Immune effector cells that stably and transiently express nucleic acids

Abstract

The present invention relates to immune effector cells comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, cell compositions and pharmaceutical compositions comprising such immune effector cells, and methods for producing such immune effector cells. The present invention also relates to particles comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, and complexes comprising these particles. The present invention also relates to methods of treating a subject with a disease, disorder, or condition associated with expression or elevated expression of an antigen using the immune effector cells, particles, or complexes.

Description

[Technical Field]

[0001] Technical Field The present invention relates to immune effector cells comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, cell compositions and pharmaceutical compositions comprising such immune effector cells, and methods for producing such immune effector cells. The present invention also relates to particles comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, and complexes comprising these particles. The present invention also relates to methods of treating a subject with a disease, disorder, or condition associated with expression or elevated expression of an antigen using the immune effector cells, particles, or complexes. [Background technology]

[0002] background Following the success of CD19 CAR-T cells in treating hematologic malignancies, hundreds of clinical trials are underway testing the efficacy of engineered T cells as cancer therapies, and the number of patients eligible for T cell-based therapeutic approaches is expected to increase significantly. However, current T cell manufacturing technologies are inadequate to meet the growing demand for CAR-T cells and related engineered T cell immunotherapies, particularly because current manufacturing methods rely on time-consuming and costly production protocols (Rafiq et al., Nat. Rev. Clin. Oncol. 2020 Mar;17(3):147-167).

[0003] Standard production protocols for engineered T cells are largely performed manually or semi-automated, limiting throughput and increasing product variability and cost. These procedures generally involve collection of patient white blood cell material and transport to a production facility, T cell stimulation and transduction (typically with a viral vector encoding an antigen receptor), expansion and cryopreservation of the T cell product under Good Manufacturing Practice (GMP) conditions, and subsequent transport back to the patient. Therefore, improving speed, scalability, and cost-effectiveness while maintaining or improving the product's biosafety and therapeutic efficacy are key priorities for optimizing CAR-T cell manufacturing (Blache et al., Nat. Commun., 2022 Sep 5;13(1):5225).

[0004] Currently approved CAR-T cell products typically rely on the use of gamma-retroviruses or lentiviruses, which have high transduction efficiencies, to deliver CAR nucleic acid sequences into patient T cells. Further development of these viral transduction methods has led to improvements in manufacturing time (Ghassemi et al., Nat. Biomed. Eng. 2022 Feb;6(2):118-128). However, viral vectors have limited gene payloads, typically require a T cell activation step that negatively impacts persistence and antitumor efficacy, entail high manufacturing costs under GMP conditions, and face a complex regulatory environment. Therefore, the development of alternative genetic engineering methods has become a focus of efforts to address the current limitations of CAR-T cell manufacturing. In this regard, transposon-based systems have emerged as a potential solution due to their superior safety, reproducibility, and speed compared to traditional viral transduction (Irving et al., Hum. Gene Ther. 2021 Oct;32(19-20):1044-1058).

[0005] However, the transposon-based protocols used to date suffer from low integration efficiency or the risk of re-excising the integrated sequence if the transposase is expressed for a long period of time. The efficiency is even lower in resting cells, such as resting T cells.

[0006] Thus, there remains an urgent need to improve the production of sufficient quantities of immune effector cells in vivo and / or in vitro, preferably in a safe and efficient manner, especially for immune effector cells that must stably express receptors.

[0007] The present invention fulfills such a need. The present invention has the particular advantage of providing immune effector cells with shorter production times, less quality testing, and / or lower costs. In particular, no additional activation step is required. Summary of the Invention

[0008] overview The present disclosure generally relates to immune effector cells comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, as well as particles and complexes useful for producing such immune effector cells. The immune effector cells, particles, and complexes can also be used in methods of treating a subject. The immune effector cells of the present disclosure are characterized in that (i) the second nucleotide sequence is not integrated into the genomic nucleic acid molecule of the immune effector cell, and / or (ii) the activator molecule is transiently expressed. If the second nucleic acid is not integrated into the genomic nucleic acid molecule, which will be evenly transmitted to daughter cells during cell division, the activator molecule is expressed for only a limited time.

[0009] Transient expression allows the activator molecule to be present for a required time period and then disappears. Transient expression can be achieved by not integrating the second nucleic acid into the genomic nucleic acid molecule, but can also be achieved by other means, such as by controlling the induction of expression from the second nucleic acid molecule.

[0010] One aspect provided by the present disclosure is an immune effector cell comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the immune effector cell, and / or (ii) the activator molecule is transiently expressed.

[0011] Without being bound by theory, providing immune effector cells with both a first antigen receptor and an activator molecule has the advantage of eliminating or incorporating an additional activation step into the production method. Furthermore, activation, particularly activation by a second antigen receptor, may aid in the overall transfection efficiency by helping the first nucleotide sequence cross the nuclear membrane and enter the nucleus, where it can be propagated by integration, particularly via transposon-based systems. Furthermore, no additional cells are required for expression of the activator molecule, providing enhanced regulatory accessibility. Because the second nucleic acid is not integrated and / or the activator molecule is transiently expressed, the activation signal is lost when no longer needed, thereby increasing the safety of the employed protocol.

[0012] In one embodiment, the immune effector cells can be isolated. The immune effector cells can be present in vitro, e.g., in cell culture or a frozen sample, or in vivo.

[0013] In one embodiment, the first nucleic acid molecule can be DNA or RNA. Preferably, the first nucleic acid molecule can be DNA. An important feature of the first nucleic acid molecule is that it can ensure stable, long-term expression of the encoded first cell surface-expressed antigen receptor.

[0014] In one embodiment, the first nucleotide sequence can be integrated into a genomic nucleic acid molecule of the immune effector cell. Preferably, the genomic nucleic acid molecule can be chromosomal, episomal, such as a non-viral episome.

[0015] In one embodiment, the first nucleotide sequence can be integrated into a genomic nucleic acid molecule, preferably a chromosome, via a DNA-based transposon system, a virus-based retrotransposon system, or a polyA-based retrotransposon system. According to this embodiment, the first nucleotide sequence can be contained within a suitable transposable element.

[0016] In one embodiment, the immune effector cell can further comprise a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, preferably a transposase.

[0017] In one embodiment, the third nucleic acid molecule may be DNA or RNA. Preferably, the third nucleic acid molecule may be RNA, more preferably mRNA.

[0018] In one embodiment, the molecule having transposase activity can be Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, Acids, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof having transposase activity. Preferably, the molecule having transposase activity is Sleeping Beauty transposase SB100X.

[0019] In one embodiment, the third nucleic acid molecule is not integrated into the genomic nucleic acid molecule of the immune effector cell, and / or the molecule encoded by the third nucleic acid molecule with transposase activity can be transiently expressed. A feature of the third nucleic acid molecule is that it is not suitable for stable long-term expression of the molecule with transposase activity. Without being bound by theory, this serves to prevent excision of the integrated first nucleotide sequence.

[0020] In one embodiment, the first cell surface expressed antigen receptor may be stably expressed.

[0021] In one embodiment, the activator molecule encoded by the second nucleotide sequence can enable the activation, expansion, differentiation, and / or proliferation of immune effector cells. Preferably, the activator molecule can be a non-coding RNA or a protein.

[0022] In one embodiment, the activator molecule can bind to the extracellular portion of a first cell surface expressed antigen receptor. Preferably, the activator molecule can be an antigen targeted / bound by the first cell surface expressed antigen receptor.

[0023] In one embodiment, the activator molecule can be a cytokine.

[0024] In one embodiment, the activator molecule can be a second cell surface expressed antigen receptor, wherein the extracellular portions of the first and second cell surface expressed antigen receptors do not bind to the same binding target.

[0025] In one embodiment, the immune effector cell can further comprise a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of a first cell surface-expressed antigen receptor, and / or the immune effector cell can further comprise a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor. In this embodiment, the fourth nucleic acid molecule or the fifth nucleic acid molecule both comprise the fourth nucleotide sequence encoding the binding target of the first cell surface-expressed antigen receptor and the fifth nucleotide sequence encoding the binding target of the second cell surface-expressed antigen receptor.

[0026] In one embodiment, the fourth and / or fifth nucleic acid molecule may be DNA or RNA. Preferably, the fourth and / or fifth nucleic acid molecule may be RNA, more preferably mRNA.

[0027] In one embodiment, the first and / or second cell surface expressed antigen receptor may be a T cell receptor (TCR), such as a chimeric antigen receptor (CAR) or an artificial T cell receptor.

[0028] In one embodiment, the binding target of the second cell surface-expressed antigen receptor can be expressed on or from a different cell than the cell expressing the binding target of the first cell surface-expressed antigen receptor.

[0029] In one embodiment, the binding target of the first cell surface-expressed antigen receptor can be a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

[0030] In one embodiment, the binding target of the second cell surface-expressed antigen receptor can be a cell surface-expressed protein or a soluble protein, or an epitope thereof. The cell surface-expressed protein can be a glycoprotein or a cell surface-expressed cytokine. Preferably, the cell surface-expressed protein can be a cluster of differentiation (CD) protein, and the soluble protein can be a soluble cytokine.

[0031] In one embodiment, the binding target of the second cell surface-expressed antigen receptor can be a cell surface protein expressed on a blood cell, which is preferably another immune effector cell. Preferably, the blood cell can be a T cell, an NK cell, a dendritic cell, a macrophage, or a B cell.

[0032] In one embodiment, the cell surface protein may be CD19 or CLDN18.2.

[0033] In one embodiment, the second nucleic acid molecule can be DNA or RNA. Preferably, the second nucleic acid molecule can be RNA, more preferably mRNA. Preferably, the RNA or mRNA comprises ribonucleobases other than A, C, G, and U. The ribonucleobase can be pseudouridine, preferably 1-methyl-pseudouridine. A characteristic of the second nucleic acid molecule is that it is not suitable for stable long-term expression of the activator molecule.

[0034] In one embodiment, the RNA can comprise a 5' cap structure. Preferably, the 5' cap structure is a naturally occurring cap or cap analog. The 5' cap structure can be one of Cap0, Cap1, Cap2, Cap3, Cap4, ARCA (Anti-Reverse Cap Analogs), modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. Preferably, the 5' cap structure is Cap0, which is m7G(5')ppp(5'), or m7G(5')ppp(5')(N1 2’-OMe In this embodiment, N1 can be selected from A, C, G, or U. Cap 1 can be a cap-proximal A, G, C, or U at position +2, and can be m7G(5')ppp(5')(N1 2’-OMe )pN2. In one embodiment, the 5' end structure is m7G(5')ppp(5')(A 2’-OMe)pGpApApU.

[0035] In one embodiment, the immune effector cells can be T cells, B cells, dendritic cells, or NK cells. Preferably, the immune effector cells are CD8+ and / or CD4+ T cells, more preferably cytotoxic T cells.

[0036] In one embodiment, the second nucleic acid may not be inherited by progeny cells of the immune effector cell in the same way that chromosomes are inherited, and / or the second nucleic acid may be diluted relative to the total number of cells in each generation of progeny cells of the immune effector cell, and / or after one cell division, the amount of the second nucleic acid molecule may be less in each daughter cell compared to the amount in the parent cell.

[0037] A further embodiment may be an immune effector cell comprising: (i) a DNA molecule comprising a transposable element comprising a nucleotide sequence encoding a first T cell receptor or chimeric antigen receptor that binds to a tumor-associated antigen; (ii) an mRNA molecule encoding a second T cell receptor or chimeric antigen receptor that binds to a target different from the tumor-associated antigen; and (iii) an mRNA molecule encoding a transposase.

[0038] A further embodiment can be an immune effector cell comprising: (i) a nucleotide sequence encoding a first T cell receptor or chimeric antigen receptor that binds to a tumor-associated antigen, the nucleotide sequence being integrated into the genome of the immune effector cell or contained within an episome present in the immune effector cell; and (ii) a nucleic acid molecule encoding a second T cell receptor or chimeric antigen receptor that binds to a target different from the tumor-associated antigen, the nucleic acid molecule not being integrated into the genomic nucleic acid molecule of the immune effector cell.

[0039] A further embodiment may be an immune effector cell comprising: (i) a DNA sequence encoding a first chimeric antigen receptor that binds to a tumor-associated antigen, the DNA sequence being integrated into the genome of the immune effector cell; and (ii) an mRNA molecule encoding a second chimeric antigen receptor that binds to a target different from the tumor-associated antigen.

[0040] In one embodiment, the immune effector cells can be CD8+ cytotoxic T cells.

[0041] In one embodiment, an immune effector cell can be activated by binding of a second T cell receptor (TCR) or chimeric antigen receptor (CAR) to its target.

[0042] In one embodiment, the immune effector cell may not comprise a DNA nucleotide sequence encoding an activator molecule.

[0043] In one embodiment, the immune effector cells can have reduced cell surface expression of an endogenous T cell receptor to a level that prevents graft-versus-host activity of the immune effector cells when administered to a subject different from the subject from which the immune effector cells were derived. Preferably, the immune effector cells do not express that endogenous T cell receptor on their cell surface.

[0044] In one embodiment, the immune effector cells are capable of reducing cell surface expression of endogenous HLA complexes to a level that prevents host-versus-graft activity in a subject to which the immune effector cells are administered, wherein the subject to which the immune effector cells are administered is different from the subject from which the immune effector cells were derived. Preferably, the immune effector cells do not express the endogenous HLA complexes on their cell surface.

[0045] A further aspect is a cell composition comprising immune effector cells according to the present disclosure. The cell composition can further comprise a cryopreservation agent.

[0046] A further aspect is a pharmaceutical composition comprising an immune effector cell according to the present disclosure or a cell composition according to the present disclosure and a pharmaceutically acceptable carrier.

[0047] In one embodiment, the immune effector cell according to the present disclosure, the cell composition according to the present disclosure, or the pharmaceutical composition according to the present disclosure is for use in a method of treating a subject having a disease, disorder, or condition associated with expression or up-regulation of a binding target of a first cell surface-expressed antigen receptor, the method comprising administering the immune effector cell, cell composition, or pharmaceutical composition to the subject. Preferably, the disease, disorder, or condition is cancer, and preferably, the cancer is a solid cancer.

[0048] In one embodiment, in the immune effector cells, cell compositions, or pharmaceutical compositions for use according to the present disclosure, the disease, disorder, or condition may be an infectious disease, and preferably, the infectious disease is a viral infection.

[0049] In one embodiment, in the immune effector cells, cell compositions, or pharmaceutical compositions for use according to the present disclosure, the immune effector cells are autologous or heterologous to the subject to which the immune effector cells, cell composition, or pharmaceutical composition is administered.

[0050] All embodiments that apply to immune effector cells equally apply to the particles, conjugates and methods disclosed herein.

[0051] A further embodiment is a particle comprising: (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, the first nucleotide sequence being comprised within a transposable element; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, the second nucleotide sequence not being comprised within a transposable element; preferably, the particle further comprises a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, preferably a transposase, the third nucleotide sequence not being comprised within a transposable element. Preferably, the first nucleic acid molecule or episome is a DNA minicircle or a linear DNA molecule.

[0052] In one embodiment, the particle can comprise (i) a DNA episome, preferably a non-viral episome, comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, the second nucleic acid molecule providing transient expression of the activator molecule when present in the cell. Preferably, the first nucleic acid molecule can be DNA or RNA.

[0053] In one embodiment of a particle according to the present disclosure, the transposable element in the first nucleic acid may be derived from a DNA-based transposon system, a viral-based transposon system, or a polyA-based retrotransposon system.

[0054] In one embodiment of the particle according to the present disclosure, the third nucleic acid molecule can be DNA or RNA. Preferably, the third nucleic acid molecule is RNA, more preferably mRNA.

[0055] In one embodiment of the particle according to the present disclosure, the molecule having transposase activity can be Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, Acids, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof having transposase activity. Preferably, the molecule having transposase activity is Sleeping Beauty transposase SB100X.

[0056] In one embodiment of the particle according to the present disclosure, the second nucleic acid molecule can be DNA or RNA. Preferably, the second nucleic acid molecule is RNA, more preferably mRNA.

[0057] In one embodiment of a particle according to the present disclosure, the first cell surface expressed antigen receptor is capable of binding to a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

[0058] In one embodiment of a particle according to the present disclosure, the first cell surface expressed antigen receptor can be a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0059] In one embodiment of a particle according to the present disclosure, the activator molecule enables the activation, expansion, differentiation, and / or proliferation of immune effector cells. The activator molecule can be a non-coding RNA or a protein.

[0060] In one embodiment of a particle according to the present disclosure, the activator molecule is capable of binding to the extracellular portion of a first cell surface-expressed antigen receptor. Preferably, the activator molecule is an antigen targeted by the first cell surface-expressed antigen receptor.

[0061] In one embodiment of a particle according to the present disclosure, the activator molecule can be a cytokine.

[0062] In one embodiment of a particle according to the present disclosure, the activator molecule can be a second cell surface-expressed antigen receptor, wherein the extracellular portions of the first and second cell surface-expressed antigen receptors do not bind to the same binding target. Preferably, the second cell surface-expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0063] In one embodiment, the particle can further comprise a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of a first cell surface-expressed antigen receptor, where the fourth nucleotide sequence is not contained within a transposable element, or the particle can further comprise a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor, where the fifth nucleotide sequence is not contained within a transposable element. Preferably, the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises both the fourth nucleotide sequence encoding the binding target of the first cell surface-expressed antigen receptor and the fifth nucleotide sequence encoding the binding target of the second cell surface-expressed antigen receptor.

[0064] In one embodiment of the particle according to the present disclosure, the fourth and / or fifth nucleic acid molecule may be DNA or RNA. Preferably, the fourth and / or fifth nucleic acid molecule is RNA, more preferably mRNA.

[0065] In one embodiment, the particle may comprise a first, second and third nucleic acid molecule as defined herein. Preferably, the particle further comprises a fourth and / or fifth nucleic acid molecule as defined herein.

[0066] In one embodiment, the particle may comprise a first, second, third and fourth nucleic acid molecule as defined herein. Preferably, the particle further comprises a fifth nucleic acid molecule as defined herein.

[0067] In one embodiment, the particles may comprise a polyalkyleneimine or a lipid. Preferably, the particles comprise a lipid, preferably a lipid with a cationic head group and / or a pH-responsive lipid and / or a pegylated lipid.

[0068] In one embodiment, the particles can be lipid particles, polymer particles, or a mixture thereof.

[0069] In one embodiment, the particles may be nanoparticles.

[0070] In one embodiment, the particle can be a lipid nanoparticle (LNP), lipoplex (LPX), polyplex (PLX), or lipopolyplex (LPLX) particle.

[0071] In one embodiment, the particles can further comprise at least one phosphatidylserine.

[0072] In one embodiment, the particles may be nanoparticles, wherein: (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) The zeta potential of the nanoparticles is less than or equal to 0. Optionally, or alternatively, the charge ratio of positive to negative charges in the nanoparticles can be less than or equal to 1.4:1.

[0073] In one embodiment, the particles may comprise a polyalkyleneimine, and preferably (a) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) may be 2.0 to 15.0, preferably 6.0 to 12.0; or (b) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) is at least about 48, optionally about 48 to 300, about 60 to 200, or about 80 to 150, or preferably, the ionic strength of the composition is 50 mM or less, and preferably, the concentration of monovalent cations is 25 mM or less and the concentration of divalent cations is 20 μM or less.

[0074] In one embodiment, the particles can be polyplex particles.

