atoms other than carbon and hydrogen. Examples include -CH3(Me) group, -CH2CH3(Et) group, -CH2CH2CH3(n-Pr or propyl) group, -CH(CH3)2(i-Pr, i Pr or isopropyl) group, -CH2CH2CH2CH3(n-Bu) group, -CH(CH3)CH2CH3(sec-butyl) group, -CH2CH(CH3)2(isobutyl) group, -C(CH3)3(tert-butyl, t-butyl, t-Bu or tThe Bu) group and the -CH2C(CH3)3(neo-pentyl) group are non-restrictive examples of alkyl groups. The term "alkanediyl," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as bond sites, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The -CH2-(methylene) group, -CH2CH2- group, -CH2C(CH3)2CH2- group, and -CH2CH2CH2- group are non-restrictive examples of alkanediyl groups. "Alkane" refers to a group of compounds having the formula HR, where R is alkyl (this term is defined above). When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-restrictive examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which the substitution of hydrogen atoms is limited to halos (i.e., -F, -Cl, -Br, or -I), and no other atoms are present except carbon, hydrogen, and halogens. The group -CH2Cl is a non-restrictive example of a haloalkyl group. The term "fluoroalkyl" is a small group of substituted alkyl groups in which the substitution of hydrogen atoms is limited to fluorosilicons, and no other atoms are present except carbon, hydrogen, and fluorine. The groups -CH2F, -CF3, and -CH2CF3 are non-restrictive examples of fluoroalkyl groups.

[0197] The term "cycloalkyl," when used without the modifier "substituted," refers to a monovalent saturated aliphatic group having a carbon atom as a bond site, where the carbon atom forms part of one or more non-aromatic ring structures, and which has no carbon-carbon double or triple bonds, and is atom-free other than carbon and hydrogen. Non-restrictive examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having two carbon atoms as bond sites, with no carbon-carbon double or triple bonds, and which is atom-free other than carbon and hydrogen. [ka] This is a non-restrictive example of a cycloalkanediyl group. The term "cycloalkane" refers to a group of compounds having the formula HR, where R is cycloalkyl (as defined above). When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0198] The term "alkenyl," when used without the modifier "substituted," refers to a monounsaturated aliphatic group having a carbon atom as a bond site, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-restrictive examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl," when used without the modifier "substituted," refers to a diunsaturated aliphatic group having two carbon atoms as a bond site, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The -CH=CH- group, -CH=C(CH3)CH2- group, -CH=CHCH2- group, and -CH2CH=CHCH2- group are non-restrictive examples of alkenediyl groups. Note that although alkenediyl groups are aliphatic, once both ends are joined, this does not prevent the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to a group of compounds having the formula HR, where R is an alkenyl (this term is defined above). Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene having only one carbon-carbon double bond, where that bond is part of a vinyl group at the end of the molecule. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The -CH=CHF group, -CH=CHCl group, and -CH=CHBr group are non-exclusive examples of substituted alkenyl groups.

[0199] The term "alkynyl," when used without the modifier "substituted," refers to a monounsaturated aliphatic group having a carbon atom as a bonding site, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-restrictive examples of alkynyl groups. "Alkyne" refers to a group of compounds having the formula HR, where R is an alkynyl. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0200] The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a bonding site, wherein the carbon atom forms part of one or more six-membered aromatic ring structures, all ring atoms are carbon, and the group is composed of no atoms other than carbon and hydrogen. If two or more rings are present, the rings may or may not be fused. As used herein, the term does not exclude the presence of one or more alkyl or aralkyl groups bonded to the first aromatic ring or any additional aromatic rings present (as far as the carbon number limit allows). Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and biphenyl. The term "arendiyl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms as bonding sites, wherein the carbon atoms form part of one or more six-membered aromatic ring structures, all ring atoms are carbon, and the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups bonded to the first aromatic ring or any additional aromatic rings present (as far as carbon number limitations allow). If two or more rings are present, the rings may or may not be fused. Unfused rings may be connected via one or more covalent bonds, alkanediyl groups, or alkenediyl groups (as far as carbon number limitations allow). Non-limiting examples of arendiyl groups include: [ka] These are some examples.

[0201] The term "arene" refers to a group of compounds having the formula HR, where R is an aryl compound (as defined above). Benzene and toluene are non-restrictive examples of arenes. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0202] The term "aralkyl," when used without the modifier "substituted," refers to a monovalent alkanediylaryl group, and the terms alkanediyl and aryl are used in a manner consistent with the above definitions. Non-restrictive examples include phenylmethyl(benzyl, Bn) and 2-phenylethyl. When the term aralkyl is used with the modifier "substituted," one or more hydrogen atoms from an alkanediyl group and / or aryl group are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-limiting examples of substituted aralkyls are (3-chlorophenyl)methyl and 2-chloro-2-phenyletho-1-yl.

[0203] The term "heteroaryl," when used without the modifier "substituted," refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as a bonding site, wherein the carbon or nitrogen atom forms one or more aromatic ring structures in which at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the heteroaryl group is not composed of any atom other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. A heteroaryl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, and sulfur. If two or more rings are present, the rings may or may not be fused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups bonded to an aromatic ring or aromatic ring system (as far as the carbon number limit allows). Non-exclusive examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a bond site. The term "heterearenediyl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two bond sites, wherein the atoms form part of one or more aromatic ring structures in which at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. If two or more rings are present, the rings may or may not be fused. Unfused rings may be connected via one or more of the following: covalent bonds, alkanediyl groups, or alkenediyl groups (as far as carbon number limitations allow). As used herein, this term does not exclude the presence of one or more alkyl groups, aryl groups, and / or aralkyl groups bonded to an aromatic ring or aromatic ring system (as far as carbon number limitations allow).Non-restrictive examples of heteroarenediyl groups include: [ka] These are some examples.

[0204] "Heterearenes" refer to a group of compounds having the formula HR, where R is a heteroaryl compound. Pyridine and quinoline are non-restrictive examples of heteroarenes. When these terms are used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0205] The term "heterocycloalkyl," when used without the modifier "substituted," refers to a monovalent non-aromatic group having a carbon or nitrogen atom as a bonding site, wherein the carbon or nitrogen atom forms one or more non-aromatic ring structures in which at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the heterocycloalkyl group is not composed of any atom other than carbon, hydrogen, nitrogen, oxygen, and sulfur. A heterocycloalkyl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, or sulfur. If two or more rings are present, the rings may or may not be fused. As used herein, the term does not exclude the presence of one or more alkyl groups bonded to the ring or ring system (as far as the carbon number limit allows). The term also does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include azilidinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranil, tetrahydrothiofuranil, tetrahydropyranil, pyranyl, oxyranil, and oxetanil. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as a bond site. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl," when used without the modifier "substituted," refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two bond sites, wherein the atoms form part of one or more ring structures in which at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. If two or more rings are present, the rings may or may not be fused. Non-condensed rings may be linked by one or more of the following: covalent bonds, alkanediyl groups, or alkenediyl groups (as far as carbon number limitations allow). As used herein, this term does not exclude the presence of one or more alkyl groups bonded to the ring or ring system (as far as carbon number limitations allow).Furthermore, this term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group remains non-aromatic. Non-restrictive examples of heterocycloalkanediyl groups include: [ka] These include: When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0206] The term "acyl," when used without the modifier "substituted," refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl (these terms are defined above). -CHO, -C(O)CH3(acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6H5, and -C(O)(imidazolyl) are non-restrictive examples of acyl groups. "Thioacyl" is similarly defined, except that the oxygen atom of the group -C(O)R is replaced by a sulfur atom (-C(S)R). The term "aldehyde" corresponds to an alkane as defined above, in which at least one of its hydrogen atoms is replaced by a -CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (including, if present, hydrogen atoms directly bonded to the carbon atom of a carbonyl group or thiocarbonyl group) are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0207] The term "alkoxy," when used without the modifier "substituted," refers to the group -OR, where R is alkyl (as defined above). Non-restrictive examples include -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy," "alkenyloxy," "alkynyloxy," "aryloxy," "aralkoxy," "heteroaryloxy," "heterocycloalkoxy," and "acyloxy," when used without the modifier "substituted," refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio," when used without the modifier "substituted," refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above in which at least one of its hydrogen atoms is replaced by a hydroxyl group. The term "ether" corresponds to an alkane as defined above in which at least one of its hydrogen atoms is replaced by an alkoxy group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0208] The term "alkylamino," when used without the modifier "substituted," refers to the group -NHR, where R is alkyl (as defined above). Non-restrictive examples include -NHCH3 and -NHCH2CH3. The term "dialkylamino," when used without the modifier "substituted," refers to the group -NRR', where R and R' may be the same alkyl group, different alkyl groups, or R and R' together to represent an alkanediyl group. Non-restrictive examples of dialkylamino groups include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocycloalkylamino," "alkoxyamino," and "alkylsulfonylamino," when used without the modifier "substituted," refer to a group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-restrictive example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to a divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amide" (acylamino), when used without the modifier "substituted," refers to a group -NHR, where R is acyl (the term is defined above). A non-restrictive example of an amide group is -NHC(O)CH3. The term "alkylimino," when used without the modifier "substituted," refers to a divalent group = NR, where R is alkyl (as defined above).When any of these terms is used with the modifier "substituted," one or more hydrogen atoms bonded to a carbon atom are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The -NHC(O)OCH3 and -NHC(O)NHCH3 groups are non-exclusive examples of substituted amide groups.

[0209] The use of the words "a" or "an" in conjunction with the term "including" in the claims and / or specification may mean "one," but also coincide with the meanings of "one or more," "at least one," and "one or two or more."

[0210] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in the errors of the apparatus or methods used to determine that value, or variations that exist among research subjects.

[0211] As used in this application, the term “average molecular weight” refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have a different polymerization level and, therefore, a different molar mass. Average molecular weight may be used to represent the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number-average molar mass, weight-average molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, average molecular weight represents either the number-average molar mass or the weight-average molar mass of the formula. In some embodiments, average molecular weight is the number-average molar mass. In some embodiments, average molecular weight may be used to represent the PEG component present in a lipid.

[0212] The term "chimeric antigen receptor (CAR)," as used herein, may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and encompasses engineered receptors that conjugate artificial specificity onto specific immune effector cells. CARs can be used to confer specificity of monoclonal antibodies onto T cells, thereby generating a large number of specific T cells, for example, for use in adoptive cell therapy. In specific embodiments, the CAR derives cell specificity to, for example, tumor-associated antigens. In some embodiments, the CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain including a tumor-associated antigen binding region. In certain embodiments, the CAR comprises a fusion of a single-strand variable fragment (scFv) derived from a monoclonal antibody, fused to the transmembrane and endodomains of CD3-zeta. Specificity of other CAR designs may derive from receptor ligands (e.g., peptides) or from pattern recognition receptors such as dectin. In some embodiments, malignant B cells can be targeted by redirecting T cell specificity using a CAR specific to the B cell lineage molecule CD19. In certain embodiments, the spatial arrangement of antigen recognition domains can be modified to reduce activation-induced cell death. In certain embodiments, the CAR may include domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules can be co-expressed with the CAR, and these include co-stimulatory molecules, imaging reporter genes (e.g., for positron emission tomography), gene products that conditionally remove T cells upon prodrug addition, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0213] The terms “tumor-associated antigen” and “cancer cell antigen” are used interchangeably herein. In either case, these terms refer to proteins, glycoproteins, or carbohydrates specifically or selectively expressed by cancer cells.

[0214] The terms “comprise,” “have,” and “include” are non-restrictive linking verbs. Any form or tense of one or more of these verbs, such as “comprises,” “comprising,” “have,” “have,” “includes,” and “including,” are also non-restrictive. For example, any way of “comprises,” “have,” or “includes” one or more steps is not limited to having only those one or more steps, but also includes other unspecified steps.

[0215] Where the term “effective” is used herein and / or in the claims, it means sufficient to achieve a desired, expected, or intended result. Where “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” is used in connection with treating a patient or subject with a compound, it means that the amount of the compound, when administered to a subject or patient to treat the disease, is sufficient to achieve such treatment for the disease.

[0216] When used herein, "IC 50 The term "inhibitory dose" refers to the inhibitory dose that represents 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to half-inhibit a given biological, biochemical, or chemical process (or its components, i.e., enzymes, cells, cell receptors, or microorganisms).

[0217] The "isomers" of the first compound are different compounds in which each molecule contains the same constituent atoms as the first compound, but the three-dimensional arrangement of those atoms is different.

[0218] As used herein, the terms “patient” or “subject” refer to living mammalian organisms, such as humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, guinea pigs, or their transgenic species. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects include adults, adolescents, infants, and fetuses.

[0219] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that, within the bounds of reasonable medical judgment, is suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit-risk ratio.

[0220] "Medically acceptable salt" means a salt of the compound of the present invention that is medically acceptable as defined above and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids, such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]octa-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphor sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopenta Examples of acid addition salts formed from propionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid are also included in pharmaceutically acceptable salts, which may be formed when the present acidic proton can react with an inorganic or organic base. Examples of acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that, as long as the salt as a whole is pharmacokinetically acceptable, the specific anions or cations that form part of any salt of the present invention are not important. Further examples of pharmacokinetically acceptable salts and methods for their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PHStahl & CGWermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0221] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle involved in the carrying or transport of a chemical, such as a liquid or solid extender, diluent, excipient, solvent, or containment agent.

[0222] "Prevention" or "prevention" includes (1) inhibiting the onset of the disease in subjects or patients who may be at risk of the disease and / or predisposed to the disease but have not yet experienced or exhibited any or all of the pathologies or overall symptoms of the disease, and / or (2) slowing the onset of the pathologies or overall symptoms of the disease in subjects or patients who may be at risk of the disease and / or predisposed to the disease but have not yet experienced or exhibited any or all of the pathologies or overall symptoms of the disease.

[0223] A "repeating unit" is the simplest structural entity of a particular material, whether organic, inorganic, or organometallic, such as a structure and / or polymer. In the case of a polymer chain, repeating units are continuously linked along the chain like beads on a necklace. For example, polyethylene, -[-CH2CH2-] n -The repeating unit is -CH2CH2-. The subscript "n" represents the degree of polymerization, i.e., the number of repeating units linked together. If the value of "n" remains undefined or "n" is absent, it simply indicates the repetition of the expression in square brackets and the polymer properties of the material. The concept of repeating units also applies when the bonding between repeating units is extended three-dimensionally, for example, in organometallic structures, modified polymers, thermosetting polymers, etc. In the context of dendrimers, repeating units may also be described as branched units, inner layers, or generations. Similarly, terminal groups may be described as surface groups.

[0224] A "stereoisomer" or "optical isomer" is an isomer of a given compound, where the same atoms are bonded to the same other atoms, but their three-dimensional arrangements are different. An "enantiomer" is a stereoisomer of a given compound that is a mirror image of each other, like a left hand and a right hand. A "diastereomer" is a stereoisomer of a given compound that is not an enantiomer. A chiral molecule contains a chiral center, also called a stereocenter or stereoisomer. A chiral center is any point in a molecule that has two groups that, when swapped, result in a stereoisomer; it does not necessarily have to be an atom. In organic compounds, the chiral center is usually a carbon, phosphorus, or sulfur atom, but other atoms can also be stereocenters in both organic and inorganic compounds. A molecule can have multiple stereocenters, giving many stereoisomers. In compounds where stereoisomerism is due to a tetrahedral chiral center (e.g., tetrahedral carbon), the hypothetically possible total number of stereoisomers is 2 n (where n is the number of stereocenters of the tetrahedron) does not exceed this number. Molecules with symmetry often have fewer stereoisomers than the maximum number of possible stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomer-enriched so that one enantiomer is present in greater than 50% of the mixture. Typically, enantiomers and / or diastereomers can be separated or decomposed using techniques known in the art. For any stereoisomer, or for an axis of opposing palmar symmetry whose stereochemistry is not defined, it is intended that its stereoisomer or axis of opposing palmar symmetry may exist as its R-form, S-form, or mixture of R-form and S-form, including racemic and non-racemic mixtures. As used herein, the expression “substantially free of other stereoisomers” means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of another stereoisomer(s).