[0075] In one embodiment, the particle can include a hydrophobic moiety having a covalently attached binding moiety, preferably, the hydrophobic moiety having a covalently attached binding moiety and the particle are non-covalently attached to each other. The hydrophobic moiety having a covalently attached binding moiety can be an integral part of the particle. Preferably, the hydrophobic moiety having a covalently attached binding moiety comprises a polymer.

[0076] In one embodiment, the hydrophobic moiety having a covalently attached binding moiety is a compound of Formula I L-X1-P-X2-B (I) and During the ceremony, P comprises a polymer; L comprises a hydrophobic moiety attached to a first end of the polymer; B comprises a linking moiety attached to the second end of the polymer; X1 is absent or a first linking moiety; X2 is absent or a second linking moiety, preferably X1 comprises a carbonyl group and / or preferably X2 comprises the reaction product of a maleimide group with a thiol or cysteine ​​group of the compound comprising the linking moiety.

[0077] In one embodiment, the hydrophobic moiety may be a lipid or may be comprised in a lipid.

[0078] In one embodiment, the polymer can provide stealth properties, can increase circulatory half-life, and / or can reduce non-specific protein binding or cell adhesion.

[0079] In one embodiment, the polymer can include polyethylene glycol (PEG).

[0080] In one embodiment of the particle according to the present disclosure, the hydrophobic moiety having a covalently attached binding moiety is a compound of formula II TIFF2026504516000002.tif28170, wherein B comprises a linking moiety, preferably B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.

[0081] In one embodiment, the binding moiety covalently attached to the hydrophobic moiety can comprise an antibody or antibody derivative.

[0082] In one embodiment, the particles are complexed with and / or encapsulate nucleic acid molecules.

[0083] A further aspect is a pharmaceutical composition comprising the particles described herein and a pharmaceutically acceptable carrier.

[0084] In one embodiment, a particle or pharmaceutical composition according to the present disclosure may be for use in a method of treating a subject having a disease, disorder or condition associated with expression or up-regulation of a binding target of a first cell surface-expressed antigen receptor, the method comprising administering the particle or pharmaceutical composition to the subject, preferably wherein the disease, disorder or condition is cancer, and the cancer is preferably a solid cancer.

[0085] In one embodiment, the particle or pharmaceutical composition may be for use according to the present disclosure and the disease, disorder or condition may be an infectious disease, preferably the infectious disease is a viral infection.

[0086] A further aspect of the present disclosure is A conjugate comprising: (a) a particle according to the present disclosure, the particle comprising a hydrophobic portion having a covalently bound binding moiety; and (b) a compound comprising (i) a moiety that binds to the binding moiety covalently bound to the hydrophobic portion, and (ii) a moiety that targets a cell surface antigen, preferably wherein the moiety that binds to the binding moiety covalently bound to the hydrophobic portion comprises an antibody or antibody derivative, preferably wherein the binding moiety covalently bound to the hydrophobic portion comprises a peptide comprising an ALFA-tag; or wherein the moiety that binds to the binding moiety covalently bound to the hydrophobic portion comprises an antibody or antibody derivative, preferably a nanobody comprising a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY. Preferably, (i) the moiety that binds to the binding moiety covalently bound to the hydrophobic portion and (ii) the moiety that targets a cell surface antigen are linked to each other.

[0087] In one embodiment, the compound in (b) can comprise a peptide or polypeptide.

[0088] In one embodiment, the moiety that targets the cell surface antigen may comprise an antibody or antibody derivative.

[0089] In one embodiment, the cell surface antigen may be characteristic of an immune effector cell. Preferably, the cell surface antigen comprises CD4 and / or CD8 and / or CD3.

[0090] In one embodiment, a conjugate according to the present disclosure may be for use in treating a subject having a disease, disorder, or condition associated with expression or up-regulation of a binding target of a first cell surface-expressed antigen receptor.

[0091] A further embodiment is a method of producing immune effector cells expressing a first antigen receptor on their cell surface, comprising contacting the immune effector cells with (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding the first cell surface-expressed antigen receptor, and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not contained within a transposable element, and the first cell surface-expressed antigen receptor is stably expressed in the cell and the activator molecule is transiently expressed in the cell. Preferably, the first nucleic acid molecule is DNA or RNA.

[0092] In one embodiment, the method can further comprise incorporating the first nucleotide sequence into a genomic nucleic acid molecule of the immune effector cell. Preferably, the first nucleotide sequence is comprised within a transposable element.

[0093] In one embodiment, the method may further comprise contacting the immune effector cell with a molecule having transposase activity, preferably a third nucleic acid molecule comprising a third nucleotide sequence encoding a transposase, wherein the third nucleotide sequence is not contained within a transposable element. Preferably, the third nucleic acid molecule is DNA or RNA. Preferably, the third nucleic acid molecule is RNA, more preferably mRNA. A characteristic of the third nucleic acid molecule is that it is not suitable for stable long-term expression of a molecule having transposase activity.

[0094] In one embodiment, the method can further comprise contacting the immune effector cell with a fourth and / or fifth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of the first and / or second cell surface-expressed antigen receptor. Preferably, the fourth or fifth nucleic acid molecule comprises both a fourth nucleotide sequence encoding a binding target of the first cell surface-expressed antigen receptor and a fifth nucleotide sequence encoding a binding target of the second cell surface-expressed antigen receptor. The fourth and / or fifth nucleic acid molecule can be DNA or RNA. Preferably, the fourth and / or fifth nucleic acid molecule is RNA, more preferably mRNA.

[0095] In one embodiment, the first cell surface expressed antigen receptor and / or the second cell surface expressed antigen receptor may be a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0096] In one embodiment of the method for producing immune effector cells, the binding target of the second cell surface-expressed antigen receptor is expressed on or from a different cell than the cell expressing the binding target of the first cell surface-expressed antigen receptor. The binding target of the first cell surface-expressed antigen receptor can be a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

[0097] In one embodiment of the method for producing immune effector cells, the second nucleic acid molecule is DNA or RNA. Preferably, the second nucleic acid molecule is RNA, more preferably mRNA.

[0098] In one embodiment of the method for producing immune effector cells, the RNA comprises a 5' cap structure, preferably the 5' cap structure is a naturally occurring cap or a cap analog.

[0099] In one embodiment of the method for producing immune effector cells, the immune effector cells are T cells, B cells, dendritic cells, or NK cells. Preferably, the immune effector cells are CD8+ and / or CD4+ T cells, preferably cytotoxic T cells.

[0100] In one embodiment of the method of producing immune effector cells, the second nucleic acid is not inherited by progeny cells of the immune effector cell in the same way that chromosomes are inherited, and / or the second nucleic acid may be diluted in each generation of progeny cells of the immune effector cell, and / or after one cell division, the amount of the second nucleic acid molecule may be less in each daughter cell compared to the amount in the parent cell.

[0101] In one embodiment, the method comprises contacting an immune effector cell with a particle or complex according to the present disclosure. Preferably, the contacting occurs in vitro. The method can further comprise, after contacting the nucleic acid molecule with the immune effector cell, contacting the immune effector cell with a binding target of, or a cell expressing, a binding target of, a second cell surface-expressed antigen receptor.

[0102] In one embodiment, the method can be a method for producing immune effector cells that express two antigen receptors on their cell surface, comprising contacting immune effector cells in vitro or ex vivo with: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is contained in a transposable element; (ii) an RNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor, wherein the second nucleotide sequence is not contained in a transposable element; and (iii) an RNA molecule comprising a third nucleotide sequence encoding a transposase, wherein the third nucleotide sequence is not contained in a transposable element; wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets, preferably, the binding target of the first cell surface-expressed antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface-expressed antigen receptor is expressed on the surface of a blood cell. Preferably, the method further comprises contacting the immune effector cell with a binding target of a second cell surface-expressed antigen receptor or with a cell expressing a binding target of a second cell surface-expressed antigen receptor.

[0103] In one embodiment, the method can be a method for producing immune effector cells expressing two antigen receptors on their cell surface, comprising contacting the immune effector cells with particles comprising: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, the first nucleotide sequence being contained in a transposable element; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets, preferably, the binding target of the first cell surface-expressed antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface-expressed antigen receptor is expressed on the surface of a blood cell. Preferably, the contacting is performed in vivo.

[0104] A further aspect of the present disclosure is a method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition, and (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of a cell of the subject or is contained within an episome present in the cell of the subject, and / or (ii) the activator molecule is transiently expressed in the subject. Preferably, the nucleic acid molecule is within a particle comprising lipid.

[0105] In one embodiment, the method may be a method for treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject particles comprising: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is contained in a transposable element, and wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; and wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets.

[0106] In one embodiment, the method may be a method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject an immune effector cell according to the present disclosure, a cell composition according to the present disclosure, or a pharmaceutical composition according to the present disclosure, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

[0107] In one embodiment, the method may be a method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a particle according to the present disclosure or a pharmaceutical composition according to the present disclosure, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

[0108] In one embodiment, the method may be a method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a conjugate according to the present disclosure, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

[0109] In one embodiment of the method of treating a subject, the antigen associated with the disease, disorder, or condition can be a tumor-associated antigen. Preferably, the method is for treating or preventing cancer in a subject.

[0110] In one embodiment of the method of treating a subject, the antigen associated with the disease, disorder, or condition comprises an antigen of an infectious agent. Preferably, the infectious agent is a virus.

[0111] In one embodiment of the method of treating a subject, the method is for treating or preventing an infection in a subject. [Brief explanation of the drawings]

[0112] [Figure 1] Figure 1 shows four potential approaches using three-component (3C) and four-component (4C) systems to generate CAR-T cells from inactivated T cells using the Sleeping Beauty transposon system. Top half: Components required for each approach. Bottom half: Expression of cargo components encoded by DNA or RNA molecules supplied to the cells at different times after delivery to the cells and potential interactions between them. 2C = Two-component system containing only a transposon and transposase. i) A 3C system further containing an RNA-encoded receptor that is stimulated by an exogenously supplied soluble ligand. ii) A 2C system (also referred to in some embodiments as a transCAR system or protocol) further containing RNA encoding a second antigen receptor used to stimulate the cells upon contact with ligand-expressing cells. iii) A 2C system further containing RNA encoding a target of the DNA-encoded CAR. iv) A 4C system: A 2C system further containing RNA encoding the CAR and RNA encoding the antigen targeted by the CAR. [Figure 2] Figure 2 shows a detailed overview of an embodiment of the transCAR protocol for generating CAR-T cells in vivo. Nanoparticles (NPs) functionalized with T cell targeting ligands trigger T cell-specific NP cargo uptake via receptor-mediated endocytosis. [Figure 3A] Figure 3 shows data generated during a study of an embodiment of the transCAR protocol. A) Non-activated primary human CD8+ T cells transfected with CD8-targeted nanoparticles (NPs) readily express the transfected mRNA. NP-mediated delivery of CAR mRNA (CLDN18.2-CAR-RNA) to CD8+ T cells allows induction of CAR-specific, antigen-mediated T cell activation and proliferation when transfected T cells are co-cultured with autologous immature dendritic cells (iDCs) expressing the CAR antigen (CLDN18.2) [black / right bar]. [Figure 3B] Figure 3 shows data generated during a study of an embodiment of the transCAR protocol. B) When a functional transposon system consisting of RNA encoding SB100X and Minicircle (MC) encoding the CLDN6 CAR is co-delivered to T cells via NP along with CAR mRNA, proliferating CD8+ T cells increase CLDN6 CAR expression over time [black bars]. The observed CLDN6 CAR expression is the result of successful transposition in CD8+ T cells, as indicated by the lack of expression in the corresponding control group in which the mRNA encoding SB100X was replaced with filler RNA (mRNA encoding Thy1.1). [Figure 3C] Figure 3 shows data generated during a study of an embodiment of the transCAR protocol. C) The generated CLDN6 CAR-T cells efficiently eradicated CLDN6-expressing 3D tumor spheroids, while CLDN6-negative tumor spheroids remained intact, demonstrating high antigen-specific anti-tumor function. [Figure 4A]Figure 4 shows a schematic of a two-day transposon-based protocol for generating CAR-T cells by electroporation, which delivers a three-component nucleic acid cocktail into cells. A) The starting substrate can be peripheral blood mononuclear cells (PBMCs) or isolated naive human T cells. The three components are transposon donor DNA carrying the CAR expression cassette within a transposable element, flanked by transposon end sequences, transposase mRNA, and mRNA encoding the CAR's cognate antigen. By day 2 after electroporation, the engineered T cells express a CAR that can recognize antigens expressed on surrounding cells. During incubation, this specific CAR-antigen interaction triggers a signaling pathway that leads to T cell activation and expansion. [Figure 4B] Figure 4 shows a schematic of a two-day transposon-based protocol for generating CAR-T cells by electroporation, delivering a three-component nucleic acid cocktail into cells. A) Comparison of CAR expression on days 2 and 8 after electroporation of naive human T cells with either a three-component (+DNA / +SB / +Ag), two transposon system components (+DNA / +SB / -Ag), or, as a negative control, a two-component protocol consisting of transposon DNA carrying the CAR and cognate antigen mRNA but without transposase (+DNA / -SB / +Ag). Equivalent CAR expression between the three-component and two-component protocols is typically observed on day 2. As the process continues, CAR expression levels in the three-component protocol exceed those in the two-component protocol by day 8. [Figure 4C]Figure 4 shows a schematic of a two-day transposon-based protocol for generating CAR-T cells by electroporation, delivering a three-component nucleic acid cocktail into cells. C) Activation of CAR-T cells in the presence of cognate antigen in a three-component setting (InstaCAR). The InstaCAR protocol, performed using isolated naive human T cells as the starting substrate, leads to antigen-specific activation of CAR-T cells on day 2, as monitored by CD25 upregulation and the resulting coexpression of CAR and CD25 on T cells. This trend persists on day 6, with T cells coexpressing CAR and CD25 electroporated with three components (+DNA / +SB / +Ag) outnumbering cells receiving only two components (+DNA / +SB / -Ag), demonstrating that stimulation with the three-component protocol increases the number of engineered T cells. The negative control does not receive transposase RNA (+DNA / -SB / +Ag) and does not express CAR. [Figure 4D] Figure 4 shows a schematic of a two-day transposon-based protocol for generating CAR-T cells by electroporation, delivering a three-component nucleic acid cocktail into cells. D) T cell activation is specifically observed in the three-component protocol. In the same experiment as in Figure 4C, CD25 expression on T cells is quantified by mean fluorescence intensity (MFI). High CD25 expression levels are observed only in the three-component protocol (+DNA / +SB / +RNA). In the two-component protocol, CD25 expression remains low and at basal levels (mock sample: -DNA / -SB / -Ag) even in the absence of transposase (+DNA / -SB / -Ag) and in the case of transposition of unrelated DNA (irrDNA, a transposon encoding the Venus fluorescent protein). [Figure 5]Figure 5 shows the InstaCAR protocol, which resulted in detectable CLDN6 CAR expression two days after electroporation and subsequent expansion of CLDN6 CAR-T cells. Freshly isolated CD3+ T cells (5 x 106) were electroporated in a 100 μL electroporation reaction. Cells were electroporated with either 3 components (+DNA / +SB / +Ag, also referred to as InstaCAR) encoding 2.5 μg of nanoplasmid encoding CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (+DNA / +SB / +Ag, also referred to as the reference protocol), or without SB100X transposase (+DNA / -SB / +Ag). Mock control cells were electroporated without nucleic acid (-DNA / -SB / -Ag). A) Efficiency of CLDN6 CAR transduction assessed by flow cytometry. CLDN6 CAR expression in live CD3+ T cells from six donors at multiple time points after electroporation is shown. B) Proliferative potential of CLDN6 CAR-T cells generated with the InstaCAR protocol (+DNA / +SB / +Ag) compared with that of CLDN6 CAR-T cells generated with the reference protocol (+DNA / +SB / -Ag). Engineered T cells were counted at multiple time points after electroporation using a Cellaca counter device and propidium iodide live / dead stain. The number of CAR+ T cells is shown. C) Determination of CAR copy number in InstaCAR protocol T cells by droplet digital PCR assay analysis of genomic DNA 13 days after electroporation. Data represent mean ± SD from different donors. CAR, chimeric antigen receptor; CLDN6, claudin 6; SB, Sleeping Beauty; Ag, antigen. [Figure 6A]Figure 6 shows that CLDN6 InstaCAR-T cells upregulate CAR expression after dilution with autologous PBMCs and cryopreservation. Freshly isolated CD3+ naive T cells (5 x 106) were electroporated in a 100 μL electroporation reaction with either 2.5 μg of nanoplasmid encoding the CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (referred to as +DNA / +SB / +Ag, also referred to as InstaCAR), or without SB100X transposase (referred to as +DNA / -SB / +Ag). Mock control cells were electroporated without nucleic acid (referred to as -DNA / -SB / -Ag). A) CAR expression after dilution with CFSE-labeled autologous PBMCs: On day 2 after electroporation, InstaCAR-T cells were cocultured with autologous PBMCs at a 1:10 ratio of InstaCAR cells:PBMCs (to mimic the dilution of InstaCAR-T cells in the blood after adoptive cell transfer, in which approximately 85–100 × 10 T cells are transferred to patients). The effect of cell dilution on CAR expression was assessed by flow cytometry at different time points using unmanipulated autologous PBMCs. PBMCs used as diluents in the coculture were identified by CFSE and excluded from the analysis. CAR expression was measured by flow cytometry at the indicated time points. Data represent the mean ± SD from two representative donors. Ag, antigen; CAR, chimeric antigen receptor; CFSE, carboxyfluorescein succinimidyl ester; CLDN6, claudin 6; DOT, day of thawing; HS, human serum; PBMC, peripheral blood mononuclear cells; SB, Sleeping Beauty. [Figure 6B]Figure 6 shows that CLDN6 InstaCAR-T cells upregulate CAR expression after dilution with autologous PBMCs and cryopreservation. Freshly isolated CD3+ naive T cells (5 x 106) were electroporated in a 100 μL electroporation reaction with either 2.5 μg of nanoplasmid encoding the CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (referred to as +DNA / +SB / +Ag, also referred to as InstaCAR), or without SB100X transposase (referred to as +DNA / -SB / +Ag). Mock control cells were electroporated without nucleic acid (referred to as -DNA / -SB / -Ag). B) CAR expression after freeze / thaw cycles. Two days after electroporation, InstaCAR-T cells were frozen and thawed. After thawing, they were cultured for an additional 11 days in X-VIVO15 medium (Lonza) supplemented with 5% human serum (HS), IL-7 (500 U / mL), and IL-15 (5,000 U / mL). CAR expression was measured by flow cytometry at the indicated time points. Data represent the mean ± SD from two representative donors. Ag, antigen; CAR, chimeric antigen receptor; CFSE, carboxyfluorescein succinimidyl ester; CLDN6, claudin 6; DOT, day of thawing; HS, human serum; PBMC, peripheral blood mononuclear cells; SB, Sleeping Beauty. [Figure 7]Figure 7 shows that CLDN6 InstaCAR-T cells retain viability and proliferative capacity after freeze / thaw cycles. Freshly isolated CD3+ naive T cells (5 x 106) from two donors were electroporated in a 100 μL electroporation reaction with either 2.5 μg of nanoplasmid encoding the CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (+DNA / +SB / +Ag, referred to as InstaCAR) or without SB100X transposase (+DNA / -SB / +Ag). Mock control cells were electroporated without nucleic acid (-DNA / -SB / -Ag). A-B) Percentage of viable cells determined by flow cytometry using a flexible live / dead marker at 48 hours after electroporation and at multiple time points after freeze / thaw. The percentage of viable cells was determined by including all events (A) or by using a clinically relevant product gating strategy (B). In the latter case, cellular debris that does not trigger clotting or an immune response is excluded, in contrast to the gating strategy used in (A). Data represent the mean ± SD from two representative donors. DOT, day of thaw. [Figure 8]Figure 8 shows that CLDN6 InstaCAR-T retains its proliferative potential even after freeze / thaw cycles. Freshly isolated CD3+ naive T cells (5 x 106) from two donors were electroporated in a 100 μL electroporation reaction with either 3 components: 2.5 μg of nanoplasmid encoding CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (referred to as +DNA / +SB / +Ag, also referred to as InstaCAR), only 2 components (referred to as +DNA / +SB / -Ag, reference protocol), or without SB100X transposase (referred to as +DNA / -SB / +Ag). A) Kinetics of CLDN6 InstaCAR-T cell expansion after 1.5 x 106 cells were frozen 2 days after electroporation, subsequently thawed, and cultured for an additional 16 days in X-VIVO15 medium supplemented with 5% human serum, IL-7 (500 U / mL), and IL-15 (5,000 U / mL). T cells were counted at the indicated time points using Cellaca and propidium iodide live / dead staining. B) Proliferative potential after co-culture of frozen CDLN6 InstaCAR-T cells with target tumor cells at a 1:1 effector-to-target ratio without exogenous cytokines on the day of thawing. Absolute InstaCAR-T cell numbers were calculated by flow cytometry using counting beads. Data represent the mean ± SD from two representative donors. DOF, day of freezing; DOT, day of thawing; CAR, chimeric antigen receptor; CLDN6, claudin 6; Ag, antigen. [Figure 9]Figure 9 shows that CLDN6 InstaCAR-T cells exhibit antigen-specific, sequential tumor-killing activity in vitro after thawing. Freshly isolated CD3+ naive T cells (5 x 106) from two donors were electroporated in a 100 μL electroporation reaction with either 2.5 μg of nanoplasmid encoding the CLDN6 CAR, 12.5 μg of mRNA-encoded SB100X transposase, and 2.5 μg of mRNA-encoded CLDN6 antigen (referred to as +DNA / +SB / +Ag, also referred to as InstaCAR), or with only the two components (referred to as +DNA / +SB / -Ag, reference protocol), or without the SB100X transposase (referred to as +DNA / -SB / +Ag). Cells were frozen two days after electroporation. On the day of thawing, cells were cocultured with PA-1 / CLDN6+ target tumor cells stably expressing firefly luciferase at different effector:target (E:T) ratios. T cells were used without normalization to CAR expression levels. Cells were challenged with tumor cells on the day of thawing (A), re-challenged with fresh tumor cells 7 days after the first challenge (B), and challenged with fresh tumor cells again 120 hours after the second challenge (C). Killing efficacy at the indicated time points (A), (B), and (C) is shown. Cytotoxicity was assessed using a bioluminescence-based assay. Data points represent the average % tumor cell killing using CLDN6 CAR-T cells from two donors. CLDN6, Claudin 6; SB, Sleeping Beauty; Ag, antigen. DETAILED DESCRIPTION OF THE INVENTION

[0113] Detailed Description Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0114] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0115] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature of the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0116] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of elements described in this application should be deemed to be disclosed by this description unless the context dictates otherwise.

[0117] The term "about" means approximately or roughly, and in the context of numerical values ​​or ranges described herein, preferably means + / - 10% of the stated or claimed numerical value or range.

[0118] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," and "the" and similar references are intended to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0119] Unless expressly stated otherwise, the term "comprising" is used in the context of this specification to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising." However, as a specific embodiment of the present invention, the term "comprising" is intended to encompass the possibility that no further members are present, i.e., for the purposes of this embodiment, "comprising" is understood to have the meaning of "consisting of."

[0120] The expression of a relative amount of a component characterized by a general term is intended to refer to the total amount of all specific variants or members encompassed by that general term. When a specific component defined by a general term is specified to be present in a certain relative amount, and this component is further characterized as a specific variant or member encompassed by that general term, it means that there are no additional variants or members encompassed by that general term such that the sum of the relative amounts of the components encompassed by that general term exceeds the specified relative amount; more preferably, it means that there are no additional variants or members encompassed by that general term at all.

[0121] Several documents are cited throughout the body of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure.

[0122] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, and most preferably by 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0123] Terms such as "increase" or "enhancement" preferably relate to an increase or enhancement of at least about 10%, preferably at least about 20%, preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 80%, and most preferably at least about 100%.

[0124] The term "net charge" refers to the overall charge of an object, such as a compound or particle.

[0125] Ions with an overall net positive charge are cations, and ions with an overall net negative charge are anions. Thus, according to the present invention, anions are ions with more electrons than protons, giving them a net negative charge, and cations are ions with fewer electrons than protons, giving them a net positive charge.

[0126] Terms such as "charged," "net charge," "negatively charged," or "positively charged," in reference to a given compound or particle, refer to the net electrical charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0127] The term "nucleic acid" according to the present invention also includes nucleic acids chemically derivatized at the nucleotide base, sugar, or phosphate, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Generally, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid that is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules, and chemically synthesized molecules. According to the present invention, nucleic acids can be in the form of single- or double-stranded linear molecules or covalently closed circular molecules.

[0128] According to the present invention, a "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to an entire nucleic acid molecule (e.g., a single strand of an entire nucleic acid molecule) or to a portion thereof (e.g., a fragment).

[0129] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues, preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material to the end or internally of the RNA, for example, to one or more nucleotides of the RNA. Nucleotides within an RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs, particularly analogs of naturally occurring RNA.

[0130] According to the present invention, RNA can be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule that is not bound to a complementary nucleic acid molecule (typically a complementary RNA molecule). Single-stranded RNA can contain self-complementary sequences that allow portions of the RNA to fold back and form secondary structural motifs, such as, but not limited to, base pairs, stems, stem-loops, and bulges. Single-stranded RNA exists as a minus strand [(-) strand] or a plus strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. Genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can directly serve as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules that combine with each other to form double-stranded RNA ("duplex RNA").

[0131] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the period required to remove half of the activity, amount, or number of a molecule. In the context of the present invention, the half-life of an RNA is an indicator of the stability of that RNA. The half-life of an RNA can affect the "expression period" of the RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0132] The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a specific period of time.

[0133] The term "fragment" in reference to a nucleic acid sequence refers to a portion of the nucleic acid sequence, i.e., a sequence representing a nucleic acid sequence truncated at the 5' and / or 3' end. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention, fragments of RNA molecules that retain the stability and / or translation efficiency of the RNA are preferred.

[0134] The term "fragment" in reference to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame (as long as the truncated open reading frame contains an initiation codon that serves to initiate translation). A fragment of an amino acid sequence may, for example, contain at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence.

[0135] The term "variant" according to the present invention, e.g., with respect to nucleic acid and amino acid sequences, includes any variant, particularly mutants, viral strain variants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unknown. Complete gene sequencing often identifies multiple allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, which according to the present invention are nucleic acids whose codon sequence differs from that of a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence derived from a different species than the given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence derived from a different virus than the given nucleic acid or amino acid sequence.

[0136] Nucleic acid variants include deletions, additions, mutations, substitutions, and / or insertions of single or multiple nucleotides compared to a reference nucleic acid. Deletions include removing one or more nucleotides from a reference nucleic acid. Addition variants include 5'- and / or 3'-terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. Substitutions involve removing at least one nucleotide in a sequence and inserting at least one other nucleotide in its place (e.g., transversions and transitions). Mutations include abasic sites, crosslinked sites, and chemically altered or modified bases. Insertions involve adding at least one nucleotide to a reference nucleic acid.

[0137] According to the present invention, a "nucleotide change" can refer to a deletion, addition, mutation, substitution, and / or insertion of a single or multiple nucleotides compared to a reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a single nucleotide deletion, a single nucleotide addition, a single nucleotide mutation, a single nucleotide substitution, and / or a single nucleotide insertion compared to the reference nucleic acid. According to the present invention, a nucleic acid variant can contain one or more nucleotide changes compared to the reference nucleic acid.

[0138] A variant of a particular nucleic acid sequence preferably has at least one functional property of the particular sequence, and is preferably functionally equivalent to the particular sequence, e.g., a nucleic acid sequence that exhibits properties that are identical or similar to those of the particular nucleic acid sequence.

[0139] As described below, some embodiments of the present invention are characterized, inter alia, by nucleic acid sequences that are homologous to other nucleic acid sequences. These homologous sequences are variants of the other nucleic acid sequences.

[0140] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence will be at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably provided over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In preferred embodiments, the degree of identity is provided over the entire length of the reference nucleic acid sequence.

[0141] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.

[0142] The term "% identical" is intended to refer in particular to the percentage of nucleotides that are identical in optimal alignment between the two sequences being compared, said percentage being purely statistical; the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the compared sequence may contain additions or deletions compared to the reference sequence in order to obtain optimal alignment between the two sequences. Comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2:482, the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, or computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0143] The percentage identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions compared, and multiplying this result by 100.

[0144] For example, the BLAST program "BLAST 2 sequences" available at the website (http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi) can be used.

[0145] A nucleic acid is "capable of hybridizing" or "hybridizes" to another nucleic acid if the two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if the two sequences can form a stable duplex with each other. According to the present invention, hybridization is preferably performed under conditions that allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in "Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989" or "Current Protocols in Molecular Biology, F.M.A.usubel et al., Editors, John Wiley & Sons, Inc., New York," and refer to hybridization at 65°C in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride / 0.15M sodium citrate, pH 7. After hybridization, the membrane onto which the DNA has been transferred is washed, for example, with 2xSSC at room temperature, and then washed with 0.1 to 0.5xSSC / 0.1xSDS at a temperature of up to 68°C.

[0146] Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, or 100% complementary). "Fully complementary" or "fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0147] The term "derivative" includes any chemical derivatization of a nucleic acid at a nucleotide base, sugar, or phosphate. The term "derivative" also includes nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs. Preferably, derivatization of a nucleic acid increases its stability.

[0148] A "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid derived from that nucleic acid sequence. Preferably, a sequence that is variant to a particular sequence retains RNA stability and / or translation efficiency when substituted for the particular sequence in an RNA molecule.

[0149] "nt" is an abbreviation for a nucleotide; or multiple nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0150] According to the present invention, the term "codon" refers to a triplet of bases in a coding nucleic acid that specifies which amino acid is to be added next during protein synthesis in the ribosome.

[0151] The terms "transcription" and "transcribe" refer to the process in which a nucleic acid molecule ("nucleic acid template") having a specific nucleic acid sequence is read by an RNA polymerase, which produces a single-stranded RNA molecule. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template can be DNA, but, for example, in the case of transcription from an alphavirus nucleic acid template, the template is typically RNA. The transcribed RNA can then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied to produce the transcript. These cloning vectors are generally named transcription vectors and are encompassed by the term "vector" according to the present invention. The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter controlling the transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0152] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complement of the template.

[0153] According to the present invention, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed.

[0154] "A nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer, where appropriate, to a nucleic acid sequence as part of an entire RNA molecule that is the product of transcription of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0155] According to the present invention, the "3' end of a nucleic acid" refers to the end having a free hydroxyl group. When a double-stranded nucleic acid, particularly DNA, is depicted in a diagram, the 3' end is always on the right side. According to the present invention, the "5' end of a nucleic acid" refers to the end having a free phosphate group. When a double-stranded nucleic acid, particularly DNA, is depicted in a diagram, the 5' end is always on the left side. 5' end 5'--P-NNNNNNN-OH-3' 3' end 3'-HO-NNNNNNN-P--5'

[0156] "Upstream" describes the relative positioning of a first element of a nucleic acid molecule relative to a second element of the nucleic acid molecule, where both elements are contained within the same nucleic acid molecule and the first element is located closer to the 5' end of the nucleic acid molecule than the second element of the nucleic acid molecule. The second element is said to be "downstream" of the first element of the nucleic acid molecule. An element located "upstream" of a second element is synonymously referred to as being located "5'" of the second element. In the case of double-stranded nucleic acid molecules, designations such as "upstream" and "downstream" are given relative to the (+) strand.

[0157] According to the present invention, "operably linked" or "operably linked" refers to a linkage within a functional relationship. A nucleic acid is "operably linked" when it is functionally associated with another nucleic acid sequence; for example, a promoter is operably linked to a coding sequence if it affects the transcription of the promoter coding sequence. Operatively linked nucleic acids are typically contiguous to each other, separated, if appropriate, by additional nucleic acid sequences, and in certain embodiments, transcribed by RNA polymerase to give a single RNA molecule (common transcript).

[0158] In a particular embodiment, according to the invention, the nucleic acid is operably linked to expression control sequences which may be homologous or heterologous to the nucleic acid.

[0159] The term "expression control sequence," according to the present invention, includes promoters, ribosomal binding sequences, and other control elements that control the transcription of a gene or the translation of an induced RNA. In certain embodiments of the present invention, the expression control sequence can be regulated. The exact structure of an expression control sequence can vary depending on the species and cell type, but typically includes a 5' non-transcribed sequence and 5'- and 3'-non-translated sequences, which are involved in the initiation of transcription and translation, respectively. More specifically, a 5' non-transcribed expression control sequence includes a promoter region, which encompasses a promoter sequence, for transcriptional control of an operably linked gene. An expression control sequence can also include an enhancer sequence or upstream activator sequence. Expression control sequences for DNA molecules typically include a 5' non-transcribed sequence, as well as 5'- and 3'-non-translated sequences, such as a TATA box, capping sequence, CAAT sequence, etc. An expression control sequence for an alphavirus RNA can include a subgenomic promoter and / or one or more conserved sequence elements. A specific expression control sequence according to the present invention is an alphavirus subgenomic promoter, as described herein.

[0160] The nucleic acid sequences defined herein, in particular transcribable and coding nucleic acid sequences, may be combined with any expression control sequence, in particular a promoter, which may be homologous or heterologous to said nucleic acid sequence, wherein the term "homologous" refers to the fact that the nucleic acid sequence is also naturally operably linked to an expression control sequence, and the term "heterologous" refers to the fact that the nucleic acid sequence is not naturally operably linked to an expression control sequence.

[0161] A transcribable nucleic acid sequence, particularly a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "operably" linked to each other when they are covalently linked to each other in such a way that the transcription or expression of the transcribable, particularly encoding, nucleic acid sequence is under the control or influence of the expression control sequence. When a nucleic acid sequence is to be translated into a functional peptide or protein, induction of an expression control sequence operably linked to a coding sequence results in the transcription of the coding sequence without causing a frameshift of the coding sequence or rendering the coding sequence unable to be translated into the desired peptide or protein.

[0162] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript, e.g., a transcript containing a coding sequence, by providing recognition and binding sites for RNA polymerase. A promoter region may further include recognition or binding sites for additional factors involved in controlling transcription of the gene. A promoter may control the transcription of a gene in a prokaryotic or eukaryotic organism. A promoter may be "inducible," initiating transcription in response to an inducer, or "constitutive," in which transcription is not controlled by an inducer. An inducible promoter is expressed very little or not at all in the absence of an inducer. In the presence of an inducer, the gene is "switched on," or transcription levels increase, usually mediated by the binding of specific transcription factors. Specific promoters according to the present invention are, for example, alphavirus subgenomic promoters as described herein. Other specific promoters are, for example, alphavirus genomic plus-strand or minus-strand promoters.

[0163] The term "core promoter" refers to a nucleic acid sequence contained in a promoter. A core promoter is typically the minimal portion of a promoter required to properly initiate transcription. A core promoter typically contains a transcription initiation site and an RNA polymerase binding site.

[0164] "Polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymer molecule from monomer building blocks. "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA and RNA polymerases, the molecular entities are typically proteins or assemblies or complexes of multiple proteins. Typically, DNA polymerases synthesize DNA molecules based on a template nucleic acid. Typically, RNA polymerases synthesize RNA molecules based on a template nucleic acid, which is either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0165] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, the sequential synthesis of the (-) strand complement of the genomic RNA and the (+) strand genomic RNA results in RNA replication. Therefore, RNA-dependent RNA polymerase is synonymously referred to as "RNA replicase" or simply "replicase." In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Representative viruses that encode RNA-dependent RNA polymerases are alphaviruses.

[0166] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a predetermined RNA molecule (template RNA molecule). The synthesized RNA molecule may be, for example, identical to or complementary to the template RNA molecule. Generally, RNA replication can occur via the synthesis of a DNA intermediate or directly by RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication is performed by RNA-dependent RNA polymerase (RdRP) without a DNA intermediate. In other words, the template RNA strand (first RNA strand) or a portion thereof serves as a template for the synthesis of a second RNA strand complementary to the first RNA strand or a portion thereof. The second RNA strand or a portion thereof can then optionally serve as a template for the synthesis of a third RNA strand complementary to the second RNA strand or a portion thereof. Thus, the third RNA strand is identical to the first RNA strand or a portion thereof. Thus, RNA-dependent RNA polymerase can either directly synthesize an RNA strand complementary to the template or indirectly synthesize an identical RNA strand (via a complementary intermediate strand).

[0167] According to the present invention, the term "template RNA" refers to an RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0168] According to the present invention, the term "gene" refers to a specific nucleic acid sequence responsible for producing one or more cellular products and / or for achieving one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid segment (typically DNA; except in the case of RNA viruses, RNA) that comprises a nucleic acid encoding a specific protein or a functional or structural RNA molecule.

[0169] As used herein, an "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. According to the present invention, the term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, e.g., by cleavage and gel electrophoretic fractionation, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is a nucleic acid that is amenable to manipulation by recombinant techniques.

[0170] The term "vector" is used herein in its most general sense and includes, for example, any intermediate vehicle for a nucleic acid that allows said nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell and, where appropriate, integrated into the genome. Such vectors are preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, viral genomes, and fragments thereof.

[0171] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant subject," such as a recombinant cell in the context of the present invention, is not naturally occurring.

[0172] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes not only known objects, but also objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from nature in the future.

[0173] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or protein. It also includes partial expression of a nucleic acid. Furthermore, with respect to RNA, expression can be transient or stable. The term "expression" or "translation" refers to the process in a cell's ribosomes by which a chain of coding RNA (e.g., messenger RNA) directs the assembly of a sequence of amino acids to make a peptide or protein.

[0174] According to the present invention, the term "mRNA" refers to "messenger RNA" and relates to a transcript that is typically produced by using a DNA template and encodes a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. Methodologies for in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilizing modifications and capping.

[0175] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to an uninterrupted or interrupted sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. An uninterrupted sequence is characterized by consecutive adenylic acid residues. Uninterrupted poly(A) sequences are typical in nature. Poly(A) sequences are not usually encoded by eukaryotic DNA and are attached to the free 3' end of RNA by template-independent RNA polymerase after transcription in the cell nucleus during eukaryotic transcription, but the present invention encompasses poly(A) sequences encoded by DNA.

[0176] According to the present invention, the term "primary structure" in relation to a nucleic acid molecule refers to the linear sequence of nucleotide monomers.

[0177] According to the present invention, the term "secondary structure" in reference to a nucleic acid molecule refers to the two-dimensional representation of the nucleic acid molecule, reflecting base pairing; for example, in the case of a single-stranded RNA molecule, particularly intramolecular base pairing. Although each RNA molecule has only a single polynucleotide strand, the molecule is typically characterized by regions of (intramolecular) base pairing. According to the present invention, the term "secondary structure" includes structural motifs, including, but not limited to, base pairs, stems, stem-loops, bulges, internal loops, and loops such as multi-branched loops. The secondary structure of a nucleic acid molecule can be represented by a two-dimensional drawing (planar graph) showing base pairs (for details on the secondary structure of RNA molecules, see Auber et al., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structure of a particular RNA molecule is relevant in the context of the present invention.