[0225] "Treatment" or "to treat" includes (1) inhibiting the disease in a subject or patient experiencing or exhibiting the pathology or overall symptoms of the disease (e.g., halting further progression of the pathology and / or overall symptoms), (2) improving the disease in a subject or patient experiencing or exhibiting the pathology or overall symptoms of the disease (e.g., improving the pathology and / or overall symptoms), and / or (3) achieving any measurable reduction of the disease in a subject or patient experiencing or exhibiting the pathology or overall symptoms of the disease.

[0226] The definitions above supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be taken as implying that undefined terms are undefined. Rather, all terms used are intended to describe the invention in a manner that enables those skilled in the art to understand the scope of the invention and to practice it. [Examples]

[0227] J. Examples The following embodiments are included to demonstrate preferred embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in the implementation of the Disclosure, and therefore may constitute a preferred mode for its implementation. However, those skilled in the art will understand that, in light of the Disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the Disclosure, and similar or comparable results can still be obtained.

[0228] Example 1: Synthesis and Characterization of LNPs Four-component LNPs (0% 18:1 PA) known in the art can be prepared from 5A2-SC8, DOPE, cholesterol, and PEG-DMG (Figure 2A). Synthetic details and characterization of 5A2-SC8 are shown in Figures 3A and 3B. A 10% 18:1 PA five-component Spleen SORT formulation was used to achieve in-situ CAR T cell production. Both formulations were prepared with a total lipid to mRNA ratio of 30:1 (wt:wt). Details of the formulation molar ratios are shown in Figure 2B. Bioluminescence imaging (BLI) 24 hours after luciferase mRNA delivery revealed that the known 0% 18:1 PA four-component formulations primarily delivered mRNA to the liver, as expected for conventional LNPs (Figures 2C and 2E). When comparing delivery efficacy and organ targeting with 5-component 10% 18:1 PA Spleen SORT LNPs, organ targeting shifted primarily to the spleen, with minimal or no protein activity in the liver (Figures 2D and 2E). LNP size was unaffected by the inclusion of 10% 18:1 PA, and the frequency-particle size distributions were nearly identical (Figure 4A). LNPs formed with and without 18:1 PA formed a homogeneous LNP population, as indicated by the low polydispersity index (Figure 4B). Zeta potential remained neutral despite the addition of negatively charged lipids (Figure 4C). However, the inclusion of 10% 18:1 PA reduced the LNP pKa from 6.55 to 5.88 (Figure 4D). These results, including the low pKa that induces spleen targeting, were consistent with the mechanism of action of the characterized SORT LNPs (Dilliard et al., 2021).

[0229] Example 2: Determination of transfect cell type To characterize the cell types transfected within the spleen, lox-stop-lox TdTom mice were delivered twice at 48-hour intervals to a dose of 0.5 mg / kg of Cre recombinase mRNA using Spleen SORT LNP containing 10% 18:1 PA, resulting in stop codon deletion and tdTom fluorescence expression. This dosing regimen transfected 7% of all T cells in the spleen (Figure 2F). To further characterize specific T cells, additional antibodies were used in flow cytometry analysis, which showed that 5.8% of CD8+ T cells and 5.5% of CD4+ T cells were transfected (Figure 2G). Spleen-targeted LNP also transfected immune cell types such as macrophages and neutrophils (Figure 2H). The gating strategy used to determine cell types is described in Figure 5.

[0230] Example 3: In-situ production of CAR T cells After confirming that CD8+ and CD4+ T cells can be transfected by Spleen SORT LNP, their ability to produce CAR T cells in situ was investigated using an established lymphocyte-rich model of B-cell lymphoma (Kueberuwa et al., 2018c, Kochenderfer et al., 2010). To construct this model, Balb / c mice were pre-treated with a low dose of cyclophosphamide (100 mg / kg) to reduce immune cell levels and enable tumor engraftment. The number of intravenously injected A20 luciferase-expressing cells (A20-Luc) was manipulated to mimic various stages of tumor load (from less aggressive to highly aggressive). After tumor engraftment, mice were treated with Spleen SORT LNP containing mRNA encoding anti-CD19 CAR mRNA with a co-stimulatory molecule (41BB or CD28) and a CD3ζ signaling domain. All mRNA was transcribed in vitro (IVT) using the SP6 IVT protocol, and the resulting mRNA was analyzed by TapeStation to confirm its length and integrity (Figure 6A). Tumor growth was then monitored by whole-body BLI and waist circumference (AC). Survival analysis was performed to determine the therapeutic benefit.

[0231] In the first model, after pretreatment with cyclophosphamide, 1 × 10 6A20-Luc cells were injected (Figure 7A). Rapid tumor growth mimicking more aggressive cancer occurred in many cells. Fourteen days after A20-Luc injection, mice were randomly divided into three groups: a control group injected with saline, a control group injected with Cre mRNA Spleen SORT LNP, and a treatment group injected with Spleen SORT LNP containing mRNA encoding an anti-CD19 CAR having a 41BB costimulatory molecule and a CD3ζ signaling domain (CAR19-41BBz) (Figure 7B). All mRNAs were formulated with Spleen SORT LNP before injection. The resulting LNPs were characterized, and their size, polydispersity index, and surface charge were determined (Figures 6B-6D). LNPs formulated with CAR19-41BBz mRNA (approximately 2,000 nt) were slightly larger than LNPs formulated with Cre mRNA (approximately 1,000 nt). Differences in LNP size may be related to differences in mRNA length. The treatment regimen was 0.5 mg / kg twice weekly.

[0232] BLI images showed rapid tumor growth and metastasis in all three groups (Figure 7C). The livers of mice treated with mRNA CAR19-41BBz had fewer metastatic tumor lesions than the saline group and the Cre mRNA control group (Figure 7D). Furthermore, the AC of mice treated with CAR19-41BBz was smaller than that of the control group (Figure 7E). Although no improvement in overall survival was observed in treated mice (Figure 8), a delay in tumor growth was seen up to day 18, as quantified by abdominal circumference. By days 21 and 24, the tumors had metastasized, and thereafter, the effect of the treatment likely disappeared. Tumor engraftment was heterogeneous, with some mice starting with higher tumor loads than others, making it difficult to assess the efficacy of in-situ produced CAR T cells. Nevertheless, the significant difference between the Cre mRNA LNP control group and the CAR19-41BBz mRNA LNP group is promising and prompts further evaluation of the in-situ CAR-T method using Spleen SORT LNP.

[0233] To manage the heterogeneity observed in the previous model, the mouse BLI was set to 1 × 10⁻⁶ 7 Treatment was initiated when p / s was reached (Figure 9A). As a result, although the initiation date differed for each mouse, all were within 2-3 weeks of IV injection of A20-Luc cells. In this study, mice were divided into two groups: a saline control group and a treatment group with CAR19-41BBz mRNA. The treatment regimen consisted of IV injections twice weekly at a dose of 0.5 mg / kg (Figure 9B). BLI follow-up of the mice showed a reduction in tumor growth rate in the treatment group, and some mice showed reduced whole-body luminescence on day 14 (Figure 9C). By day 21, this effect was more pronounced, and AC was smaller and tumor growth was less in the treatment group (Figure 9D). On day 28, all mice in the saline group reached the endpoint. AC at the endpoint was compared for each group, corresponding to day 21 for the saline group and day 28 for the CAR19-41BBz treated mice. At the endpoint, tumor burden was significantly lower in treated mice (p=0.0010) (Figure 9E). Compared to induction in the previous model, AC in the model in Figure 7 lost statistical significance before reaching the endpoint. After day 28, tumors grew rapidly, leading to metastasis and loss of therapeutic efficacy. Although not statistically significant (p=0.09), more than half of the treated mice lived longer than the saline group (Figure 9F). While tumor heterogeneity was resolved in the second model, tumor aggressiveness persisted, making it difficult to observe the survival benefit. Nevertheless, the increased reduction in abdominal circumference in treated mice was promising and prompted further model modification and evaluation of the in-situ CAR-T method using Spleen SORT LNP in further modified models.

[0234] To reduce the aggression of the model, pretreatment with cyclophosphamide followed by 5 × 10 5 A20-Luc cells were injected (Figure 10A). Injecting fewer cells resulted in slower tumor engraftment, indicating a less aggressive lymphoma model. Mouse BLI was 1 × 10 7Treatment was initiated when p / s reached a certain level. The initiation date was always within 2-3 weeks after IV injection of A2O-Luc cells. The endpoint of this experiment was tumor bioluminescence (1 × 10⁻¹⁴). 9 This was based on p / s. This was done to reduce variability in tumor volume that did not appear in the abdomen (e.g., intracerebral and hind limbs). Treatment groups consisted of saline, mRNA CAR19-41BBz, and an additional mRNA (CAR19-28z) encoding an anti-CD19 CAR with a CD28 costimulatory molecule and a CD3ζ signaling domain, in order to study the effect of the signaling domain (Figure 10B). The treatment regimen involved weekly treatment with either CAR19-41BBz mRNA LNP or CAR19-28z mRNA LNP at a dose of 0.5 mg / kg. In tumor BLI follow-up, the group treated with CAR19-41BBz mRNA showed delayed tumor growth compared to the other groups (Figures 10C and 10D). When quantified, tumor growth was significantly less compared to saline and CAR19-28z (Figure 10E). Furthermore, mice in the CAR19-41BBz mRNA LNP group showed a statistically significant extension of survival compared to the other two treatment groups (p=0.0017) (Figure 10F). The results suggest that CD28 costimulatory signaling may not be powerful enough to combat invasive models such as B-cell lymphoma used in this study. CARs with CD28 have been reported to cause increased T-cell exhaustion (Cappell & Kochenderfer, 2021; Long et al., 2015), which may explain the lack of response observed in this treatment group.

[0235] Despite these lymphocyte-rich B-cell lymphoma models exhibiting aggressive and rapid tumor growth, physical signs of a response to treatment were observed in the CAR19-41BBz-treated group. Since rapid metastasis and large liver tumor lesions occurred in all stages of aggressive lymphoma, the effect of in situ CAR T cells on reducing metastatic lesions and abdominal circumference was investigated. For this purpose, tumors from the livers of aggressive model mice were examined (Figures 7 and 9). Tumors were thin-sectioned, stained for CD3+ T cells, and imaged using a confocal microscope (Figures 11A, 11B, and 12–15). Interestingly, a quantitative increase in tumor-infiltrating lymphocytes (TILs) was observed in CAR19-41BBz mRNA LNP-treated livers compared to saline-treated and Cre mRNA LNP-treated livers (Figure 10C). A similar increase was observed in the number of TILs from livers treated with mRNA CAR19-41BBz in a second model (Figure 10D). The results confirm that treatment with Spleen SORT LNP containing mRNA CAR19-41BBz produces in situ CAR T cells that can infiltrate metastatic lesions in the liver.

[0236] Example 4: Experimental Procedure LNP lipids 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-Dioleoyl-sn-glycero-3-phosphate (18:1 PA) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Sigma Aldrich. PEG-DMG (Sunbright GM-020) was purchased from NOF America Corporation. Ionizable aminolipid 5A2-SC8 was synthesized in the applicant's laboratory according to a previously reported protocol (Zhou et al., 2016).

[0237] Reagents for biological testing We purchased the Slide-A-Lyzer MINI dialysis machine, 3.5K MWCO, and QUANT-iT Ribogreen reagent from ThermoFisher. We purchased the MEGAscript SP6 transcription kit from ThermoFisher. We purchased N1-methylpsoiduridine-5'-triphosphate from TriLink. We purchased the ScriptCap Cap1 capping system from Cellscript Inc. Cre mRNA was provided by Recode Therapeutics. We purchased cyclophosphamide (NSC-26271) monohydrate from Selleckchem. We obtained MSGV-1D3-28Z All ITAMs intact (plasmid #107226) from Addgene. We purchased 4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazole carboxylate sodium salt (D-luciferin) from Goldbio. For flow cytometry, FITC anti-mouse CD3 antibody and PerCP anti-mouse CD4 antibody were purchased from BioLegend, and CD8a monoclonal antibody (53-6.7) and Alexa Fluor 700 were purchased from Thermo Fisher. For immunofluorescence of frozen tissue sections, Alexa Fluor 647 anti-mouse CD3 antibody was purchased from BioLegend. Triton X-100 and Dulbecco's phosphate-buffered saline were purchased from Sigma Aldrich.

[0238] cell culture Dulbecco's Modified Eagle Medium (DMEM), containing high glucose, sodium pyruvate, L-glutamine, and phenol red, as well as Roswell Park Memorial Institute (RPMI) 1640 medium, were purchased from ThermoFisher Scientific. Trypsin-EDTA (0.25%) and fetal bovine serum (FBS) (10%) were purchased from Sigma-Aldrich. Penicillin-streptomycin was purchased from Fisher Scientific. Blastodicin-selective antibiotics were purchased from InvivoGen. A20 cells were donated by Professor Yang-Xin Fu's laboratory.

[0239] Animal testing. C57 / BL6 mice were used in in vivo luciferase mRNA delivery studies. Ai14 mice were used in cell tracking experiments after administration of LNPs containing Cre mRNA. Balb / c mice were used in tumor studies. All experiments were approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Texas Southwestern Medical Center and complied with local, state, and federal regulations where applicable.

[0240] Synthesis of 5A2-G1. A 20 mL vial equipped with a stirring bar was used to add tetraethylenepentamine (5A2, 4.0 g, 1.0 equivalent), ethyl 2-(acryloyoxy)methacrylate (AEMA, 20.4 g, 5.25 equivalents), and butylated hydroxytoluene (BHT, 838 mg, 0.18 equivalents). The resulting reaction mixture was stirred under N2 at 50°C and 500 rpm for 24 hours. The crude product was purified by flash column (silica, with 30%-50% acetone / hexane and 3% triethylamine added) to obtain 5A2-G1 (43% yield). Rf = 0.15 (50% acetone / hexane, 3% triethylamine added, silica).

[0241] Synthesis of 5A2-SC8. A 20 mL vial was equipped with a stirring bar, and 5A2-G1 (3.9 g, 1.0 equivalent), dimethylphenylphosphine (DMPP, 193 μL, 0.45 equivalents), and 1-octanthiol (SC8, 4.71 mL, 6 × 1.5 equivalents) were added. The resulting reaction mixture was stirred under N2 at 55°C and 500 rpm for 48 hours. The crude product was dissolved in a minimal amount of CH2Cl2 and purified by flash column (neutral Al2O3, 20% to 100% ethyl acetate / hexane) to obtain 5A2-SC8 (yield 59%). Rf = 0.2 (70% ethyl acetate / hexane, neutral Al2O3). HRMS C 110 H 203 N5O 24 Calculated value for S6: 2170.3142, Measured value: 2170.2966.

[0242] Formulation of 5A2-SC8 SORT LNP. 5A2-SC8 LNPs were prepared by quickly mixing an acidic aqueous solution and an ethanol solution by hand. The ethanol solution contained 5A2-SC8, DOPE, cholesterol, PEG-DMG, and 18:1 PA in a molar ratio of 15:15:30:3:7, with a total lipid:mRNA weight ratio of 30:1 (wt / wt). The mRNA was dissolved in 10 mM citrate buffer (pH 3.0). The aqueous and ethanol solutions were rapidly mixed in a 3:1 volume ratio for 30 seconds and incubated at room temperature for 15 minutes to allow for LNP assembly. For in vitro experiments, PBS solution was added until the final concentration was reached. For in vivo studies, the LNPs were purified by dialysis in sterile PBS for 2 hours at a 3.5 kD cutoff. The volume was then adjusted to the desired concentration for subsequent experiments.

[0243] Characterization of LNPs. LNP size and zeta potential were measured using a Malvern Zetasizer. Size and polydispersity index were measured by dynamic light scattering (He-Ne laser, λ=632nm; detection angle=173°) using 100 μL of fresh LNP dispersion. Zeta potential was measured after diluting LNP to 800 μL in 1×PBS. mRNA mounting was measured using QUANT-iT. The overall / apparent pKa of each LNP formulation was measured using a TNS assay. LNP formulations were incubated individually for 5 minutes with 5 μM TNS [6-(p-toluidino)-2-naphthalenesulfonic acid] fluorescent probe dissolved in a series of different buffers with pH ranges of 2–11 [10 mM HEPES, 10 mM MES (4-morpholine ethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl], normalized to 75 μM total lipids. The mean fluorescence intensity was measured using a Tecan plate reader (λ¬Ex=324, λEm=435) in a triple technical repeat. The overall / apparent pKa was estimated by the pH at which the half-maximal fluorescence was recorded after nonlinear regression line fitting (GraphPad Prism).