[0178] According to the present invention, the secondary structure of nucleic acid molecules, particularly single-stranded RNA molecules, is determined by prediction using a web server for RNA secondary structure prediction (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html). Preferably, according to the present invention, the term "secondary structure" in relation to nucleic acid molecules specifically refers to the secondary structure determined by said prediction. Predictions can also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0179] According to the present invention, a "base pair" is a structural motif in a secondary structure in which two nucleotide bases are bound to each other through hydrogen bonds between the donor and acceptor sites on the bases. Complementary bases A:U and G:C form stable base pairs through hydrogen bonds between the donor and acceptor sites on the bases; A:U and G:C base pairs are called Watson-Crick base pairs. A weaker base pair (called a wobble base pair) is formed by the bases G and U (G:U). A:U and G:C base pairs are called canonical base pairs. G:U (which occurs quite frequently in RNA) and other rare base pairs (e.g., A:C;U:U) are called non-canonical base pairs.

[0180] According to the present invention, "nucleotide pairing" refers to two nucleotides that associate with each other such that the bases form a base pair (canonical or non-canonical, preferably a canonical base pair, most preferably a Watson-Crick base pair).

[0181] According to the present invention, the terms "stem-loop" or "hairpin" or "hairpin loop" in reference to nucleic acid molecules refer interchangeably to a specific secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule such as single-stranded RNA. The specific secondary structure represented by a stem-loop is composed of a continuous nucleic acid sequence including a stem and a (terminal) loop, also called a hairpin loop, in which the stem is formed by two adjacent fully or partially complementary sequence elements; these are separated by a short sequence (e.g., 3 to 10 nucleotides) that forms the loop of the stem-loop structure. The two adjacent fully or partially complementary sequences are defined, for example, as stem 1 and stem 2 of a stem-loop element. A stem-loop is formed when two adjacent fully or partially reverse-complementary sequences, such as stem 1 and stem 2 of the stem-loop element, base-pair with each other, resulting in a double-stranded nucleic acid sequence containing an unpaired loop at the end formed by the short sequence located between stem 1 and stem 2 of the stem-loop element. Thus, a stem-loop comprises two stems (stem 1 and stem 2) that are separated by a short sequence that base pairs with each other at the level of the secondary structure of the nucleic acid molecule and is not part of stem 1 or stem 2 at the level of the primary structure of the nucleic acid molecule. For illustrative purposes, a two-dimensional representation of a stem-loop resembles a lollipop structure. Formation of the stem-loop structure requires the presence of a sequence that can fold back on itself to form a paired duplex; the paired duplex is formed by stem 1 and stem 2. The stability of a paired stem-loop element is typically determined by its length, the number of nucleotides in stem 1 that can form base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides in stem 2, and the number of nucleotides in stem 1 that cannot form such base pairs with the paired nucleotides in stem 2 (mismatches or bulges). According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, e.g., 4, 5, 6, or 7 nucleotides. If a given nucleic acid sequence is characterized by a stem-loop, then each complementary nucleic acid sequence will typically also be characterized by a stem-loop.Stem loops are typically formed by single-stranded RNA molecules. For example, several stem loops exist in the 5' replication recognition sequence of alphavirus genomic RNA.

[0182] According to the present invention, with respect to a specific secondary structure (e.g., a stem-loop) of a nucleic acid molecule, "disruption" or "disrupt" means that the specific secondary structure is absent or altered. Typically, the secondary structure can be disrupted as a result of changing at least one nucleotide that is part of the secondary structure. For example, a stem-loop can be disrupted by changing one or more nucleotides that form the stem, thereby making nucleotide pairing impossible.

[0183] According to the present invention, with respect to a nucleic acid molecule, the term "tertiary structure" refers to the three-dimensional structure of a nucleic acid molecule as defined by its atomic coordinates.

[0184] According to the present invention, nucleic acids such as RNA, e.g., rRNA, can encode peptides or proteins. Thus, a transcribable nucleic acid sequence or a transcript thereof can comprise an open reading frame (ORF) that encodes a peptide or protein.

[0185] According to the present invention, the term "nucleic acid encoding a peptide or protein" means that the nucleic acid, when present in an appropriate environment, preferably in a cell, is capable of directing the assembly of amino acids to produce a peptide or protein during translation. Preferably, the coding RNA according to the present invention is capable of interacting with the cellular translation machinery, such that the coding RNA can be translated to obtain the peptide or protein.

[0186] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, more preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably six or more, preferably twenty or more, up to a maximum of preferably fifty, preferably one hundred or preferably one hundred and fifty consecutive amino acids linked together via peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, although the terms "peptide" and "protein" are generally used synonymously herein.

[0187] The terms "peptide" and "protein" according to the present invention include substances which contain not only amino acid components but also non-amino acid components such as sugar and phosphate structures, and also substances which contain bonds such as ester bonds, thioether bonds or disulfide bonds.

[0188] According to the present invention, the terms "initiation codon" and "start codon" refer synonymously to a codon (base triplet) of an RNA molecule that may be the first codon translated by a ribosome. Such codons typically encode the amino acid methionine in eukaryotes and a modified methionine in prokaryotes. The most common initiation codon in eukaryotes and prokaryotes is AUG. Unless otherwise specified herein, the terms "initiation codon" and "start codon" with respect to an RNA molecule refer to the AUG codon. According to the present invention, the terms "initiation codon" and "start codon" are also used to refer to the corresponding base triplet of a deoxyribonucleic acid, i.e., the base triplet that encodes the start codon of an RNA. When the start codon of a messenger RNA is AUG, the base triplet that encodes AUG is ATG. According to the present invention, the terms "initiation codon" and "start codon" preferably refer to a functional initiation codon or start codon, i.e., an initiation codon or start codon that is or will be used as a codon by a ribosome to initiate translation. Within an RNA molecule, there may be AUG codons that are not used by the ribosome to initiate translation, for example, due to the short distance from the codon to the cap. These codons are not encompassed by the term functional initiation codon or start codon.

[0189] Below, specific and / or preferred variations of individual features of the invention are provided. The invention also contemplates, as particularly preferred embodiments, embodiments produced by combining two or more of the specific and / or preferred variations described for two or more features of the invention.

[0190] "Isolated" means changed or removed from its natural state. For example, a cell, nucleic acid, or peptide that is naturally present in a living animal is not "isolated," but the same cell, nucleic acid, or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." Preferably, the isolated cell, nucleic acid, or peptide exists in a purified or substantially purified state. An isolated cell or cell population preferably exists free of cells of a different cell type; for example, isolated T cells exist free of other blood cells, such as dendritic cells. Preferably, the isolated cell exists only with allogeneic cells of the same cell type.

[0191] The term "syngeneic" is used to describe cells that have the same genetic information as another cell or population of cells.

[0192] The term "autologous" is used to describe something that is derived from the same subject. For example, "autologous transplant" refers to the transplantation of tissue or organs derived from the same subject. Such treatment is advantageous because it overcomes immunological barriers that would otherwise cause rejection.

[0193] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical.

[0194] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0195] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. For example, transplanting bone marrow from one individual into another constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0196] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant subject," such as a recombinant cell in the context of the present invention, is not naturally occurring.

[0197] As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0198] As used herein, "lentivirus" refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells, and can deliver large amounts of genetic information into the DNA of host cells, making it one of the most efficient gene transfer vectors.HIV, SIV, and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0199] As used herein, the term "specifically binds" refers to a molecule, such as an antibody or CAR, that recognizes a specific antigen in a sample or subject but does not substantially recognize or bind other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. However, such cross-species reactivity does not in itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than a general protein. If an antibody is specific for epitope "A," in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0200] The term "minicircle" as used herein refers to a vector that is a supercoiled DNA molecule lacking a bacterial replication origin and an antibiotic resistance gene, and is primarily composed of a eukaryotic expression cassette (see, for example, F. Jia et al., Nature Methods, Vol. 7, No. 3, pp. 197-199, March 2010).

[0201] Immune effector cells Cells used in connection with the present invention and into which nucleic acids (DNA and / or RNA) can be introduced are lytic cells, particularly immune effector cells such as lymphoid cells, preferably T cells, particularly cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes causes destruction of target cells. For example, cytotoxic T cells cause destruction of target cells by one or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, allowing granzymes to enter the cell and trigger the caspase cascade in the cytoplasm, inducing apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between T cells and target cells. The cells used in connection with the present invention are preferably autologous cells, although xenogeneic or allogeneic cells may also be used.

[0202] The term "immune effector cell" or "immunoreactive cell" in the context of the present invention relates to a cell which exerts an effector function during an immune response.

[0203] The term "effector function" in the context of the present invention includes any function mediated by a component of the immune system that results in, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor growth and / or tumor progression (including the inhibition of tumor dissemination and metastasis). Preferably, effector function in the context of the present invention is a T-cell mediated effector function. Such a function can be mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + It involves activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, removal of cells, i.e., cells characterized by antigen expression, by, for example, apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of antigen-expressing target cells.

[0204] In one embodiment, an "immune effector cell" is capable of binding to an antigen, such as an antigen presented in the context of MHC on a cell or an antigen expressed on the surface of a cell, and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, an "immune effector cell" is a T cell, preferably a CD4 + and / or CD8 + According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, particularly T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are characterized by CD34 + They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells upon exposure to antigen resembles clonal selection in the immune system.

[0205] Preferably, "immune effector cells" recognize antigens with some degree of specificity, particularly when presented in the context of MHC or when present on the surface of diseased cells such as cancer cells. Preferably, said recognition enables the cells that recognize the antigen to become responsive or reactive. The cells are called helper T cells (CD4 + In the case of CD8 T cells, such responsiveness or reactivity may be mediated by cytokine release and / or CD8 +This may involve activation of lymphocytes (CTLs) and / or B cells. If the cells are CTLs, such response or reactivity may include elimination of the cells, i.e., cells characterized as expressing the antigen, by, for example, apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing the antigen. CTL responsiveness may also be determined using artificial reporters that accurately represent CTL responsiveness. Such CTLs that recognize and respond to an antigen are also referred to herein as "antigen-responsive CTLs."

[0206] In one embodiment, the immune effector cell is a CAR-expressing immune effector cell. In one embodiment, the immune effector cell is a TCR-expressing immune effector cell.

[0207] Immune effector cells used in accordance with the present invention may express an endogenous antigen receptor, such as a T cell receptor or a B cell receptor, or may lack expression of an endogenous antigen receptor.

[0208] "Lymphoid cells" are cells, or precursors of such cells, that can generate an immune response, such as a cellular immune response, optionally after appropriate modification, e.g., after introduction of an antigen receptor such as a TCR or CAR, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells, which can be modified to express an antigen receptor on the cell surface. In one embodiment, lymphoid cells lack endogenous expression of a T cell receptor.

[0209] The terms "T cell" and "T lymphocyte" are used interchangeably herein and refer to T helper cells (CD4 + T cells) and cytotoxic T cells (CTL, CD8 +The term "antigen-specific T cells" or similar terms refers to T cells that recognize the antigen targeted by the T cell and preferably exert T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. The specificity of a T cell can be assessed using any of a variety of standard techniques, such as, for example, a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0210] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a specialized receptor on their surface, called the T cell receptor (TCR). The thymus is the primary organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0211] T helper cells assist other white blood cells in immunological processes, such as the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore CD4 + Also known as T cells, helper T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that control or support the active immune response.

[0212] Cytotoxic T cells destroy virus-infected and tumor cells and are involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore CD8 + Also known as T cells, these cells recognize targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0213] "Regulatory T cells" or "Tregs" are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0214] As used herein, the term "naive T cells" refers to mature T cells that, unlike activated or memory T cells, have not encountered cognate antigen in the periphery. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isoform.

[0215] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that have previously encountered and responded to a cognate antigen. Upon a second encounter with the antigen, memory T cells can proliferate and mount a more rapid and potent immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + These can be either CD45 or CD45R, and usually express CD45RO.

[0216] According to the present invention, the term "T cells" includes cells that can mature into T cells upon appropriate stimulation.

[0217] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is produced by independent T cell receptor α and β (TCRα and TCRβ) genes and consists of two separate peptide chains called the α-TCR chain and the β-TCR chain. γδ T cells (γδ T cells) represent a small subset of T cells that possess different T cell receptors (TCRs) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This T cell population is much smaller than αβ T cells (2% of all T cells).

[0218] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore double-negative (CD4 - CD8 - As development progresses, double-positive thymocytes (CD4 + CD8 + ) and eventually became single positive (CD4 + CD8 - or CD4 - CD8 + ) and are released from the thymus into peripheral tissues.

[0219] T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood from a mammal, such as a patient, using a commercially available cell separation system. Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures. A sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0220] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of a T cell receptor. As provided herein, NK cells can also be differentiated from stem or progenitor cells.

[0221] nucleic acid As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0222] The nucleic acid may be contained within a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, retroviral vectors, adenoviral vectors, viral vectors such as baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0223] In one embodiment of all aspects of the invention, a nucleic acid, such as a nucleic acid encoding an antigen receptor or a nucleic acid encoding an immune effector cell activator molecule, is expressed in an immune effector cell to provide the antigen receptor or activator molecule.

[0224] The nucleic acids of the present invention can be introduced into immune effector cells by various means, for example, by the particles or complexes of the present disclosure, or any composition comprising either or both of the particles and complexes, or by electroporation, or by viral-based systems, or by any particle, particularly lipid- or polymer-based particle, capable of introducing nucleic acid into cells. In some embodiments, the nucleic acid is introduced into or taken up by a cell, where the cell can be present in a subject, e.g., a patient. Thus, according to the present invention, the cell into which the nucleic acid described herein is introduced can be present in vitro or in vivo, e.g., the cell can form part of an organ, tissue, and / or organism of a patient.

[0225] In some embodiments, the first nucleic acid molecule is a DNA or RNA molecule introduced into a cell. In other embodiments, the first nucleic acid molecule is a DNA molecule into which the first nucleotide sequence has been incorporated, and is DNA that does not contain the first nucleotide sequence but was previously present in the cell. In this embodiment, the first nucleotide sequence was previously introduced into the cell along with another first nucleic acid molecule, which can be DNA or RNA, and is then removed and integrated into a DNA molecule, such as genomic DNA, already present in the cell. The DNA molecule into which the first nucleotide sequence has been incorporated becomes the first nucleic acid molecule upon integration.

[0226] In some embodiments, the first nucleic acid is an RNA molecule that is integrated into the genome of the immune effector cell via a retrotransposon-based system, preferably a viral-based retrotransposon system or a polyA-based retrotransposon system.

[0227] The terms "genome" or "genomic DNA" or "genomic nucleic acid molecule" are meant to refer to any type of DNA molecule that is transmitted and evenly distributed from mother cells to daughter cells. Genomic DNA refers to both chromosomal DNA and extrachromosomal DNA, such as episomes, preferably non-viral episomes.

[0228] The term "episome" should be understood to refer to a DNA molecule that remains part of the eukaryotic genome without integration. Episomes accomplish this by being replicated along with the rest of the genome and then distributed equally to each daughter cell, like chromosomes.

[0229] In some embodiments, the second nucleic acid molecule is not integrated into the genomic nucleic acid molecule of the immune effector cell, and in particular is not contained within an episome present in the immune effector cell. Preferably, the second nucleic acid is an RNA molecule, preferably an mRNA, more preferably a modified RNA or mRNA. In some embodiments, the RNA molecule may be degraded or lost during cell division.

[0230] In another embodiment, the second nucleic acid molecule is a DNA molecule, preferably a plasmid, which is preferably degraded in the immune effector cell, or epigenetically silenced, or not evenly propagated during cell division.

[0231] In some embodiments, the second nucleic acid is transiently expressed.

[0232] The term "transiently expressed" is understood to mean that a nucleic acid or transcript is expressed for only a limited period of time. Preferably, this means that the transcript, non-coding RNA, or protein-encoding nucleic acid is lost from the cell by degradation or unequal distribution of nucleic acids during cell division, and in particular, that the nucleic acid is not replicated within the cell to replenish the lost nucleic acid. A transiently expressed nucleic acid is preferably not part of the cell's genome. For example, a DNA plasmid encoding a specific transcript without a eukaryotic origin of replication (preferably a human origin, not a mammalian origin of replication) is only transiently expressed because, even if the encoding transcript is expressed, the plasmid is lost because it does not have an origin of replication and is not replenished after degradation or replicated before cell division. For example, a protein is understood to be transiently expressed if it is expressed for only a limited period of time due to the loss and non-replication of the nucleic acid encoding that protein. Another example of a transiently expressed nucleic acid is an mRNA molecule introduced into a cell, for example, by electroporation.

[0233] In some embodiments, the third nucleic acid molecule is DNA or RNA. The third nucleic acid molecule provides the enzymes, such as transposase, reverse transcriptase, or integrase, necessary to integrate the first nucleotide sequence into the genomic nucleic acid molecule when the first nucleotide sequence is integrated into the genome of the immune effector cell. Providing a means for integrating the first nucleotide sequence via a separate nucleic acid has the particular advantage of helping to prevent loss of the integrated first nucleotide sequence because it provides a limited-time means for integration that can also remove the integrated nucleotide sequence. The third nucleic acid molecule can be one or more nucleic acid molecules. If more than one enzyme is required for integration, providing separate nucleic acids encoding the required enzymes can help provide additional flexibility, for example, regarding the amount of enzyme produced in the cell or when combining different enzymes.

[0234] In some embodiments, the third nucleic acid molecule is not integrated into the genomic nucleic acid molecule of the immune effector cell, and in particular is not contained within an episome present in the immune effector cell. Preferably, the third nucleic acid is an RNA molecule, preferably an mRNA, more preferably a modified RNA or mRNA. In some embodiments, the RNA molecule can be degraded or lost during cell division.

[0235] In another embodiment, the third nucleic acid molecule is a DNA molecule, preferably a plasmid, which preferably is not propagated during cell division or is not degraded in the immune effector cell, or is epigenetically silenced.

[0236] In alternative embodiments, the enzymes required for integration may be provided in other forms as opposed to being encoded on the nucleic acid molecule.

[0237] In some embodiments, the immune effector cells or particles of the present disclosure comprise a fourth and / or fifth nucleic acid molecule. In some embodiments, the fourth and / or fifth nucleic acid molecule is one or more nucleic acid molecules.

[0238] In some embodiments, the fourth nucleic acid molecule encodes one or more antigens that are bound by the first cell surface-expressed antigen receptor.

[0239] In some embodiments, the fifth nucleic acid molecule encodes one or more antigens bound by a second cell surface-expressed antigen receptor.

[0240] In some embodiments, the fourth nucleic acid molecule is not integrated into the genomic nucleic acid molecule of the immune effector cell, and in particular is not contained within an episome present in the immune effector cell. Preferably, the fourth nucleic acid is an RNA molecule, preferably an mRNA, more preferably a modified RNA or mRNA. In some embodiments, the RNA molecule can be degraded or lost during cell division.

[0241] In another embodiment, the fourth nucleic acid molecule is a DNA molecule, preferably a plasmid, which preferably is not propagated during cell division or is not degraded in the immune effector cell or is epigenetically silenced.

[0242] In some embodiments, the fifth nucleic acid molecule is not integrated into the genomic nucleic acid molecule of the immune effector cell, and in particular is not contained within an episome present in the immune effector cell. Preferably, the fifth nucleic acid is an RNA molecule, preferably an mRNA, more preferably a modified RNA or mRNA. In some embodiments, the RNA molecule can be degraded or lost during cell division.