[0244] In vivo luciferase mRNA delivery All experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and followed local, state, and federal regulations where applicable. Normal wild-type C57BL / 6 female mice were maintained as colonies. When the mice reached a body weight of 18–20 g, LNP formulated with Fluc mRNA was administered intravenously (IV) at a dose of 0.3 mg / kg. 24 hours later, D-luciferin was administered intraperitoneally at a dose of 150 mg / kg. Five minutes later, organs (lungs, liver, kidneys, and spleen) were dissected, and luciferase luminescence was imaged using AMI-HTX (Spectral Imaging systems). Luminescence was quantified as total luminescence (p / s).

[0245] Cell tracking. All experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and followed local, state, and federal regulations where applicable. Ai14 mice were maintained as colonies. When the mice reached a body weight of 18–20 g, they were intravenously injected with LNP formulated with Cre mRNA at a dose of 0.5 mg / kg. 48 hours later, the mice received a second intravenous injection of LNP-Cre mRNA at a dose of 0.5 mg / kg. 48 hours later, organs were collected and processed by flow cytometry.

[0246] Flow cytometry After LNP injection, the spleen was harvested and passed through a 100 μm cell strainer (BD-Biosciences) to obtain a single-cell suspension. Blood was then collected in an EDTA-containing tube to prevent coagulation. The single-cell suspension and blood were treated with 1× red blood cell (RBC) lysis buffer (BioLegend) on ice for 5 and 10 minutes, respectively. The lysis buffer was neutralized by adding twice the volume of cell staining buffer (BioLegend). Approximately 1 × 10⁻⁶ cells were obtained. 6 Cells were stained on ice for 20-30 minutes with 1:100 dilutions of anti-mouse CD45, CD4, and CD3 antibodies (all from BioLegend) and anti-mouse CD8 antibody (ebiosciences). Live and dead cells were distinguished using Ghost Red 780 (Tonbo Biosciences) or Live Dead Aqua (Invitrogen). Finally, the cells were washed, resuspended in 500 μL of cell staining buffer, and analyzed using an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo version 10.8.1 software (BD Biosciences).

[0247] Plasmid cloning The mouse anti-CD19 (CD28 costimulatory region and CD3 zeta signaling domain) chimeric antigen receptor CAR19-28z was cloned from the MSGV-1D3-28Z Addgene plasmid and inserted into a pCS2 vector by restriction end cloning. For CAR19-41BBz, the CD28 costimulatory region of 1D3-28Z was replaced with the 41BB coding region (1D3-41BBz), and the gene was purchased from Azenta (genewiz). Subsequently, the purchased DNA was cloned into a pCS2 vector by restriction end cloning. The mRNA and DNA sequences of the chimeric antigen receptors CAR19-28z and CAR19-41BBz are shown in Table 1. [Table 5] TIFF2026529161000139.tif184159TIFF2026529161000140.tif184159TIFF2026529161000141.tif182159

[0248] mRNA synthesis The CAR19-41BBz mRNA and CAR19-28z mRNA listed in Table 1 were prepared by in vitro transcription (IVT) as described above (Cheng et al., 2018). Simply put, the coding region was cloned into a pCS2+MT plasmid (Addgene), and then the 5' and 3' untranslated regions and polyA were cloned into a template. Finally, linearized pDNA was obtained by XhoI restriction immediately following the polyA sequence. In vitro transcription was performed according to the SP6 promoter kit's IVT protocol. UTP was replaced with N1-methylpsoiduridine-5'-triphosphate in the IVT reaction, and cap-1 mRNA was used. The final product was purified by the LiCl precipitation method described in the kit. mRNA size was verified using TapeStation (Agilent).

[0249] Aggressive lymphocyte-rich A20 lymphoma model A20 cells were transduced using a lentivirus to express luciferase, and luciferase-positive A20-Luc cells were selected and proliferated by 72-hour incubation with blastidicine and frozen in 10% DMSO until in vivo testing. A lymphocyte-rich model was created using previously reported protocols (Kueberuwa et al., 2018a, Kueberuwa et al., 2018b). Briefly, normal wild-type balb / c female mice were purchased from Invigo at 5-6 weeks of age. When the mice reached 6-8 weeks of age, they were injected with cyclophosphamide at 100 mg / kg to briefly weaken the immune system and promote tumor cell engraftment. 1 × 10⁶ cells were observed 24 hours after cyclophosphamide injection. 6 Individual cells were intravenously injected into mice. Treatment was initiated 14 days after cyclophosphamide injection to restore the immune system. Abdominal circumference (AC) was 75 mm, as measured by the mean of the product of the sagittal diameter (SAD) and transverse abdominal diameter (TAD) multiplied by the constant π (AC = π(SAD + TAD) / 2). 2 The endpoint was reached when the target value was reached. The tumor was also monitored using bioluminescence imaging (BLI) with a Spectral Imaging Systems AMI-HTX exposure of 30 seconds.

[0250] Second invasive lymphocyte-rich A20 lymphoma model The same protocol as above was followed to establish the tumor. The same dose of cells was administered: 1 x 10⁶ cells 24 hours after cyclophosphamide injection. 6 Individual cells. BLI signaling 1 × 10 7 Treatment was initiated after p / s reached 75 mm. 2 The endpoint was reached when it reached that point.

[0251] A low-aggression lymphocyte-rich A20 lymphoma model The same protocol described above was followed to establish the tumor. The dose of cells to be administered was 5 × 10⁶. 5 The cells were modified. The bioluminescent signal was 1 × 10⁻⁶ 7Treatment was initiated after p / s reached the target. The BLI signal was 1 × 10⁻⁶ just before image saturation. 9 The endpoint was reached when the p / s (pages per second) was reached.

[0252] Immunofluorescence Tumor tissue was collected at the endpoint of the tumor model and frozen at -80°C in Tissue-Plus® OCT compound. The tissue was frozen-sectioned by the Simmons Cancer Center Core facility using a Cryostat machine (Leica Biosystems). Tissue slides were fixed with 4% PFA and then washed three times with 1×PBS for 5 minutes each. The tissue was then blocked for 1 hour with 10% BSA solution containing 0.25% Triton-X100, followed by staining with 2% BSA containing 0.25% Triton-X100 containing Alexa Fluor® 647 anti-mouse CD3 antibody (1:100) for 48 hours. Tissue slices were washed three times with 1×PBS containing 0.25% Triton-X100 for 5 minutes each and mounted with DAPI-FluoroShield. Slides were imaged at 20x magnification using a confocal microscope (LSM 700, Zeiss).

[0253] Sirius Red staining Tissue sections were deparaffinized and rehydrated using the following steps: First, the slides were placed on a rack and gently placed in a staining jar containing 100% xylene, and washed twice (for 10 minutes each). Then, they were placed in a staining jar containing 50% xylene (v / v in ethanol) and washed twice separately (for 10 minutes each). Subsequently, the slides were washed with ethanol of different concentrations (95%, 75%, and 50%, each for 5 minutes twice). After that, the slices were washed by immersion in distilled water for 10 minutes twice, and then Weigert hematoxylin was added to the samples and the nuclei were stained by incubation for 8 minutes. The slides were washed under running tap water for 10 minutes, then immersed in picrosirius red staining solution (0.5 g of Sirius Red per 500 mL of picric acid (1.3% in water, Sigma-Aldrich)) for 1 hour, followed by washing with acidified water (5 mL of acetic acid in 1 L of water), with the washing solution changed twice. Finally, the slides were dehydrated by changing the 100% ethanol three times and removed with xylene. The sections were scanned under a microscope equipped with a 10x lens.

[0254] Flow cytometry analysis The optimized flow cytometry protocol was based on published methods (Cheng et al, 2018, Cheng et al, 2020, Zhu et al, 2014). Fresh tumor tissue was excised and placed in a 10 cm tissue culture dish, and cut into small pieces with a sterile razor blade. The tissue was transferred to a 50 mL tube containing a 100 μm cell strainer, washed with PBS (20 mL), and then centrifuged at 2000 rpm for 3 minutes. After removing the supernatant, tumor digestion buffer (5 mL RPMI with 1% FBS and 0.25 mL of 10× digestion buffer (2 mg / mL collagenase D, 250 units / μL DNAse I, Sigma Aldrich)) was added to the tube containing the pelleted tissue. The tube was placed on a shaker at 37°C and shaken for 1 hour. The sample was filtered using a 100 μm cell strainer, washed with 35 mL of PBS, and rotated at high speed at 2000 rpm for 3 minutes. The pellet was resuspended in 2 mL of ACK lysis buffer, and the erythrocytes were lysed by incubation on ice for 5 minutes. After adding 30 mL of PBS, the pellet was washed again by centrifugation at 2000 rpm for 3 minutes. Next, the cells (5 × 10) were lysed. 6Cells (per mL) were incubated in a light-shielded environment at 2–8°C for 40 minutes with 0.5 μL of Ghost Dye Red 780 (Tonbo Bioscience) antibody cocktail solution (1 μL of each antibody per 100 μL of cell staining buffer). The labeled samples were washed twice with 1.5 mL of cell staining buffer (BioLegend). The samples were resuspended in 500 μL of cell staining buffer. Data acquisition was performed using LSRFortessa (BD Biosciences). Furthermore, a single color compensation control was performed using LSRFortessa before sample data acquisition. FlowJo was used for data analysis. All fluorophore-conjugated anti-mouse antibodies used for flow cytometry were purchased from BioLegend: Pacific Blue anti-mouse CD45 (Biolegend, catalog number 1031266), APC anti-mouse CD3 antibody (Biolegend, catalog number 100236), PE anti-mouse CD8a (Biolegend, catalog number 162304), PerCP / Cyanine 5.5 anti-mouse CD4 (Biolegend, catalog number 116012), Alexa Fluor 488 anti-mouse / human CD11b (Biolegend, catalog number 101217), and Alexa Fluor® 594 anti-mouse F4 / 80 (Biolegend, catalog number 123140).

[0255] Measurement of compressive modulus The compressive modulus of tumor tissue was measured using an unconstrained compression experimental protocol, following published studies (Voutouri et al, 2018, Rashid et al, 2012). Fresh tumors were collected from mice in each group after treatment and mechanically tested within 4 hours. Each tumor tissue was stored in PBS, and all samples were kept on ice during transport and measurement. All samples were cut into small sizes for testing (the size of each sample is detailed in Table 2) and measured at room temperature of approximately 22°C. Tumor specimens (n=8) were loaded into a mechanical testing system equipped with a 5.6 lbf load cell (TestResources, MN, USA, 250 lbf actuator). Compressive measurements of tumor tissue were performed up to a final strain of 30% at a compression rate of 0.1 mm / min. The compressive modulus was calculated from the slope of the stress-strain curve in the strain range of 25–30% (Voutouri et al, 2018). [Table 6]

[0256] Statistics and Reproducibility Data are reported as mean ± standard deviation. Statistical analysis was performed using a two-tailed t-test or a one-way ANOVA with multiple comparison tests. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, no significant difference (ns) was observed using GraphPad Prism software (GraphPad Software, USA). Exact p-values ​​less than 0.0001 were obtained from Excel using the same statistical analysis. Data obtained from microscopic images, T7E1, and Western blots are representative images from three biologically independent samples (n=3).

[0257] Any of the compounds, materials, compositions, and methods disclosed and claimed herein can be prepared and carried out without undue experimentation in view of this disclosure. While this disclosure may focus on certain embodiments or be described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications and alterations may be applied to the compounds, compositions, and methods without departing from the spirit, scope, and concept of the invention. Modifications and alterations that are apparent to those skilled in the art will be deemed to fall within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference insofar as they provide exemplary procedures or other details that supplement those described herein. Amini et al., Nat. Rev. Clin. Oncol, 19, 342-355, 2022. Cappell & Kochenderfer, Nat. Rev. Clin. Oncol., 18, 715-727, 2021. Carpenito et al.,Proc.Natl.Acad.Sci.USA,106,3360-3365,2009. Cheng et al.,Adv Mater,30,e1805308,2018. Cheng et al.,Nat.Nanotechnol.,15,313-320,2020. Choi et al.,Res.Public Health,19,12366,2022. Dilliard et al.,Proc.Natl.Acad.Sci.USA,118,e2109256118,2021. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- Kymriah”,www.fda.gov / media / 106989 / download 2017. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- Yescarta”,www.fda.gov / media / 108458 / download,2017. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- Tecartus ”,www.fda.gov / media / 140415 / download 2020. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- ABECMA”,www.fda.gov / media / 147062 / download,2021. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- Breyanzi”,www.fda.gov / media / 145712 / download 2021. Food Drug Administration,“Center for Biologics Evaluation and Research.Approval Letter- CARVYKTI”,www.fda.gov / media / 156572 / download,2022. Gu et al.,Zhejiang Univ.Sci.B,23,793-811,2022. Hernandez et al.,JAMA Oncol.,4,994-996,2018. Huang et al.,Mol.Ther.,16,580-589,2008. Kochenderfer et al.,Blood,116,3875-3886,2010. Kueberuwa et al.,Molecular Therapy-Oncolytics,8,41-51,2018a. Kueberuwa et al.,J Vis Exp,e58492,2018b. Kueberuwa et al.,J.Vis.Exp.,e58492,2018c. Liang et al.,Medicine(Baltimore),99,e22510,2020. Long et al.,Nat.Med.,21,581-590,2015. Milone et al.,Mol.Ther.,17,1453-1464,2009. Owen et al.,Cancer Immunol.Immunother.,72,805-814,2023. Parayath & Stephan,Annu.Rev.Biomed.Eng.,23,385-405,2021. Xin et al.,Front.Oncol.,12,809754,2022. Zhou et al.,Proceedings of the National Academy of Sciences,113,520-525,2016.

Claims

1. A method for preparing chimeric antigen receptor (CAR) T cells in a patient, comprising administering mRNA encapsulated within lipid nanoparticles to the patient, wherein the lipid nanoparticles selectively bind to spleen cells, and the administration results in the formation of the CAR T cells in vivo.

2. A method for preparing chimeric antigen receptor (CAR) T cells in a patient, comprising administering mRNA encapsulated within lipid nanoparticles to the patient, wherein the lipid nanoparticles selectively internalize in spleen cells, and the administration results in the formation of the CAR T cells in vivo.

3. The method according to either claim 1 or claim 2, wherein the spleen cells are lymphocytes.

4. The method according to claim 3, wherein the lymphocyte is a T cell.

5. The method according to claim 4, wherein the T cell is a CD4+ T cell.

6. The method according to claim 4, wherein the T cell is a CD8+ T cell.

7. The lipid nanoparticles have a pK of less than 6. ａ The method according to any one of claims 1 to 6, comprising:

8. The pK ａ The method according to claim 7, wherein the value is approximately 1 to approximately 6.

9. The pK ａ The method according to claim 8, wherein the value is approximately 3 to approximately 6.

10. The method according to any one of claims 1 to 9, wherein the lipid nanoparticles include an ionizable cationic lipid.