[0243] In another embodiment, the fifth nucleic acid molecule is a DNA molecule, preferably a plasmid, which preferably is not propagated during cell division or is not degraded in the immune effector cell or is epigenetically silenced.

[0244] In some embodiments, at least one or all of the first, second, third, fourth and fifth nucleic acid molecules are modified RNA molecules.

[0245] modified RNA In some aspects, the RNA or RNA molecules described herein are modified RNA. In some embodiments, the modified RNA comprises at least one functional analog of A, C, G, and / or U.

[0246] In one embodiment, the RNA described herein may have modified nucleotide / nucleoside / backbone modifications. As used herein, the term "RNA modification" may refer to chemical modifications, including backbone modifications as well as sugar or base modifications.

[0247] In this context, modified RNA molecules as defined herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. A backbone modification in the context of the present disclosure is a modification in which the phosphate of the backbone of a nucleotide contained in an RNA molecule as defined herein is chemically modified. A sugar modification in the context of the present disclosure is a chemical modification of the sugar of a nucleotide of an RNA molecule as defined herein. Furthermore, a base modification in the context of the present invention is a chemical modification of the base moiety of a nucleotide of an RNA molecule. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0248] Sugar Modifications: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be modified in the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CHCHO)CHCHOR; "locked" nucleic acids (LNAs) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by a methylene bridge; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. A "deoxy" modification can include hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar via a linker, where the linker comprises one or more of C, N, and O atoms. The sugar group can also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified RNA molecules can include nucleotides that contain, for example, arabinose as the sugar.

[0249] Backbone Modification: The phosphate backbone may be further modified in modified nucleosides and nucleotides, which may be incorporated into the modified RNA molecules described herein. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can include complete replacement of the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, both non-linked oxygens are replaced with sulfur. The phosphate linker can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates).

[0250] Base Modification: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be further modified at the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified at the major groove surface. In some embodiments, the chemical modification at the major groove can include an amino group, a thiol group, an alkyl group, or a halo group.

[0251] In certain embodiments of the present disclosure, the nucleotide analog / modification is selected from base modifications, preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxy-cytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate , 4-thio-uridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo 2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deaza-adenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-aza The base-modifying nucleotide may be selected from adenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, and xanthosine-5'-triphosphate. Particularly preferred is a base-modifying nucleotide selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.In some embodiments, the modified nucleoside is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl- Examples of functional analogs of uridine include N-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In a preferred embodiment, the functional analog for uridine is N-methyl-pseudouridine (mΨ).

[0252] In some embodiments, the modified nucleoside is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl -pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0253] In other embodiments, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N Includes 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methyl-thio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, uiosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0254] In some embodiments, the nucleotide can be modified on the major groove face and can include replacing the hydrogen on C-5 of uracil with a methyl or halo group. In certain embodiments, the modified nucleoside is 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine, 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine.

[0255] In further embodiments, the modified RNA is selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a nucleoside modifications selected from -thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0256] In certain preferred embodiments, the RNA comprises a modified nucleoside in place of at least one (eg, all) uridines.

[0257] The term "uracil" as used herein describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: The file is TIFF2026504516000003.tif26170.

[0258] As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is: The file is TIFF2026504516000004.tif36170.

[0259] UTP (uridine 5'-triphosphate) has the following structure: JPEG2026504516000005.jpg31170.

[0260] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: JPEG2026504516000006.jpg32170.

[0261] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0262] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the following structure: I have TIFF2026504516000007.tif34170.

[0263] N1-methyl-pseudo-UTP has the following structure: JPEG2026504516000008.jpg30170.

[0264] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure: I have TIFF2026504516000009.tif42170.

[0265] In certain preferred embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0266] In certain preferred embodiments, the RNA contains a modified nucleoside in place of at least one uridine, hi some embodiments, the RNA contains a modified nucleoside in place of each uridine.

[0267] In certain preferred embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise multiple types of modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0268] In certain preferred embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-azauridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyluridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine], or any one or more of other modified uridines known in the art.

[0269] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidines as described above. For example, in one embodiment, 5-methylcytidine is partially or completely substituted for cytidine in the RNA, preferably completely. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA contains pseudouridine (5-) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0270] cap In some embodiments, the RNA or RNA molecules described herein may optionally include a 5' cap, a 5' UTR, a coding sequence, a 3' UTR, and / or a poly(A) tail. In some embodiments, the coding sequence or open reading frame may be optimized with respect to codon usage.

[0271] "RNA containing a 5'-cap" or "RNA with a 5'-cap" or "RNA modified with a 5'-cap" or "capped RNA" refers to RNA that includes a 5'-cap. For example, providing RNA with a 5'-cap can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap, where the 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or RNA can be generated, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme, e.g., vaccinia virus capping enzyme. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphodiester bond.

[0272] In this disclosure, naturally occurring caps typically refer to unmethylated cap dinucleotides (G(5')ppp(5')N; also referred to as GpppN) and methylated cap dinucleotides ((m 7 G(5')ppp(5')N;m 7 GpppN) m 7 GpppN (wherein N is G) has the following formula: Represented as TIFF2026504516000010.tif41170.

[0273] The capped RNAs of the present disclosure can be prepared in vitro and are therefore independent of the capping machinery within the host cell. Co-transcriptional capping involves the use of all four ribonucleoside triphosphates or functional analogs thereof, and m 7 G(5')ppp(5')G(m 7 It works by transcribing a DNA template in vitro using either a bacterial or bacteriophage nucleic acid polymerase in the presence of a capping agent such as GpppG. The nucleic acid polymerase then catalyzes the transcription of the m-phosphate of the α-phosphate of the template nucleoside triphosphate (pppN). 7 Transcription is initiated by nucleophilic attack of the 3'-OH of the guanosine moiety of GpppG, forming intermediate m 7 This results in GpppGpN, where N is the second base of the RNA molecule.

[0274] In a preferred embodiment of the present disclosure, the RNA molecule comprises a 5'-cap analog, which was originally described to facilitate the synthesis of large amounts of RNA transcripts by in vitro transcription.

[0275] Several cap analogs (also called synthetic caps) have been generally described for messenger RNA, all of which can be used in the context of the present disclosure. Ideally, a cap analog is selected that is associated with higher translation efficiency and / or increased resistance to degradation in vivo and / or increased resistance to degradation in vitro.

[0276] Preferably, a cap analog is used that can be incorporated into an RNA strand in only one direction. Pasquinelli et al. (1995, RNA J. 1:957-967) showed that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit to initiate transcription, such that approximately 40-50% of capped transcripts have the reverse cap dinucleotide (i.e., the initial reaction product is Gpppm). 7 Compared to RNA with a correct cap, RNA with a reverse cap does not function in translating nucleic acid sequences into proteins. Therefore, incorporating the cap in the correct orientation, i.e., m 7 It would be desirable to obtain RNA with a structure essentially corresponding to GpppGpN, etc. Reverse incorporation of cap dinucleotides has been shown to be inhibited by substitution of either the 2'- or 3'-OH group of the methylated guanosine unit (Stepinski et al., 2001, RNA J. 7:1486-1495; Peng et al., 2002, Org. Lett. 24:161-164). RNA synthesized in the presence of such "anti-reverse cap analogs" retains the traditional 5'-capped m 7 It is translated more efficiently than in vitro-transcribed RNA in the presence of GpppG. To this end, one cap analog, in which the 3'OH group of the methylated guanosine unit is replaced with OCH3, has been described, for example, by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is a suitable cap dinucleotide according to the present disclosure. TIFF2026504516000011.tif39170

[0277] In one embodiment, a cap has the effect that RNA bearing such a cap is essentially resistant to decapping. This is important because the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is generally limited by spontaneous degradation of the mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase comprising a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the alpha and beta phosphate groups of the triphosphate bridge. In the present disclosure, caps can be selected that are not or are not susceptible to such types of cleavage. A suitable cap analog for this purpose is represented by formula (I): TIFF2026504516000012.tif40170, In the formula, R 1 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; R 2 and R 3 is independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2 and R 3 together form OXO, where X is selected from the group consisting of optionally substituted CH, CHCH, CHCHCH, CHCH(CH); C(CH3)2 or R 2 is R 2 is bonded to the hydrogen atom at the 4'-position of the ring to form -O-CH2- or -CH2-O-, R 5 is selected from the group consisting of S, Se, and BH3; R 4 and R 6 is independently selected from the group consisting of O, S, Se, and BH3; n is 1, 2 or 3.

[0278] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 Preferred embodiments of the above are disclosed in WO 2011 / 015347 (A1) and may be appropriately selected in the present disclosure.

[0279] For example, in one embodiment, an RNA molecule of the present disclosure comprises a phosphorothioate cap analog, wherein one of the three non-bridging O atoms in the triphosphate chain is replaced with an S atom, i.e., R 4 , R 5 or R 6 is a specific cap analog in which one of the R is S. Phosphorothioate-cap analogs have been described by Kowalska et al., 2008, RNA, 14:1119-1131, as a solution to the undesired decapping process and thus increasing RNA stability in vivo. In particular, replacing the sulfur atom of the beta-phosphate group of the 5'-cap with an oxygen atom results in stabilization against Dcp2. In a preferred embodiment of the present disclosure, R in formula (I) 5 is S;R 4 and R 6 is O.

[0280] In a further embodiment, the RNA molecules of the present disclosure comprise phosphorothioate-cap-analogs in which a phosphorothioate modification of the RNA 5'-cap is combined with an "anti-reverse cap analog" (ARCA) modification. The various ARCA-phosphorothioate-cap-analogs are described in WO 2008 / 157688 A2, all of which can be used in the RNA molecules of the present disclosure. In that embodiment, R in formula (I) 2 or R 3 At least one of is not OH, preferably R 2 and R 3One of the groups is methoxy (OCH3), and R 2 and R 3 The other of the two is preferably OH. In a preferred embodiment, the sulfur atom of the beta-phosphate group is replaced by an oxygen atom (hence, R 5 is S;R 4 and R 6 (The symbol O is O). The phosphorothioate modification of ARCA is thought to ensure that the α, β, and γ phosphorothioate groups are correctly positioned within the active sites of cap-binding proteins in both the translational and uncapping machinery. At least some of these analogs are inherently resistant to the pyrophosphatases Dcp1 / Dcp2. Phosphorothioate-modified ARCA has been described to have a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate groups.

[0281] Particularly preferred caps in the present disclosure, namely, m2 7,2’-O Gpp s pG is referred to as beta-S-ARCA (WO 2008 / 157688 (A2); Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in one embodiment of the present disclosure, the RNA of the present disclosure is modified with beta-S-ARCA. Beta-S-ARCA has the following structure: Represented by TIFF2026504516000013.tif39170.

[0282] Generally, substitution of an oxygen atom for a sulfur atom in the bridging phosphate results in phosphorothioate diastereomers, designated D1 and D2, based on their elution patterns in HPLC. Briefly, the "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time than the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). Determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0283] In a first particularly preferred embodiment of the present disclosure, the RNA of the present disclosure is modified with the beta-S-ARCA (D2) diastereomer. The two diastereomers of beta-S-ARCA differ in their susceptibility to nucleases. RNA bearing the D2 diastereomer of beta-S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of an unmodified ARCA 5'-cap), while RNA bearing a beta-S-ARCA (D1) 5'-cap has been shown to be moderately susceptible to Dcp2 cleavage (71% cleavage). Furthermore, increased stability against Dcp2 cleavage has been shown to correlate with increased protein expression in mammalian cells. In particular, RNA bearing a beta-S-ARCA (D2) cap has been shown to be translated more efficiently in mammalian cells than RNA bearing a beta-S-ARCA (D1) cap. Thus, in one embodiment of the present disclosure, the RNA of the present disclosure comprises a substituent R of formula (I): 5 The stereochemical configuration at the P atom constituting the D2 diastereomer of beta-S-ARCA is P β In this embodiment, the R 5 is S;R 4 and R 6 is O. Furthermore, R in formula (I) 2 or R 3At least one of is preferably not OH, and preferably R 2 and R 3 One of the groups is methoxy (OCH3), and R 2 and R 3 The other of is preferably OH.

[0284] In a second particularly preferred embodiment, the RNA of the present disclosure is modified with beta-S-ARCA(D1) diastereomer. This embodiment is particularly suitable for introducing capped RNA into immature antigen-presenting cells, such as for vaccination purposes. It has been demonstrated that beta-S-ARCA(D1) diastereomers are particularly suitable for increasing RNA stability, increasing RNA translation efficiency, elongating RNA translation, increasing total protein expression of RNA, and / or increasing immune responses to antigens or antigenic peptides encoded by the respective capped RNAs upon introduction into immature antigen-presenting cells (Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in an alternative embodiment of the present disclosure, the RNA of the present disclosure is modified with a beta-S-ARCA(D1) diastereomer of formula (I): 5 The stereochemical configuration at the P atom constituting the D1 diastereomer of beta-S-ARCA is P β The substituent R is modified with a cap analog according to formula (I), characterized in that the stereochemical configuration at the atoms corresponds to the cap analog. Respective cap analogs and their embodiments are described in WO 2011 / 015347 (A1) and Kuhn et al., 2010, Gene Ther. 17:961-971. 5 The stereochemical configuration at the P atom constituting the D1 diastereomer of beta-S-ARCA is P β Any cap analog described in WO 2011 / 015347 A1 that corresponds to the arrangement of atoms in formula (I) can be used in the present disclosure. 5 is S;R 4 and R 6 is O. Furthermore, R in formula (I) 2 or R 3At least one of is preferably not OH, and preferably R 2 and R 3 One of the groups is methoxy (OCH3), and R 2 and R 3 The other of is preferably OH.

[0285] In one embodiment, the RNA of the present disclosure is modified with a 5'-cap structure according to formula (I), where any one phosphate group is replaced with a boranophosphate group or a phosphoroselenoate group. Such caps increase stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (wherein the alkyl is preferably methyl); each cap analog is referred to as BH3-ARCA or Se-ARCA. Compounds particularly suitable for mRNA capping include β-BH3-ARCA and β-Se-ARCA, as described in WO 2009 / 149253 (A2). In these compounds, the substituent R of formula (I) 5 The stereochemical configuration of the P atom in the D1 diastereomer of beta-S-ARCA is β It is preferred that the stereochemical configuration at the atoms corresponds.

[0286] In some embodiments, the RNA comprises a cap, which may be appropriate in the context of the present disclosure, including Cap 0 (methylation of the first nucleobase, e.g., m7 GpppN), Cap 1 ( m7 GpppN (additional methylation of the ribose of the adjacent nucleotide), Cap 2 ( m7 Additional methylation of the ribose of the second nucleotide downstream of GpppN), Cap 3 ( m7 Additional methylation of the ribose of the third nucleotide downstream of GpppN), cap 4 ( m7Additional methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA, e.g., beta-S-ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0287] In some embodiments, the RNA comprises a cap that is Cap-0 (also referred to herein as "Cap0"), Cap-1 (also referred to herein as "Cap1"), or Cap-2 (also referred to herein as "Cap2"). See, e.g., Figure 1 of Ramanathan A et al. and Figure 1 of Decroly E et al.

[0288] In some embodiments, Cap0 is a guanosine nucleoside methylated at the 7-position of the guanine ( m7 In some embodiments, Cap0 is linked to the RNA via a 5'-5' triphosphate linkage, as defined herein. m7 Gppp or m7 Also called G(5')ppp(5').

[0289] In some embodiments, Cap1 is a guanosine nucleoside methylated at the 7-position of the guanine ( m7 G or 7m G) and the 2'O-methylated first nucleotide in the RNA ( 2’OMe N1 or N12'OMe or N1 2’OMe In some embodiments, Cap1 is linked to the RNA via a 5'-5' triphosphate linkage; in some embodiments, Cap1 comprises m7 Gppp(N1 2’OMe ) or m7 G(5')ppp(5')(N1 2’OMe ) or 7m G(5')ppp(5')N1 2’-OMe) In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0290] In some embodiments, m7 G(5')ppp(5')(N1 2’OMe ) Cap1 includes a second nucleotide N2 that is A, G, C, or U proximal to the cap at position +2. In some embodiments, such a Cap1 is m7 G(5')ppp(5')(N1 2’OMe )pN2). In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0291] In some embodiments, Cap1 is m7 G(5')ppp(5')(A1 2’OMe ) pG2, wherein A1 is a cap-proximal A at the +1 position and G2 is a cap-proximal G at the +2 position, and has the following structure: I have TIFF2026504516000014.tif69170.

[0292] In some embodiments, Cap1 is m7 G(5')ppp(5')(A1 2’OMe ) pU2, wherein A1 is a cap-proximal A at the +1 position and U2 is a cap-proximal U at the +2 position, and has the following structure: I have TIFF2026504516000015.tif71170.

[0293] In some embodiments, Cap1 is m7 G(5')ppp(5')(G1 2’OMe ) pG2, wherein G1 is a cap-proximal G at the +1 position and G2 is a cap-proximal G at the +2 position, and has the following structure: I have TIFF2026504516000016.tif65170.

[0294] In some embodiments, Cap1 is a guanosine nucleoside methylated at the 7-position of the guanine ( m7 In some embodiments, Cap1 comprises a guanosine nucleoside methylated at the 7-position of the guanosine and a 3'O methylation at the ribose (m7G3'OMe or 7m G 3’OMe ), as well as the 2'O-methylated first nucleotide in the RNA (N1 2’OMe In some embodiments, Cap1 is linked to the RNA via a 5'-5' triphosphate linkage, referred to herein as (m7G3'OMe)ppp(2'OMeN1) or ( m7 G 3’OMe )(5')ppp(5')( 2’OMe In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0295] In some embodiments, m7 G 3’OMe )(5')ppp(5')(N1 2’OMe ) Cap1 is the cap-proximal nucleotide at position 2 and includes a second nucleotide N2 selected from A, G, C, or U ( m7 G 3’OMe )(5')ppp(5')(N1 2’OMe )pN2). In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0296] In some embodiments, Cap1 is m7 G 3’OMe)(5')ppp(5')(A1 2’OMe ) pG2, wherein A1 is a cap-proximal A at the +1 position and G2 is a cap-proximal G at the +2 position, and has the following structure: I have TIFF2026504516000017.tif72170.

[0297] In some embodiments, Cap1 is m7 G 3’OMe )(5')ppp(5')(G1 2’OMe ) pG2, wherein G1 is a cap-proximal G at the +1 position and G2 is a cap-proximal G at the +2 position, and has the following structure: I have TIFF2026504516000018.tif75170.

[0298] In some embodiments, the second nucleotide of Cap1 can include one or more modifications, such as methylation. In some embodiments, Cap1 including a second nucleotide that includes a 2'O methylation is a Cap2 structure.

[0299] In some embodiments, an RNA polynucleotide comprising Cap1 has increased translation efficiency, increased translation rate, and / or increased expression of the encoded payload compared to a suitable reference comparator. m7 G 3’OMe )(5')ppp(5')(A1 2’OMe ) pG2 (wherein A1 is the cap-proximal nucleotide at the +1 position and G2 is the cap-proximal nucleotide at the +2 position) m7 G 3’OMe )(5')ppp(5')(G1 2’OMe )pG2, where G1 is the cap-proximal nucleotide at position 1 and G2 is the cap-proximal nucleotide at position 2. In some embodiments, the increased translation efficiency is assessed by administering the RNA polynucleotide to a cell or organism.

[0300] In some embodiments, the cap analog used in the RNA polynucleotide is m7 G 3’OMe Gppp(m1 2’-OMe )ApG(m2 7,3’-OMe G(5')ppp(5')m 2’-OMe ApG or ( m7 G 3’OMe )(5')ppp(5')(A 2’OMe )pG) and has the following structure: JPEG2026504516000019.jpg61170.

[0301] The following are RNA and m2 7,3’OMe G(5')ppp(5')m 2’-OMe An exemplary Cap1 RNA containing ApG. JPEG2026504516000020.jpg74170

[0302] Below is another exemplary Cap1 RNA. JPEG2026504516000021.jpg73170

[0303] UTR The term "untranslated region" or "UTR" refers to a region of a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region of an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0304] If present, the 3'-UTR is located at the 3' end of a gene, downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include the poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) tail (if present), e.g., immediately adjacent to the poly(A) tail.

[0305] When present, a 5'-UTR is located at the 5' end of a gene, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5'-cap, e.g., immediately adjacent to the 5'-cap.

[0306] According to the present disclosure, a 5'-untranslated region and / or a 3'-untranslated region can be operably linked to an open reading frame such that these regions are associated with the open reading frame in a manner that increases the stability and / or translation efficiency of the RNA comprising the open reading frame.

[0307] In some embodiments, an RNA molecule according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR.

[0308] UTRs are involved in RNA stability and translation efficiency. Both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs), in addition to the structural modifications of the 5'-cap and / or 3' poly(A)-tail described herein. Sequence elements within UTRs are generally understood to affect translation efficiency (mainly the 5'-UTR) and RNA stability (mainly the 3'-UTR). To increase the translation efficiency and / or stability of RNA, an active 5'-UTR is preferably present. To independently or additionally increase the translation efficiency and / or stability of RNA molecules, an active 3'-UTR is preferably present.

[0309] The terms "active to increase translation efficiency" and / or "active to increase stability," with respect to a first nucleic acid sequence (e.g., a UTR), mean that the first nucleic acid sequence, in a co-transcript with a second nucleic acid sequence, is capable of modifying the translation efficiency and / or stability of the second nucleic acid sequence such that the translation efficiency and / or stability is increased compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0310] A 5'-UTR according to the present disclosure can include any combination of two or more nucleic acid sequences, optionally separated by a linker. A 3'-UTR according to the present disclosure can include any combination of two or more nucleic acid sequences, optionally separated by a linker.

[0311] The term "linker" according to the present disclosure relates to a nucleic acid sequence that is added between two nucleic acid sequences and connects the two nucleic acid sequences. There are no particular limitations on the sequence of the linker.

[0312] The 3'-UTR typically has a length of 200 to 2000 nucleotides, e.g., 500 to 1500 nucleotides. The 3'-untranslated regions of immunoglobulin mRNAs are relatively short (less than approximately 300 nucleotides), while the 3'-untranslated regions of other genes are relatively long. For example, the 3'-untranslated region of tPA is approximately 800 nucleotides long, that of factor VIII is approximately 1800 nucleotides long, and that of erythropoietin is approximately 560 nucleotides long. The 3'-untranslated regions of mammalian mRNAs usually contain a homologous region known as the AAUAAA hexanucleotide sequence. This sequence is likely a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeats and can fold to form a stem-loop structure that functions as a barrier to exonucleases or interacts with proteins known to increase RNA stability (e.g., RNA-binding proteins).

[0313] Human beta-globin 3'-UTR, particularly two consecutive identical copies of human beta-globin 3'-UTR, contributes to high transcriptional stability and translation efficiency (Holtkamp et al., 2006, Blood 108:4009-4017). Thus, in one embodiment, an RNA molecule according to the present disclosure comprises two consecutive identical copies of human beta-globin 3'-UTR. Thus, in the 5'□3' direction, it comprises: (a) an optional 5'-UTR; (b) an open reading frame; and (c) a 3'-UTR; the 3'-UTR comprises two consecutive identical copies of human beta-globin 3'-UTR, a fragment thereof, or a variant or fragment of human beta-globin 3'-UTR.

[0314] In one embodiment, an RNA molecule according to the present disclosure comprises a 3'-UTR that is active to increase translation efficiency and / or stability, but is not the human beta-globin 3'-UTR, a fragment thereof, or a variant of the human beta-globin 3'-UTR or a fragment thereof.

[0315] In one embodiment, an RNA molecule according to the present disclosure comprises an active 5'-UTR to increase translation efficiency and / or stability.

[0316] Poly(A) sequence In some embodiments, an RNA molecule according to the present disclosure comprises a 3'-poly(A) sequence.

[0317] According to the present invention, in one embodiment, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, preferably at most 500, preferably at most 400, preferably at most 300, preferably at most 200, particularly at most 150 A nucleotides, in particular about 120 A nucleotides. In this context, "consisting essentially of" means that the majority of nucleotides in the poly(A) sequence, typically at least 50% and preferably at least 75% of the number of nucleotides in the "poly(A) sequence", are A nucleotides (adenylic acid), while allowing the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), and C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0318] Indeed, it has been demonstrated that a 3'-poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on the levels of RNA in transfected eukaryotic cells, as well as on the levels of proteins translated from open reading frames located upstream (5') of the 3'-poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0319] The present disclosure provides a 3'-poly(A) sequence that is attached during RNA transcription, i.e., during the preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

[0320] In some embodiments of the present disclosure, the 3'-poly(A) cassette present in the coding strand of the DNA template molecule consists essentially of dA nucleotides but is interrupted by random sequences with an even distribution of the four nucleotides (dA, dC, dG, dT). Such random sequences can be 5 to 50, preferably 10 to 30, and more preferably 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005004 A1. Any poly(A) cassette disclosed in WO 2016 / 005004 A1 can be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an even distribution of the four nucleotides (dA, dC, dG, dT), for example, 5 to 50 nucleotides in length, exhibit consistent propagation of plasmid DNA in E. coli at the DNA level and are still associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level.

[0321] As a result, in some embodiments of the present disclosure, the 3'-poly(A) sequences contained in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences with an even distribution of the four nucleotides (A, C, G, and U). Such random sequences can be 5 to 50, preferably 10 to 30, and more preferably 10 to 20 nucleotides in length.

[0322] Codon Usage Generally, the degeneracy of the genetic code allows certain codons (base triplets that code for amino acids) present in an RNA sequence to be substituted with other codons (base triplets) while maintaining the same coding capacity (the replacing codon codes for the same amino acid as the replaced codon). In some embodiments of the present disclosure, at least one codon of an open reading frame contained in an RNA molecule differs from each codon in the respective open reading frame in the species from which the open reading frame is derived. In such embodiments, the coding sequence of the open reading frame is said to be "adapted" or "modified." The coding sequence of an open reading frame contained in an RNA molecule can be adapted.

[0323] For example, when the coding sequence of an open reading frame is adapted, frequently used codons can be selected: WO 2009 / 024567 A1 describes the adaptation of the coding sequence of a nucleic acid molecule, including replacing rare codons with more frequently used codons. Because the frequency of codon usage depends on the host cell or host organism, this type of adaptation is suitable for adapting a nucleic acid sequence for expression in a specific host cell or host organism. Generally speaking, more frequently used codons are typically translated more efficiently in the host cell or host organism, although adaptation of all codons of an open reading frame is not always necessary.

[0324] For example, when the coding sequence of an open reading frame is adapted, the content of G (guanylic acid) and C (cytidylic acid) residues can be altered by selecting the codon with the highest GC-rich content for each amino acid. It has been reported that RNA molecules with GC-rich open reading frames have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther. 23:1457-1465).

[0325] antibody The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, with all four chains interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically contains a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region (herein referred to as C H The heavy chain constant region is usually composed of three domains: CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is very flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain usually contains a light chain variable region (herein referred to as V L or VL) and a light chain constant region (herein referred to as C L The light chain constant region is usually composed of one domain, CL. The VH and VL regions are further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs) (or hypervariable regions that may be hypervariable in sequence and / or in the form of structurally defined loops), and regions interspersed with more conserved regions called framework regions (FRs). Each VH and VL usually consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[0326] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, capable of binding, preferably specifically, to an antigen. In some embodiments, binding occurs under typical physiological conditions with a half-life of a significant period, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant, functionally defined period (a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to an antigen). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with an antigen. As used herein, the terms "antigen-binding region," "binding region," or "binding domain" refer to the region or domain that interacts with an antigen and typically includes both the VH and VL regions. As used herein, the term "antibody" includes not only monospecific antibodies but also multispecific antibodies containing two or more, e.g., three or more, different antigen-binding regions. The constant regions of antibodies (Abs) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. As noted above, the term "antibody" as used herein includes, unless otherwise specified or clearly contradicted by context, fragments of antibodies that are antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to antigens, and antibody derivatives, i.e., constructs derived from antibodies. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), which consist essentially of the VH domain and are also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21(11):484-90); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(1):111-24) and (vii) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be recombinantly produced as a single protein chain that pairs with the VL and VH regions to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs)—see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless otherwise specified or clearly indicated by context. While such fragments are generally included within the meaning of antibodies, they collectively and independently exhibit distinct biological properties and utilities and are a unique feature of the present disclosure. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein.Furthermore, unless otherwise specified, the term "antibody" should be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to antigens, which are provided by known techniques such as enzymatic cleavage, peptide synthesis, and recombinant technology.

[0327] The term "single-chain Fv" or "scFv" refers to an antibody in which the heavy and light chain variable domains (VH and VL) of a traditional two-chain antibody are combined to form a single chain, optionally with a linker (usually a peptide) inserted between the two chains to allow proper folding and creation of an active binding site.

[0328] Single-domain antibodies, also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. In some embodiments, single-domain antibodies are the variable domains (VH) of heavy-chain antibodies. These are called VHH fragments. Like intact antibodies, single-domain antibodies can selectively bind to specific antigens. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids. Cartilaginous fish also possess heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called VNAR fragments can be derived. Another approach is to split the dimeric variable domain from common human or mouse immunoglobulin G (IgG) into monomers. Currently, most research on single-domain antibodies is based on heavy-chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes.

[0329] An antibody can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody has a sequence that is closer to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, an IgG1 antibody can be a sequence variant of a naturally occurring IgG1 antibody, including variations in the constant region.

[0330] In various embodiments, the antibody is an IgG1 antibody, more specifically an IgG1, kappa or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

[0331] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse whose genome contains human heavy chain and light chain transgenes, fused to an immortalized cell.

[0332] The term "chimeric antibody" as used herein refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic use have been developed to reduce the immunogenicity of antibodies. The term "variable region" or "variable domain" used in the context of a chimeric antibody refers to the region containing the CDRs and framework regions of both the heavy and light chains of an immunoglobulin. Chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies can be recombinant antibodies engineered genetically or enzymatically. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibody production may be performed by methods other than those described herein.

[0333] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology with the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs) that form the antigen-binding site into homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to replace framework residues of the parent antibody (i.e., non-human antibody) with human framework regions (backmutation). Structural homology modeling can help identify amino acid residues within the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations relative to the non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, can be applied to obtain humanized antibodies with favorable properties, such as affinity and biochemical properties.

[0334] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization method of Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization is preferred, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display using libraries of human antibody genes, can in principle also be employed. The animal system suitable for preparing hybridomas secreting human monoclonal antibodies is the murine system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (such as murine myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be generated using, for example, transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat immune system. Thus, in some embodiments, human antibodies are obtained from transgenic animals, such as mice or rats, that have human germline immunoglobulin sequences in place of the animal's immunoglobulin sequences.In such embodiments, the antibody is derived from human germline immunoglobulin sequences introduced into the animal, with the final antibody sequence being the result of further modification of said human germline immunoglobulin sequences by somatic hypermutation and affinity maturation by the endogenous animal antibody machinery (see, e.g., Mendez et al. 1997 Nat Genet. 15(2):146-56).

[0335] As used herein, unless contradicted by context, the terms "Fab-arm," "binding arm," or "arm" comprise one heavy chain-light chain pair and are used interchangeably herein with "half molecule."

[0336] When used in the context of antibodies, the term "full length" indicates that the antibody is not a fragment, but rather contains all of the domains of a particular isotype that are normally found in that isotype in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0337] As used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, wherein said Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain.

[0338] The present disclosure also contemplates antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies described herein. A functional variant of a VL, VH, or CDR used in the context of an antibody still enables the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the "reference" or "parent" antibody, and in some cases, such an antibody may have higher affinity, selectivity, and / or specificity than the parent antibody.

[0339] Such functional variants typically retain significant sequence identity with the parent antibody.

[0340] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of a parent antibody sequence primarily by conservative substitutions; for example, up to 10 of the substitutions in the variant, e.g., 9, 8, 7, 6, 5, 4, 3, 2 or 1, are conservative amino acid residue substitutions.

[0341] Functional variants of the antibody sequences described herein, such as the VL or VH regions, or antibody sequences that have a degree of homology or identity to the antibody sequences described herein, such as the VL or VH regions, preferably contain modifications or mutations in non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0342] The term "specificity," as used herein, unless contradicted by context, is intended to have the following meaning: Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope.

[0343] Antibodies or fragments useful herein may compete with the specific antibodies or fragments described herein.

[0344] The terms "compete" and "competition" may refer to the competition between a first antibody and a second antibody for the same antigen. Methods for testing antibody competition for binding to target antigens are well known to those skilled in the art. An example of such a method is the so-called cross-competition assay, which can be carried out, for example, as ELISA or by flow cytometry. Alternatively, competition can be determined using biolayer interferometry.

[0345] Antibodies that compete for binding to a target antigen may bind to different epitopes on the antigen, and these epitopes may be close to each other, so that a first antibody that binds to one epitope prevents a second antibody from binding to the other epitope. However, in other situations, two different antibodies may bind to the same epitope on an antigen and compete for binding in a competitive binding assay. Such antibodies that bind to the same epitope are considered to have the same specificity herein. Thus, in some embodiments, antibodies that bind to the same epitope are considered to bind to the same amino acid on the target molecule. The binding of antibodies to the same epitope on a target antigen can be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to those skilled in the art. Preferably, antibodies or binding domains that bind to different epitopes do not compete with each other for binding to their respective epitopes.

[0346] Naturally occurring antibodies are generally monospecific, i.e., they bind to a single antigen. Described herein are binding agents, e.g., docking compounds, that bind to different epitopes, e.g., on a primary target and a connector compound. Such binding agents are at least bispecific or multispecific, e.g., trispecific, tetraspecific, etc. Thus, a binding agent can comprise two or more antibodies described herein or fragments thereof. In particular, the binding agents described herein can be two different antibodies, antibodies and fragments of different antibodies, and artificial proteins consisting of fragments of two different antibodies, where the fragments of two different antibodies form two binding domains.

[0347] According to the present disclosure, bispecific binding agents, particularly bispecific proteins such as bispecific antibodies, are molecules that have two different binding specificities and can therefore bind to two epitopes. In particular, the term "bispecific antibody" as used herein refers to an antibody that comprises two antigen-binding sites, i.e., a first binding site that has affinity for a first epitope and a second binding site that has binding affinity for a second epitope that is different from the first epitope.

[0348] As used herein, the term "bispecific" refers to an agent that has two different antigen-binding regions that bind to different epitopes.

[0349] A "multispecific binding agent" is a molecule that has two or more different binding specificities.

[0350] Many different formats and uses of bispecific antibodies are known in the art and are reviewed by Kontermann; Drug Discovery Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-Apr;4(2):182-97.

[0351] Bispecific binding agents according to the present disclosure are not limited to a particular bispecific format or method of manufacture.

[0352] Examples of bispecific antibody molecules that can be used herein include: (i) a single antibody with two arms containing different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an extra peptide linker; and (iii) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)"). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010); (iv) chemically linked bispecific (Fab') fragments; (v) Tandabs, which are fusions of two single-chain diabodies resulting in tetravalent bispecific antibodies with two binding sites for each target antigen; (vi) Flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fabs, can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which comprise, for example, two scFvs fused to both ends of human Fab-arms; and (ix) diabodies.

[0353] The term "bispecific antibody" includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains on another chain and forming two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Bispecific antibodies also include bispecific single-chain antibodies. The term "bispecific single-chain antibody" refers to a single polypeptide chain containing two binding domains. In particular, the terms "bispecific single-chain antibody" or "single-chain bispecific antibody" or related terms as used herein refer to an antibody construct obtained by linking at least two antibody variable regions into a single polypeptide chain, preferably lacking the constant and / or Fc portions present in intact immunoglobulins. For example, a bispecific single-chain antibody can be a construct having two antibody variable regions, e.g., two VH regions, each capable of specifically binding to a different epitope, linked to each other via a short polypeptide spacer, such that the two antibody variable regions present as a single continuous polypeptide chain. Another example of a bispecific single-chain antibody can be a single polypeptide chain having three antibody variable regions, where two antibody variable regions, e.g., one VH and one VL, can constitute an scFv, where the two antibody variable regions are linked to each other via a synthetic polypeptide linker, the latter often being genetically engineered to minimize immunogenicity while maintaining maximum resistance to proteolysis. This scFv can specifically bind to a particular epitope and is linked to an additional antibody variable region, such as a VH region, that can bind to an epitope different from that bound by the scFv. Yet another example of a bispecific single-chain antibody can be a single polypeptide chain having four antibody variable regions.Here, two first antibody variable regions, e.g., a VH region and a VL region, can form one scFv capable of binding to one epitope, while a second VH region and a VL region can form a second scFv capable of binding to a different epitope. Within a single continuous polypeptide chain, individual antibody variable regions of one specificity can be advantageously separated by a synthetic polypeptide linker, while each scFv can be advantageously separated by a short polypeptide spacer, as described above. According to some embodiments, the first binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to some embodiments, the first binding domain of the bispecific antibody comprises two antibody variable domains, preferably scFvs, i.e., VH-VL or VL-VH. According to some embodiments, the second binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. In some embodiments, the second binding domain of the bispecific antibody comprises two antibody variable domains, preferably scFv, i.e., VH-VL or VL-VH. In a minimal form, the total number of antibody variable regions in the bispecific antibody is only two. For example, such an antibody may comprise two VH domains or two VHH domains. In some embodiments, the first and second binding domains of the bispecific antibody each comprise one antibody variable domain, preferably a VHH domain. In some embodiments, the first and second binding domains of the bispecific antibody each comprise two antibody variable domains, preferably scFv, i.e., VH-VL or VL-VH. In this embodiment, the binding agent preferably comprises (i) the heavy chain variable domain (VH) of the first antibody, (ii) the light chain variable domain (VL) of the first antibody, (iii) the heavy chain variable domain (VH) of the second antibody, and (iv) the light chain variable domain (VL) of the second antibody.

[0354] In some embodiments, a bispecific molecule comprises two Fab regions, each directed against a different epitope. In some embodiments, the molecule of the present disclosure is an antigen-binding fragment (Fab)2 complex. A Fab2 complex is composed of two Fab fragments, one of which contains an Fv domain, i.e., a VH domain and a VL domain, specific for one epitope, and the other of which contains an Fv domain specific for another epitope. Each Fab fragment may be composed of two single chains, a VL-CL module and a VH-CH module. Alternatively, each individual Fab fragment may be arranged as a single chain, preferably VL-CL-CH-VH, with the individual variable and constant domains connected by a peptide linker.

[0355] In some embodiments, binding agents according to the present disclosure include various types of bivalent and trivalent single-chain variable fragments (scFvs), which are fusion proteins that mimic the variable domains of two antibodies. Bivalent (or divalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in tandem scFvs. The present disclosure also includes multispecific molecules comprising two or more scFv binding domains.