11. The method according to claim 10, wherein the ionizable cationic lipid is a dendrimer or a dendron.

12. The aforementioned dendrimer or dendron is given by formula: Core - Repeating unit - Terminal group (I) [In the formula, the core is connected to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeating unit, The aforementioned core is given by the formula: 【Chemistry 1】 It has, During the ceremony, X １ is amino or alkylamino （Ｃ≦１２） , dialkylamino （Ｃ≦１２） heterocycloalkyl （Ｃ≦１２） heteroaryl （Ｃ≦１２） , or their replacement forms, R １ is amino, hydroxy, or mercapto, or alkylamino （Ｃ≦１２） , dialkylamino （Ｃ≦１２） , or a substituted form of either of these groups, and a is 1, 2, 3, 4, 5, or 6, or The aforementioned core is given by the formula: 【Chemistry 2】 It has, During the ceremony, X ２ N(R) ５ ) ｙ And, R ５ hydrogen, alkyl （Ｃ≦１８） , or substituted alkyl （Ｃ≦１８） and y is 0, 1, or 2, provided that the sum of y and z is 3. R ２ is amino, hydroxy, or mercapto, or alkylamino （Ｃ≦１２） , dialkylamino （Ｃ≦１２） , or a substitutional form of either of these groups, b is 1, 2, 3, 4, 5, or 6, and z is 1, 2, or 3, provided that the sum of z and y is 3, or The aforementioned core is given by the formula: 【Transformation 3】 It has, During the ceremony, X ３ -NR ６ - and here, R ６ hydrogen, alkyl （Ｃ≦８） , or substituted alkyl （Ｃ≦８） , -O-, or alkylaminodiyl （Ｃ≦８） alkoxydiyl （Ｃ≦８） , Allenziil （Ｃ≦８） , heteroalene zil （Ｃ≦８） heterocycloalkanediyl （Ｃ≦８） , or a substitutional form of any of these groups, R ３ and R ４ Each of these is independently amino, hydroxy, or mercapto, or alkylamino. （Ｃ≦１２） , dialkylamino （Ｃ≦１２） , or a substitution of either of these groups, or formula: -N(R ｆ ) ｆ (CH ２ CH ２ N(R) ｃ )) ｅ R ｄ It is the basis of, During the ceremony, e and f are independently 1, 2, or 3, provided that the sum of e and f is 3. R ｃ , R ｄ , and R ｆ These are, independently, hydrogen and alkyl. （Ｃ≦６） , or substituted alkyl （Ｃ≦６） And, c and d are independently 1, 2, 3, 4, 5, or 6, or The aforementioned core is an alkylamine （Ｃ≦１８） , dialkylamine （Ｃ≦３６） heterocycloalkanes （Ｃ≦１２） , or a substitutional form of any of these groups, The aforementioned repeating unit includes a degradable diacyl and a linker. The aforementioned degradable diacyl group is of formula: 【Chemistry 4】 It has, During the ceremony, A １ and A ２ These are, independently, -O- or -NR ａ -And here, R ａ hydrogen, alkyl （Ｃ≦６） , or substituted alkyl （Ｃ≦６） And, Y ３ is Arcanziel （Ｃ≦１２） , Alkenzil （Ｃ≦１２） , Allenziil （Ｃ≦１２） , or a substitution form of any of these bases, or formula: 【Transformation 5】 It is the basis of, During the ceremony, X ３ and X ４ is Arcanziel （Ｃ≦１２） , Alkenzil （Ｃ≦１２） , Allenziil （Ｃ≦１２） , or a substitutional form of any of these groups, Y ５ Covalent bonds, alkanediyl （Ｃ≦１２） , Alkenzil （Ｃ≦１２） , Allenziil （Ｃ≦１２） , or a substitutional form of any of these groups, R ９ is alkyl （Ｃ≦８） or substituted alkyl （Ｃ≦８） And, The linker group is, formula: 【Transformation 6】 It has, During the ceremony, Y １ is Arcanziel （Ｃ≦１２） , Alkenzil （Ｃ≦１２） , Allenziil （Ｃ≦１２） , or a substitutional form of any of these groups, If the repeating unit includes a linker group, the linker group includes independent degradable diacyl groups bonded to both the nitrogen atom and the sulfur atom of the linker group (when n is greater than 1), the first group of the repeating unit is a degradable diacyl group, and for each linker group, the next repeating unit includes two degradable diacyl groups bonded to the nitrogen atom of the linker group, where n is the number of linker groups present in the repeating unit. The terminal group is given by formula: 【Transformation 7】 It has, During the ceremony, Y ４ is Arcanziel （Ｃ≦１８） is or alkandiil （Ｃ≦１８） One or more of the hydrogen atoms above are -OH, -F, -Cl, -Br, -I, -SH, -OCH ３ , -OCH ２ CH ３ , -SCH ３ , or -OC(O)CH ３ Arcandil has been replaced by （Ｃ≦１８） And, R １０ is hydrogen, carboxy, hydroxy, or Aryl （Ｃ≦１２） alkylamino （Ｃ≦１２） dialkylamino （Ｃ≦１２） N-heterocycloalkyl （Ｃ≦１２） -C(O)N(R １１ )-alkanediyl （Ｃ≦６） -heterocycloalkyl （Ｃ≦１２） -C(O)-alkylamino （Ｃ≦１２） -C(O)-dialkylamino （Ｃ≦１２） -C(O)-N-heterocycloalkyl （Ｃ≦１２） wherein R １１ is hydrogen, alkyl （Ｃ≦６） or substituted alkyl （Ｃ≦６） and The last degradable diacyl in the chain is attached to the terminal group, n is further defined by [0, 1, 2, 3, 4, 5, or 6], The method according to claim 11, wherein the salt is pharmaceutically acceptable or a pharmaceutically acceptable salt thereof.

13. The terminal group is, formula: 【Transformation 8】 [In the formula, Y ４ is Arcanziel （Ｃ≦１８） and R １０ The method according to claim 12, further defined by [is hydrogen].

14. The aforementioned core is given by the formula: 【Chemistry 9】 [In the formula, X ２ N(R) ５ ) ｙ And, R ５ is hydrogen or alkyl （Ｃ≦８） , or substituted alkyl （Ｃ≦１８） and y is 0, 1, or 2, provided that the sum of y and z is 3. R ２ is amino, hydroxy, or mercapto, or alkylamino （Ｃ≦１２） , dialkylamino （Ｃ≦１２） , or a substitutional form of either of these groups, b is 1, 2, 3, 4, 5, or 6, and The method according to either claim 12 or claim 13, further defined by [z is 1, 2, or 3, provided that the sum of z and y is 3].

15. The aforementioned core is given by the formula: 【Chemistry 10】 [In the formula, X ３ -NR ６ - and here, R ６ hydrogen, alkyl （Ｃ≦８） , or substituted alkyl （Ｃ≦８） , -O-, or alkylaminodiyl （Ｃ≦８） alkoxydiyl （Ｃ≦８） , Allenziil （Ｃ≦８） , heteroalene zil （Ｃ≦８） heterocycloalkanediyl （Ｃ≦８） , or a substitutional form of any of these groups, R ３ and R ４ Each of these is independently amino, hydroxy, or mercapto, or alkylamino. （Ｃ≦１２） , dialkylamino （Ｃ≦１２） , or a substitution of either of these groups, or formula: -N(R ｆ ) ｆ (CH ２ CH ２ N(R) ｃ )) ｅ R ｄ It is the basis of, During the ceremony, e and f are independently 1, 2, or 3, provided that the sum of e and f is 3. R ｃ , R ｄ , and R ｆ These are, independently, hydrogen and alkyl. （Ｃ≦６） , or substituted alkyl （Ｃ≦６） And, The method according to either claim 12 or claim 13, further defined by [c and d being independently 1, 2, 3, 4, 5, or 6].

16. The aforementioned core, 【Chemistry 11】 The method according to any one of claims 12 to 15, as further defined as

17. The aforementioned dendrimer 【Chemistry 12】 【change】 It may be further defined as, The method according to any one of claims 12 to 16, wherein the salt is pharmaceutically acceptable or a pharmaceutically acceptable salt thereof.

18. The method according to any one of claims 1 to 17, wherein the lipid nanoparticles further comprise permanent anionic lipids.

19. The method according to claim 18, wherein the permanent anionic lipid comprises a phosphate group.

20. The aforementioned permanent anionic lipid is 【Chemistry 13】 [In the formula, R １ and R ２ Each of them is independently alkyl （Ｃ８－Ｃ２４） Alkenil （Ｃ８－Ｃ２４） , or a substitutional form of either base, R ３ hydrogen, alkyl （Ｃ≦６） , or substituted alkyl （Ｃ≦６） is or -Y １ -R ４ And here, Y １ is Arcanziel （Ｃ≦６） or substitute alkanediyl （Ｃ≦６） and R ４ is acyloxy （Ｃ≦８－２４） or substituted acyloxy （Ｃ≦８－２４） The method according to any one of claims 1 to 19, further defined as:

21. The aforementioned permanent anionic lipid is 【Chemistry 14】 The method according to claim 20, further defined as:

22. The method according to any one of claims 1 to 21, wherein the lipid nanoparticles further comprise phospholipids.

23. The method according to any one of claims 1 to 22, wherein the lipid nanoparticles further comprise a steroid.

24. The method according to claim 23, wherein the steroid is cholesterol.

25. The method according to any one of claims 1 to 24, wherein the lipid nanoparticles further comprise polymer composite lipids.