[0356] Another possibility is to create scFvs with a linker peptide that is too short (about 5 amino acids) for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Even shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. These show even higher affinity for their targets than diabodies.

[0357] A particularly preferred example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small bivalent bispecific antibody fragment. Diabodies contain a heavy chain variable domain (VH) and a light chain variable domain (VL) on the same polypeptide chain, linked by a peptide linker that is too short to allow pairing between the two domains on the same chain (VH-VL). This forces pairing with complementary domains on another chain, promoting the assembly of a dimeric molecule with two functional antigen-binding sites.

[0358] In some embodiments, bispecific or multispecific molecules according to the present disclosure comprise immunoglobulin variable domains (VH, VL) and constant domains (C). In some embodiments, the bispecific molecule is a minibody, preferably a minibody comprising two single VH-VL-C chains linked together via the constant domains (C) of each chain. According to this embodiment, the corresponding variable heavy chain region (VH), the corresponding variable light chain region (VL), and the constant domains (C) are arranged from N- to C-terminus in the order VH(epitope 1)-VL(epitope 1)-(C) and VH(epitope 2)-VL(epitope 2)-C, where C is preferably a CH3 domain, and epitope 1 refers to the first epitope and epitope 2 refers to the second epitope. Pairing of the constant domains results in the formation of a minibody.

[0359] According to another aspect, the bispecific binding agent of the present disclosure is in the format of a bispecific single-chain antibody construct, whereby the construct comprises or consists of at least two binding domains. In some embodiments, each binding domain comprises one variable region from an antibody heavy chain ("VH region"), where the VH region of the first binding domain specifically binds epitope 1 and the VH region of the second binding domain specifically binds epitope 2. The two binding domains are optionally linked to each other by a short polypeptide spacer. Each binding domain may further comprise one variable region from an antibody light chain ("VL region"), where the VH and VL regions in each of the first and second binding domains are linked to each other via a polypeptide linker of sufficient length to allow pairing between the VH and VL regions of the first binding domain and the VH and VL regions of the second binding domain.

[0360] In some embodiments, the binding agents described herein comprise an antibody, e.g., a full-length antibody, comprising a first binding domain. In some embodiments, the binding agents described herein comprise an antibody fragment, such as an scFv or VHH, comprising a second binding domain covalently linked to an antibody comprising the first binding domain. In some embodiments, the binding agent comprises an antibody fragment, such as an scFv or VHH, covalently linked to the N-terminus or C-terminus of the antibody light chain or heavy chain.

[0361] In some embodiments, the binding moiety described herein, e.g., the binding moiety included in a docking compound that binds to a primary target, comprises a DARPin. In some embodiments, the binding moiety binds the particle to immune effector cells, particularly CD8 + It targets T cells, such as T cells.

[0362] The term "DARPin" refers to a designed ankyrin repeat protein. DARPins are based on naturally occurring ankyrin repeat proteins but contain one or more amino acid mutations that may affect, for example, binding affinity to target molecules, cell surface expression, etc. DARPins preferably contain two to three ankyrin repeat modules flanked by N-capping and C-capping repeats. Each ankyrin repeat module contains approximately 33 amino acid residues.

[0363] Ankyrin repeat proteins were identified in 1987 by sequence comparison of four proteins from Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. Breeden and Nasmyth reported multiple copies of a repeat unit of approximately 33 residues in the sequences of swi6p, cddOp, notch, and lin-12 (Breeden et al., Nature 329, 651-654 (1987)). Later, 24 copies of this repeat unit were discovered in the ankyrin protein, and this repeat unit was named ankyrin repeat (Lux et al., Nature 344, 36-42 (1990)). This repeat unit has since been identified in hundreds of proteins from various organisms and viruses (Bork, Proteins 17(4), 363-74 (1993)). These proteins are present in the nucleus, cytoplasm, or extracellular space. This is consistent with the fact that the ankyrin repeat domain of these proteins does not depend on disulfide bridges and therefore does not depend on the oxidative state of the environment. The number of repeat units per protein varies from two to more than 20. The tertiary structure of ankyrin repeat units shares a characteristic fold consisting of a β-hairpin followed by two antiparallel α-helices, terminating in a loop connecting the repeat unit to the next (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6 (1999)). Domains constructed from ankyrin repeat units are formed by stacking the repeat units into extended, curved structures. Proteins containing ankyrin repeat domains often contain additional domains. Although the latter domains have various functions, the function of the ankyrin repeat domain is mostly binding to other proteins. Analysis of the repeat units of these proteins reveals that the target interaction residues are mainly found in the exposed parts of the β-hairpin and the first α-helix. Therefore, these target interaction residues form a large contact surface on the ankyrin repeat domain.This interface is exposed on a framework constructed from stacked units of α-helix 1, α-helix 2, and a loop.

[0364] DARPins that bind to specific targets can be identified by screening combinatorial libraries of DARPins and selecting those with desirable binding properties to the target. Such screening methods are described, for example, in Muench et al., Molecular Therapy, 16(4), 686-693, 2011. For example, target-specific DARPins can be selected from diverse libraries using ribosome display or phage display methods.

[0365] The term "repeat protein" refers to a (poly)peptide / protein comprising one or more repeat domains. In one embodiment, the repeat protein comprises up to four repeat domains. In one embodiment, the repeat protein comprises up to three repeat domains. In one embodiment, the repeat protein comprises up to two repeat domains. In a most preferred embodiment, the repeat protein comprises one repeat domain.

[0366] The individual domains of the repeat proteins can be linked to each other directly or via (poly)peptide linkers. The term "(poly)peptide linker" refers to an amino acid sequence that can link two protein domains. Such linkers include, for example, glycine-serine linkers of variable length, which are known to those skilled in the relevant art.

[0367] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat units (modules). In one embodiment, said repeat units are structural units with identical or similar folding structures, preferably tightly stacked to form a coiled-coil structure, preferably with a common hydrophobic core.

[0368] The term "structural unit" refers to a locally ordered portion of a (poly)peptide formed by three-dimensional interactions between two or more segments of secondary structure that are adjacent to each other along the (poly)peptide chain. Such structural units constitute a structural motif.

[0369] The term "structural motif" refers to the three-dimensional arrangement of secondary structural elements present in at least one structural unit. Structural motifs are well known to those skilled in the relevant art. The structural units may not be able to achieve a defined three-dimensional arrangement by themselves. However, their sequential arrangement as repeat modules within a repeat domain may result in mutual stabilization of adjacent units, resulting in a coiled-coil structure.

[0370] The term "repeat module" refers to a repeated amino acid sequence of a repeat protein derived from a repeat unit of a naturally occurring protein. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family of naturally occurring repeat proteins, such as ankyrin repeat proteins.

[0371] The term "set of repeat modules" refers to the total number of repeat modules present in a repeat domain. Such a "set of repeat modules" present in a repeat domain may comprise two or more consecutive repeat modules and may contain only one type of repeat module in two or more copies, or may contain two or more different types of modules, each present in one or more copies. For example, such a set of repeat modules comprising three repeat modules may comprise, consecutively from N- to C-terminus, repeat module 1, repeat module 2 and repeat module 3.

[0372] Different repeat domains may have the same number of repeat modules per repeat domain or may have different numbers of repeat modules per repeat domain.

[0373] Preferably, the repeat modules comprised in a set are homologous repeat modules. In the context of the present disclosure, the term "homologous repeat module" refers to a repeat module in which 70% or more of the framework residues of said repeat module are homologous. Preferably, 80% or more of the framework residues of said repeat module are homologous. Most preferably, 90% or more of the framework residues of said repeat module are homologous. Computer programs for determining the percentage of homology between polypeptides, such as Fasta, Blast or Gap, are known to those skilled in the relevant art.

[0374] The term "repeat unit" refers to an amino acid sequence comprising one or more naturally occurring protein sequence motifs, said "repeat unit" occurring in multiple copies and exhibiting a defined folding topology common to all said motifs that determines the folding of the protein. Such a repeat unit comprises framework residues and interacting residues.

[0375] An example of such a repeat unit is the ankyrin repeat unit. Naturally occurring proteins comprising two or more such repeat units are referred to as "naturally occurring repeat proteins". The amino acid sequences of the individual repeat units of a repeat protein, when compared to each other, may have a significant number of mutations, substitutions, additions, and / or deletions while substantially retaining the general pattern or motif of the repeat units.

[0376] The term "repeat sequence motif" or "repeat consensus sequence" refers to an amino acid sequence deduced from one or more repeat units. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of the repeat unit. The target interaction residue positions correspond to the target interaction residue positions of the repeat unit. Such a repeat sequence motif comprises fixed positions and randomized positions. The term "fixed position" refers to an amino acid position within a repeat sequence motif, which position is set to a specific amino acid. In many cases, such fixed positions correspond to framework residue positions.

[0377] The term "randomized position" refers to an amino acid position within a repeat motif where two or more amino acids are allowed. Often, such randomized positions correspond to the positions of target interaction residues. However, some positions of framework residues may also be randomized.

[0378] The term "folding topology" refers to the tertiary structure of said repeat units. The folding topology is determined by stretches of amino acids forming at least part of alpha helices or beta sheets, or stretches of amino acids forming linear polypeptides or loops, or any combination of alpha helices, beta sheets and / or linear polypeptides / loops.

[0379] The term "series" refers to an arrangement in which the modules are arranged in tandem.

[0380] Repeat proteins have at least 2, frequently 6 or more, 10 or more, or 20 or more repeat units, and usually about 2 to 6. Most often, repeat proteins are structural and / or adhesive proteins and are found in prokaryotes and eukaryotes, including vertebrates and invertebrates.

[0381] In most cases, the repeat units show a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (different amino acid residues but with similar physicochemical properties), and some of the amino acid residues may be key residues that are highly conserved in different repeat units found in naturally occurring proteins.

[0382] However, a high degree of sequence variability may be possible through insertion and / or deletion and / or substitution of amino acids between the different repeat units found in natural proteins, as long as the common folding topology is maintained.

[0383] The term "framework residue" relates to amino acid residues of a repeat unit or corresponding amino acid residues of a repeat module that contribute to the folding topology, i.e. contribute to the folding of said repeat unit (or module) or contribute to interactions with neighboring units (or modules). Such contributions can be interactions with other residues within the repeat unit (module), or influences on the backbone conformation of the polypeptide, such as found in an alpha helix or a beta sheet, or influences on stretches of amino acids forming a linear polypeptide or a loop.

[0384] The term "target interacting residue" refers to an amino acid residue of a repeat unit, or a corresponding amino acid residue of a repeat module, that contributes to the interaction with the target substance. Such contribution may be a direct interaction with the target substance or an influence on other directly interacting residues, e.g., by stabilizing the conformation of the (poly)peptide of said repeat unit (module), thereby enabling or enhancing the interaction of said directly interacting residue with said target.

[0385] A "target" may be an individual molecule, such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule, and includes any portion of such an individual molecule, or a complex of two or more such molecules. A target may specifically be a molecule on an immune effector cell, in particular CD8.

[0386] In one embodiment, the repeat modules are directly linked. In the context of the present invention, the term "directly linked" refers to repeat modules that are arranged as direct repeats within the repeat protein, without any intervening amino acid sequences.

[0387] In another embodiment, the repeat modules are linked by (poly)peptide linkers. Thus, the repeat modules may be indirectly linked via (poly)peptide linkers as intervening sequences separating the individual modules. An "intervening sequence" may be any amino acid sequence that allows linking the individual modules without interfering with the folding topology or stacking of the modules. Preferably, said intervening sequence is a short (poly)peptide linker of less than 10, more preferably less than 5 amino acid residues.

[0388] In one embodiment, the repeat protein further comprises an N- and / or C-terminal capping module having an amino acid sequence different from any one of said repeat modules. The term "capping module" refers to a polypeptide fused to a repeat module at the N- or C-terminus of a repeat domain, wherein said capping module forms tight tertiary interactions with said repeat module, thereby providing a cap that shields from solvent the hydrophobic core of said repeat module on the side not in contact with consecutive repeat modules.

[0389] Said N-terminal and / or C-terminal capping modules may be or be derived from capping units or other domains found in naturally occurring repeat proteins that flank the repeat units.

[0390] The term "capping unit" refers to a naturally occurring, folded (poly)peptide, which defines a specific structural unit fused to the N- or C-terminus of a repeating unit, with which it forms tight tertiary interactions, thereby providing a cap that shields the hydrophobic core of the repeating unit from solvent on one side. Such capping units may have sequence similarity to the repeating sequence motif.

[0391] antigen receptor Immune effector cells according to the present disclosure express at least one antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that binds to an antigen or its processing product, particularly when present on or presented by a target cell. The immune effector cells can be modified (e.g., ex vivo / in vitro or in vivo in the subject to be treated) to express the antigen receptor. In one embodiment, the modification to express the antigen receptor is performed ex vivo / in vitro. The modified cells can then be administered to a patient. In one embodiment, the modification to express the antigen receptor is performed in vivo. The cells can be endogenous to the patient or can be administered to the patient.

[0392] In one embodiment of all aspects of the invention, the expression of the antigen receptor is on the cell surface.

[0393] The immune effector cell, particularly a T cell, has a first cell surface-expressed antigen receptor. In some embodiments, the immune effector cell, particularly a T cell, has a first and a second cell surface-expressed antigen receptor. The terms "cell surface-expressed antigen receptor" and "antigen receptor" are used synonymously throughout this application.

[0394] In one embodiment, the antigen receptor of the present disclosure may also bind to a cell surface or soluble cytokine, hi this embodiment, the antigen receptor is a cytokine receptor.

[0395] T cell receptor As used herein, the term "T cell receptor" or "TCR" refers to a protein receptor on T cells consisting of a heterodimer of an alpha (α) and a beta (β) chain, although in some cells the TCR consists of a gamma (γδ) and a delta (γδ) chain. In some embodiments, the TCR can be derived from any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells. Each α, β, γ, and δ chain consists of two Ig-like domains: a variable domain (V) that confers antigen recognition via complementarity-determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a connecting peptide and a transmembrane region (TM). The TM region associates with the invariant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with the complex between antigenic peptides bound to proteins encoded by the major histocompatibility complex (MHC).

[0396] Chimeric Antigen Receptor Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors (CARs) is a promising anticancer treatment because CAR-engineered T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells are genetically engineered to express a CAR specifically directed against an antigen on the patient's tumor cells and then reinfused into the patient.

[0397] In some embodiments, the first antigen receptor is a CAR. In some embodiments, the first and second antigen receptors are CARs.

[0398] According to the present invention, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, that expresses the CAR on its cell surface. Such cells do not necessarily require antigen processing and presentation to recognize the target cell, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by a CAR results in activation of the immune effector cell that expresses the CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0399] CARs contain a target-specific binding element, generally referred to as an antigen-binding moiety or domain, which is part of the extracellular domain of the CAR. The antigen-binding domain recognizes a ligand that serves as a cell surface marker on target cells associated with a particular disease state. Specifically, the CARs of the present disclosure target antigens, such as tumor antigens, on diseased cells, such as tumor cells.

[0400] In one embodiment, the binding domain in the CAR specifically binds to an antigen. In one embodiment, the antigen to which the binding domain in the CAR binds is expressed in cancer cells (tumor antigen). In one embodiment, the antigen is expressed on the surface of cancer cells. In one embodiment, the binding domain binds to the extracellular domain of the antigen or an epitope within the extracellular domain. In one embodiment, the binding domain binds to a natural epitope of the antigen present on the surface of a living cell.

[0401] In one embodiment of the present invention, the antigen-binding domain comprises a variable region of an immunoglobulin heavy chain (VH) with specificity for an antigen and a variable region of an immunoglobulin light chain (VL) with specificity for an antigen. In one embodiment, the immunoglobulin is an antibody. In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked via a peptide linker. Preferably, the antigen-binding moiety in the CAR is an scFv.

[0402] The CAR is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain is not naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is modified by amino acid substitution to avoid binding of the same or different surface membrane proteins to the transmembrane domain and to minimize interaction with other members of the receptor complex. The transmembrane domain may be derived from natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region of particular use in the present invention may be derived from (i.e., include at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0403] In some cases, a CAR of the present disclosure comprises a hinge domain that forms the link between the transmembrane domain and the extracellular domain.

[0404] The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include a truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0405] It is known that signals generated via the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0406] In one embodiment, the CAR comprises a primary cytoplasmic signaling sequence derived from CD3-ζ. Additionally, the cytoplasmic domain of the CAR can comprise a CD3-ζ signaling domain combined with a costimulatory signaling region.

[0407] The identity of the costimulatory domain is limited only by its ability to enhance cell proliferation and survival upon binding of the targeting moiety by the CAR. Suitable costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR) superfamily, CD134 (OX40), a member of the TNFR superfamily receptor, and CD278 (ICOS), a CD28 superfamily costimulatory molecule expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described costimulatory domains can be used without adversely affecting the present invention, provided that the variant has the same or similar activity as the domain on which it is modeled. Such variants have at least about 80% sequence identity to the amino acid sequence of the domain from which they are derived. In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Specific combinations include all possible variations of the four described domains, with specific examples including CD28 + CD137 (4-1BB) and CD28 + CD134 (OX40).

[0408] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0409] In one embodiment, the CAR comprises a signal peptide that directs the nascent protein to the endoplasmic reticulum. In one embodiment, the signal peptide is located before the antigen-binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin, such as IgG.

[0410] CARs can contain the above domains together in the form of a fusion protein. Such fusion proteins will generally comprise an antigen-binding domain, one or more costimulatory domains, and a signal transduction sequence linked in an N- to C-terminal direction. However, the CARs of the present invention are not limited to this configuration, and other configurations are also permissible, including a binding domain, a signal transduction domain, and one or more costimulatory domains. Since the binding domain must be free to bind to the antigen, it will be understood that the configuration of the binding domain in the fusion protein will generally be such that the domain is displayed on the outside of the cell. Similarly, since the costimulatory domain and the signal transduction domain play a role in inducing the activity and proliferation of cytotoxic lymphocytes, the fusion protein will generally display these two domains inside the cell.

[0411] In one embodiment, the CAR molecule is i) a target antigen (e.g., CLDN6 or CLDN18.2) binding domain; ii) a transmembrane domain; and iii) an intracellular domain comprising a 4-1BB costimulatory domain and a CD3-ζ signaling domain; Includes.

[0412] In one embodiment, the antigen binding domain comprises an scFv. In one embodiment, the transmembrane domain is selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19 , IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The antigen-binding domain comprises a transmembrane domain of a protein selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof. In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In one embodiment, the antigen-binding domain is linked to the transmembrane domain by a hinge domain. In one embodiment, the hinge domain is a CD8α hinge domain.

[0413] In one embodiment, the CAR molecule of the invention comprises: i) a target antigen-binding domain; ii) CD8α hinge domain; iii) the CD8α transmembrane domain; and iv) an intracellular domain comprising a 4-1BB costimulatory domain and a CD3-ζ signaling domain; Includes.

[0414] According to the present disclosure, when a CAR is present on a T cell, it recognizes an antigen, such as on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, such that the T cell is stimulated and / or expanded or exerts an effector function as described above.

[0415] immune effector cell activator molecule The immune effector cells or particles of the invention comprise a second nucleic acid encoding an immune effector activator molecule, also referred to throughout this application as an "activator molecule."

[0416] In some embodiments, the activator molecule is transiently expressed.

[0417] A wide variety of molecules can be activator molecules, as long as they enable the activation, expansion, differentiation, and / or proliferation of immune effector cells, particularly T cells.