26. The method according to claim 25, wherein the polymer composite lipid is a PEG-modified lipid.

27. The aforementioned polymer composite lipid is given by formula: 【Chemistry 15】 [In the formula, R １２ and R １３ Each of them is independently alkyl （Ｃ≦２４） Alkenil （Ｃ≦２４） , or a substitutional form of either of these groups, R ｅ hydrogen, alkyl （Ｃ≦８） , or substituted alkyl （Ｃ≦８） and The method according to either claim 25 or claim 26, further defined by [x is 1 to 250].

28. The polymer composite lipid is dimyristoyl-sn-glycerol or formula: 【Chemistry 16】 [In the formula, n １ is 5 to 250, and n ２ and n ３ The method according to claim 25 or claim 26, wherein each is independently a compound of [2 to 25].

29. The method according to any one of claims 1 to 28, wherein the mRNA encodes a chimeric antigen receptor (CAR).

30. The method according to claim 29, wherein the mRNA encodes two or more chimeric antigen receptors.

31. The method according to any one of claims 1 to 30, wherein the mRNA further encodes a co-stimulatory molecule.

32. The method according to claim 31, wherein the mRNA encodes two or more co-stimulatory molecules.

33. The method according to any one of claims 1 to 32, wherein the mRNA further encodes a signal transduction domain.

34. The method according to claim 33, wherein the mRNA encodes two or more signal transduction domains.

35. The method according to any one of claims 1 to 34, wherein the mRNA further encodes one or more cytokines.

36. The aforementioned mRNA (i) One or more chimeric antigen receptors, (ii) One or more signaling domains, (iii) The method according to any one of claims 1 to 35, comprising encoding one or more co-stimulatory molecules.

37. The aforementioned mRNA (i) One or more chimeric antigen receptors, (ii) One or more signaling domains, (iii) One or more co-stimulatory molecules, (iv) The method according to any one of claims 1 to 36, comprising encoding one or more cytokines.

38. The method according to any one of claims 29 to 37, wherein the encoded chimeric antigen receptor is an antigen of a tumor marker.

39. The method according to claim 38, wherein the tumor marker is CD19 or CD20.

40. The method according to any one of claims 31 to 39, wherein the coded co-stimulatory molecule is CD28 or 41BB.

41. The method according to any one of claims 33 to 40, wherein the encoded signal transduction domain is CD3ζ.

42. The method according to any one of claims 1 to 41, wherein the lipid nanoparticles contain ionizable cationic lipids in a molar percentage of about 1% to about 45%.

43. The method according to claim 42, wherein the lipid nanoparticles contain about 10% to about 30% of the ionizable cationic lipid as a mole percentage of the lipid nanoparticles.

44. The method according to any one of claims 1 to 43, wherein the lipid nanoparticles contain permanent anionic lipids in a molar percentage of about 1% to about 40%.

45. The method according to claim 44, wherein the lipid nanoparticles contain the permanent anionic lipid in a molar percentage of about 5% to about 20% of the lipid nanoparticles.

46. The method according to any one of claims 1 to 45, wherein the lipid nanoparticles contain phospholipids in a molar percentage of about 1% to about 45%.

47. The method according to claim 46, wherein the lipid nanoparticles contain about 10% to about 30% of the phospholipids as a mole percentage of the lipid nanoparticles.

48. The method according to any one of claims 1 to 47, wherein the lipid nanoparticles contain a steroid in a molar percentage of about 10% to about 70% of the lipid nanoparticles.

49. The method according to claim 48, wherein the lipid nanoparticles contain the polymer composite lipid in a molar percentage of about 25% to about 60% of the lipid nanoparticles.

50. The method according to any one of claims 1 to 47, wherein the lipid nanoparticles contain polymer composite lipids in a molar percentage of about 0.01% to about 15% of the lipid nanoparticles.

51. The method according to claim 50, wherein the lipid nanoparticles contain the polymer composite lipid in an amount of about 0.1% to about 10% as a mole percentage of the lipid nanoparticles.

52. The aforementioned lipid nanoparticles (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) The method according to any one of claims 1 to 51, comprising a steroid.

53. The aforementioned lipid nanoparticles (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids and, The method according to claim 52, comprising (iv) a polymer composite lipid.

54. The aforementioned lipid nanoparticles (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids and, The method according to claim 52, comprising (iv) phospholipid.

55. The aforementioned lipid nanoparticles (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, (iii) Steroids and, (iv) Phospholipids and (v) The method according to claim 52, comprising a polymer composite lipid.

56. The method according to any one of claims 1 to 55, wherein the method comprises administering the lipid nanoparticles systemically to the patient.

57. The method according to claim 56, wherein the systemic administration is by injection.

58. The method according to either claim 56 or claim 57, wherein the systemic administration is intravenous administration.

59. A method for treating a disease or disorder in a patient, comprising administering lipid nanoparticles containing mRNA to the patient, wherein the mRNA encodes a chimeric antigen receptor and the lipid nanoparticles selectively bind to spleen cells.

60. The method according to claim 59, wherein the disease is cancer.

61. The method according to claim 60, wherein the cancer is a lymphatic system cancer.

62. The method according to claim 61, wherein the cancer of the lymphatic system is lymphoma.

63. The method according to claim 59, wherein the disease is a cardiovascular disease.

64. The method according to claim 63, wherein the cardiovascular disease is traumatic injury or heart failure.

65. The method according to claim 59, wherein the disease or disorder is a fibrous disease.

66. A method for modifying lymphocytes, comprising administering lipid nanoparticles containing mRNA encoding a chimeric antigen receptor to a patient, wherein the selectivity of the lipid nanoparticles is such that they bind to the lymphocytes.

67. The method according to claim 66, wherein the lymphocyte is a T cell.

68. The method according to claim 67, wherein the T cell is a CD4+ T cell.

69. The method according to claim 67, wherein the T cell is a CD8+ T cell.

70. A composition, (A) Lipid nanoparticles, (i) Ionizable cationic lipids, (ii) Permanent anionic lipids, and (iii) The lipid nanoparticles comprising one or more additional lipids, (B) mRNA encoding a chimeric antigen receptor, and, The mRNA is encapsulated within the lipid nanoparticles, and the lipid nanoparticles have an apparent pK of less than 6. ａ The composition having the above-mentioned properties.

71. The composition according to claim 70, wherein the additional lipids include a steroid.

72. The composition according to claim 71, wherein the steroid is cholesterol.

73. The composition according to claim 70 to 72, wherein the additional lipids include phospholipids.

74. The composition according to claim 73, wherein the phospholipid is a neutral phospholipid.

75. The composition according to any one of claims 70 to 74, wherein the additional lipids include polymer complex lipids.

76. The composition according to claim 75, wherein the polymer composite lipid is a PEG-modified lipid.

77. The composition according to any one of claims 70 to 76, wherein the lipid nanoparticles contain ionizable cationic lipids in a molar percentage of about 1% to about 45%.

78. The composition according to claim 77, wherein the lipid nanoparticles contain about 10% to about 30% of the ionizable cationic lipids as a mole percentage of the lipid nanoparticles.

79. The composition according to any one of claims 1 to 78, wherein the lipid nanoparticles contain permanent anionic lipids in a molar percentage of about 1% to about 40% of the lipid nanoparticles.

80. The composition according to claim 79, wherein the lipid nanoparticles contain the permanent anionic lipid in a molar percentage of about 5% to about 20% of the lipid nanoparticles.

81. The composition according to any one of claims 1 to 80, wherein the lipid nanoparticles contain phospholipids in a molar percentage of about 1% to about 45% of the lipid nanoparticles.

82. The composition according to claim 81, wherein the lipid nanoparticles contain about 10% to about 30% of the phospholipids as a mole percentage of the lipid nanoparticles.

83. The composition according to any one of claims 1 to 82, wherein the lipid nanoparticles contain a steroid in a molar percentage of about 10% to about 70% of the lipid nanoparticles.

84. The composition according to claim 83, wherein the lipid nanoparticles comprise the polymer composite lipid in a molar percentage of about 25% to about 60% of the lipid nanoparticles.

85. The composition according to any one of claims 1 to 84, wherein the lipid nanoparticles contain polymer composite lipids in a molar percentage of about 0.01% to about 15% of the lipid nanoparticles.

86. The composition according to claim 85, wherein the lipid nanoparticles contain the polymer composite lipid in an amount of about 0.1% to about 10% as a mole percentage of the lipid nanoparticles.

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