[0418] In some embodiments, the activator molecule is a cytokine.

[0419] Examples of cytokines include interferons such as interferon alpha (IFN-α) or interferon gamma (IFN-γ), interleukins such as IL-2, IL-7, IL-10, IL-12, IL-15, and IL-23, colony-stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factors. According to another embodiment, the immunostimulatory agent includes an adjuvant-type immunostimulatory agent such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0420] The term "cytokine" refers to proteins with molecular weights of approximately 5 to 60 kDa that are involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, cytokines, upon release, affect the behavior of cells in the vicinity of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to regulated cell surface interferon receptors, thereby inhibiting viral replication within the cell. Interferons can be classified into two types: Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factors (TNFs), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), and variants and derivatives thereof.

[0421] According to the present disclosure, the cytokine may be a naturally occurring cytokine, or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, particularly a mammal.

[0422] In some embodiments, the activator molecule is a second cell surface expressed antigen receptor described herein, preferably a CAR or a TCR.

[0423] In some embodiments, the activator molecule is an antigen, particularly an antigen to which the first cell surface-expressed antigen receptor specifically binds. In some embodiments, the antigen is an antigen described herein.

[0424] The term "antigen" may be referred to as "antigen targeted by an antigen receptor," "cognate antigen molecule," or simply "antigenic molecule."

[0425] In one embodiment, the cognate antigen molecule comprises an antigen or fragment thereof, or a variant of the antigen or fragment, expressed by the target cell targeted by the antigen receptor.

[0426] In one embodiment, expression of the cognate antigen molecule is on the cell surface.

[0427] Binding of the antigen by the antigen receptor preferably results in the stimulation, priming, and / or expansion of immune effector cells. The stimulated, primed, and / or expanded immune effector cells are preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, the antigen may include a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to the disease-associated antigen. In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the stimulation, priming, and / or expansion of immune effector cells, wherein the stimulated, primed, and / or expanded immune effector cells target the antigen, i.e., the disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, an antigen may correspond to or comprise a disease-associated antigen, or may correspond to or comprise a fragment of a disease-associated antigen, or may comprise a fragment of a disease-associated antigen, or may correspond to or comprise an antigen homologous to a disease-associated antigen or a fragment thereof. When an antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen targeted by a first and / or second antigen receptor of an immune effector cell, or a sequence homologous to an epitope of the disease-associated antigen. Thus, according to the present invention, an antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that is capable of stimulating, priming, and / or expanding immune effector cells bearing an antigen receptor that binds to the antigen or a cell expressing the antigen. Preferably, the antigen (which resembles a disease-associated antigen) provides a relevant epitope for binding by an antigen-binding domain present in the immune effector cell. In one embodiment, the antigen (which resembles a disease-associated antigen) is expressed on the surface of an immune effector cell so as to provide a relevant epitope for binding by other immune effector cells.In one embodiment, the antigen (similar to the disease-associated antigen) is expressed by and presented on the surface of immune effector cells in the context of an MHC, so as to provide relevant epitopes for binding by other immune effector cells. The antigen may be a recombinant antigen.

[0428] In one embodiment of all aspects of the invention, the second nucleic acid encoding the antigen is expressed in an immune effector cell and provides the antigen or its processing product for binding by an antigen receptor expressed by other immune effector cells, said binding resulting in stimulation, priming and / or expansion of the other immune effector cells.

[0429] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, such as a T cell that binds to the reference amino acid sequence or a cell that expresses the reference amino acid sequence, the amino acid sequence induces an immune response with specificity that reacts with the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties as the antigen targeted by T cells and / or exerts the same or essentially the same effect with respect to stimulating, priming, and / or expanding T cells.

[0430] As used herein, "activation" or "stimulation" refers to the state of an immune effector cell, such as a T cell, that has received sufficient stimulation to induce detectable cell proliferation. Activation may also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell undergoing cell division.

[0431] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0432] The term "clonal expansion" or "expansion" refers to the process by which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion leads to differentiation of lymphocytes.

[0433] The term "antigen" relates to an agent comprising an epitope against which an immune response can be generated. The term "antigen" particularly includes proteins and peptides. In one embodiment, an antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. An antigen or its processing product, e.g., a T-cell epitope, is in one embodiment bound by an antigen receptor. Thus, an antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0434] The term "disease-associated antigen" is used in the broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to elicit a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, disease-associated antigens or epitopes thereof can be used for therapeutic purposes. Disease-associated antigens may be associated with infection by a microorganism, typically a microbial antigen, or may be associated with cancer, typically a tumor.

[0435] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, it refers to an antigen produced intracellularly or produced as a surface antigen on a tumor cell. Tumor antigens are usually preferentially expressed by cancer cells (e.g., expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, are expressed exclusively by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, L DLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 Minor BCR-abL, Pml / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1. Particularly preferred tumor antigens are proteins of the claudin family, such as CLAUDIN-6 or CLAUDIN-18.2.

[0436] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0437] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0438] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as a receptor or antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, for example, by an extracellularly located antibody. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be via one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0439] "Cell surface" or "surface of a cell" is used according to its normal meaning in the art and thus includes the outside of a cell that is accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible for binding, for example, by an antigen-specific antibody added to the cell. In one embodiment, the antigen expressed on the surface of a cell is a transmembrane protein having an extracellular portion that is recognized by a CAR.

[0440] In the context of the present invention, the term "extracellular portion" or "exodomain" refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside said cell, for example by an extracellularly located binding molecule, such as an antibody. Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

[0441] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length. For example, an epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0442] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may also be effective.

[0443] In one embodiment, the target antigen of the first antigen receptor is a tumor antigen, and the antigen or a fragment thereof (e.g., epitope) as the activator molecule is derived from the tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of said neoantigen containing one or more amino acid changes.

[0444] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy relies on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host.

[0445] Peptide and protein antigens can be from 2 to 100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0446] In one embodiment of all aspects of the present invention, the antigen is expressed in a diseased cell, such as a cancer cell. In one embodiment, the antigen is expressed on the surface of a diseased cell, such as a cancer cell. In one embodiment, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In one embodiment, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In one embodiment, binding of a CAR expressed by and / or present on a T cell to an antigen present on a diseased cell, such as a cancer cell, results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0447] particle Particles according to the present disclosure comprise at least first and second nucleic acid molecules. In some embodiments, the particles may comprise additional nucleic acids. In some embodiments, the particles comprise a third nucleic acid, preferably two or more third nucleic acids. In some embodiments, the particles comprise a third and fourth nucleic acid, preferably two or more third and / or fourth nucleic acids. In some embodiments, the particles comprise a third and fifth nucleic acid, preferably two or more third and / or fifth nucleic acids. In some embodiments, the particles comprise a third, fourth, and fifth nucleic acid, preferably two or more third, fourth, and / or fifth nucleic acids.

[0448] To overcome the obstacles to safe and effective nucleic acid delivery, nucleic acid can be administered with one or more delivery vehicles, which can protect nucleic acid from degradation, maximize delivery to target cells, and minimize exposure to non-target cells.Such nucleic acid delivery vehicles can complex or encapsulate nucleic acid, and can comprise various materials, including polymers and lipids.In some embodiments, such nucleic acid delivery vehicles can form particles containing nucleic acid, preferably DNA and / or RNA.

[0449] The DNA or RNA described herein, particularly mRNA, can be present in particles comprising (i) DNA and / or RNA, and (ii) at least one cationic or cationically ionizable compound, such as a polymer or lipid, that complexes the DNA and / or RNA. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged DNA and RNA are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid, particularly DNA and / or RNA particles.

[0450] Different types of nucleic acid-containing particles have previously been described as suitable for the delivery of DNA and / or RNA in particulate form (see, e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral DNA and / or RNA delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acid from degradation and, depending on the specific chemical properties, can aid in cellular uptake and endosomal escape.

[0451] In the context of the present disclosure, the term "particle" refers to a structured entity formed by molecules or molecular complexes, particularly particle-forming compounds. In some embodiments, a particle comprises an envelope (e.g., one or more layers or lamellae) made from one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may also contain additional substances (e.g., additional lipids) that do not necessarily have to be amphiphilic. Thus, a particle may have a monolayer or multilayer structure, and the substances that make up one or more layers or lamellae comprise one or more types of amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), optionally in combination with additional substances (e.g., additional lipids) that do not necessarily have to be amphiphilic. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0452] "DNA particles," "RNA particles," or "DNA and RNA particles" can be used to deliver DNA and / or RNA to a desired target site (e.g., a cell, tissue, organ, etc.). The DNA and / or RNA particles can be formed from lipids, including at least one cationic lipid or cationically ionizable lipid. Without intending to be bound by any theory, it is believed that the cationic lipid or cationically ionizable lipid combines with the nucleic acid to form aggregates, which result in colloidally stable particles.

[0453] The DNA and / or RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0454] The lipoplexes (LPX) described herein are obtained by mixing two aqueous phases: one containing RNA and one containing a lipid dispersion. In some embodiments, the lipid phase contains liposomes.

[0455] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles, in which the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture can form a lamellar (bilayer) phase as needed in the applied aqueous environment.

[0456] In some embodiments, liposomes comprise a single or multiple phospholipid bilayer surrounding an aqueous core (also referred to herein as an aqueous lumen). They can be prepared from materials with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for DNA and / or RNA delivery are amphiphilic in nature, consisting of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0457] In some embodiments, the lipoplexes are multilamellar liposome-based formulations formed by electrostatic interactions between cationic liposomes and nucleic acids. In some embodiments, the formed lipoplexes have distinct internal arrangements of molecules that result from the conversion of the liposomal structure into compact DNA and / or RNA-lipoplexes.

[0458] In some embodiments, LPX particles comprise amphiphilic lipids, particularly cationic amphiphilic lipids or cationically ionizable amphiphilic lipids, as described herein, and DNA and / or RNA (particularly mRNA). In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic amphiphilic lipids or cationically ionizable amphiphilic lipids) and negatively charged RNA (particularly mRNA) result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can typically be synthesized using cationic or cationically ionizable amphiphilic lipids, such as DOTMA and / or DODMA, and optionally additional lipids, such as DOPE or DSPC.

[0459] In general, lipid nanoparticles (LNPs) are typically obtained by directly mixing DNA and / or RNA in an aqueous phase with lipids in a phase containing an organic solvent, such as ethanol. In this case, lipids or lipid mixtures that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0460] In some embodiments, LNPs comprise or consist of cationic lipids / cationically ionizable lipids and helper lipids such as phospholipids, cholesterol, and / or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the DNA and / or RNA LNPs described herein, the DNA and / or RNA (particularly mRNA) is bound by cationically ionizable lipids that occupy the central core of the LNP. In some embodiments, the polymer-conjugated lipids, along with the phospholipids, form the surface of the LNP. In some embodiments, charged and uncharged forms of cholesterol and cationically ionizable lipids can be distributed throughout the LNP.

[0461] In some embodiments, the DNA and / or RNA (e.g., mRNA) described herein may be non-covalently associated with a particle described herein. In embodiments, the DNA and / or RNA (particularly mRNA) may be attached to the outer surface of the particle (surface RNA (particularly surface mRNA)) and / or contained within the particle (encapsulated DNA and / or RNA (particularly encapsulated mRNA)).

[0462] In some embodiments, the particles described herein (e.g., LNP and LPX) have a diameter in the range of about 10 to about 2000 nm, e.g., at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm), and / or up to about 1900 nm (e.g., up to about 1800 nm, up to about 1700 nm, up to about 1600 nm, up to about 1500 nm, up to about 1400 nm, up to about 1300 nm, up to about 1200 nm, maximum about 1100 nm, maximum about 1000 nm, maximum about 950 nm, maximum about 900 nm, maximum about 850 nm, maximum about 800 nm, maximum about 750 nm, maximum about 700 nm, maximum about 650 nm, maximum about 600 nm, maximum about 550 nm, or maximum about 500 nm), for example, in the range of about 20 to about 1500 nm, for example, about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 9 In some embodiments, the particles described herein (e.g., LNP and LPX) have a size (e.g., diameter) ranging from about 40 nm to about 200 nm, e.g., about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0463] In some embodiments, the particles described herein (e.g., LNP and LPX) have a particle size of about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm , about 50nm to about 250nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 600nm, about 100nm to about 500nm, about 100nm to about 450nm, about 100nm to about 400nm, about 100nm to about 350nm, about 100nm to about 300nm, about 100nm to about 250nm, Approximately 100nm to approximately 200nm, approximately 150nm to approximately 1000nm, approximately 150nm to approximately 800nm, approximately 150nm to approximately 700nm, approximately 150nm to approximately 600nm, approximately 150nm to approximately 500nm m, about 150nm to about 450nm, about 150nm to about 400nm, about 150nm to about 350nm, about 150nm to about 300nm, about 150nm to about 250nm, about 150nm to about 200n In some embodiments, the particles described herein (e.g., LNP and LPX) have an average diameter in the range of about 40 nm to about 200 nm, e.g., about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0464] The DNA and / or RNA particles (particularly mRNA particles) described herein may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the DNA and / or RNA particles may exhibit a polydispersity index in the range of about 0.01 to about 0.4, or about 0.1 to about 0.3.

[0465] The N / P ratio gives the ratio of the number of nitrogen groups in lipids to the number of phosphate groups in nucleic acids. Because nitrogen atoms are usually positively charged (depending on pH) and phosphate groups are negatively charged, this correlates with the charge ratio. The N / P ratio is pH dependent when charge balance exists. Because positively charged nanoparticles are advantageous for transfection, lipid formulations can be formed with an N / P ratio greater than 4 and up to 12. In this case, DNA and / or RNA are considered to be completely bound to the nanoparticles.

[0466] The present disclosure describes compositions comprising DNA and / or RNA (especially mRNA) and at least one cationic lipid or cationic ionizable lipid that associates with DNA and / or RNA to form DNA and / or RNA particles, as well as formulations comprising such particles.DNA and / or RNA particles can comprise DNA and / or RNA complexed in different forms by non-covalent interactions with the particles.The particles described herein are not virus particles, and in particular are not infectious virus particles, i.e., they cannot infect cells with the virus.

[0467] Suitable cationic lipids or cationically ionizable lipids form DNA and / or RNA particles and are included in the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with DNA and / or RNA to form DNA and / or RNA particles. Such components include any component that can become part of a DNA and / or RNA particle.

[0468] In some embodiments, DNA and / or RNA particles (particularly mRNA particles) comprise multiple types of DNA and / or RNA molecules whose molecular parameters may be similar or different from one another in terms of basic structural elements such as molar mass or molecular structure, capping, coding regions, or other features.

[0469] In particle formulations, each DNA and / or RNA species can be formulated separately as an individual particle formulation. In that case, each individual particle formulation will contain one DNA and / or RNA species. The individual particle formulations can be present as separate entities, for example, in separate containers. Such formulations are obtained by providing each DNA and / or RNA species separately (typically in the form of a DNA- and / or RNA-containing solution) with a particle-forming agent, thereby allowing particles to form. Each particle will contain only the specific DNA and / or RNA species provided when the particle is formed (individual particle formulation). In some embodiments, a composition, such as a pharmaceutical composition, contains two or more individual particle formulations. Each pharmaceutical composition is referred to as a mixed particle formulation. A mixed particle formulation according to the present disclosure can be obtained by separately forming individual particle formulations and then mixing the individual particle formulations. The mixing step can result in a formulation containing a mixed population of DNA and / or RNA-containing particles. The individual particle populations can also be present together in a single container containing a mixed population of individual particle formulations. Alternatively, all DNA and / or RNA species of pharmaceutical composition can be formulated together as a combined particle preparation.This preparation can be obtained by providing the combined preparation (typically a combined solution) of all DNA and / or RNA species together with a particle forming agent, thereby allowing particle formation.In contrast to mixed particle preparations, combined particle preparations typically comprise particles that contain two or more DNA and / or RNA species.In combined particle compositions, different DNA and / or RNA species are typically present together in a single particle.

[0470] polymer Polymers are commonly used materials for nanoparticle-based delivery due to their high chemical flexibility. Cationic polymers are typically used to electrostatically condense negatively charged DNA and / or RNA onto particles, particularly nanoparticles. These positively charged groups often consist of amines that change protonation state at pH 5.5-7.5, which is thought to lead to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have become widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. These synthetic polymers are also suitable as cationic polymers herein.

[0471] As used herein, "polymer" is given its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or in some cases, there may be more than one type of repeating unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may also be present within the polymer.

[0472] When more than one type of repeating unit is present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer, as used herein, can be a copolymer. The repeating units forming a copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., consisting of one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block). A block copolymer can have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0473] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. In certain embodiments, biocompatible polymers are biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment, such as within the body.

[0474] In certain embodiments, the polymer can be protamine or a polyalkyleneimine.

[0475] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, specifically binding to DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and yields primarily arginine upon hydrolysis. In its purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0476] In accordance with the present disclosure, the term "protamine" as used herein is meant to include any protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and multimeric forms of said amino acid sequence or fragments thereof, as well as polypeptides that are artificial and specifically designed for a particular purpose and cannot be isolated from natural or biological sources (synthetic).

[0477] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-10 to 10 Da, preferably 1,000 to 10 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0478] According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0479] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In one embodiment, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can be associated, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is entrapped or encapsulated.

[0480] The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

[0481] lipids As used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and distinct phases. One of these phases is composed of lipid bilayers, which exist in aqueous environments as vesicles, multilamellar / unilamellar liposomes, or membranes. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and those groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, such as carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0482] As used herein, the term "hydrophobic" refers to any molecule, moiety, or group that is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. A monovalent radical of a hydrocarbon is referred to herein as hydrocarbyl. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen, provided that the group is substantially immiscible or insoluble in aqueous solution.

[0483] The term "hydrocarbon" includes acyclic, e.g., straight-chain (linear) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl, as defined herein. It is understood that one or more of the hydrogen atoms of the alkyl, alkenyl, or alkynyl can be replaced with other atoms, e.g., halogen, oxygen, or sulfur. Unless otherwise specified, hydrocarbon groups can also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0484] The term "alkyl" refers to a saturated, straight-chain or branched, monovalent hydrocarbon moiety which can have 1 to 30, typically 1 to 20, and often 6 to 18 carbon atoms. Exemplary non-polar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0485] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond and a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2; if the alkenyl group has an odd number of carbon atoms, the result of the division is rounded down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0486] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. An alkynyl group can optionally have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2; if the alkynyl group has an odd number of carbon atoms, the result of the division is rounded down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2, carbon-carbon triple bonds.

[0487] The term "alkylene" refers to a saturated, straight-chain or branched, divalent hydrocarbon moiety which can have 1 to 30, typically 2 to 20, and often 4 to 12 carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octadecamethylene, and the like.

[0488] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond and a total of 2 to 30, typically 2 to 20, and often 4 to 12 carbon atoms. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2; if the alkenylene group has an odd number of carbon atoms, the result of the division is rounded down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0489] The term "cycloalkyl" refers to the cyclic, non-aromatic forms of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclocodecyl, cyclocodecenyl, and adamantyl. Cycloalkyl groups may consist of one ring (monocyclic), two rings (bicyclic), or three or more rings (polycyclic).

[0490] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10) carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerene.

[0491] The term "aromatic" as used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this disclosure. Similarly, if a bicyclic or polycyclic aryl (such as naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this disclosure (even if one ring, such as 1,2-dihydronaphthyl, is still aromatic).

[0492] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water and the non-polar portion is insoluble in water. Furthermore, the polar portion can have either a formal positive or a formal negative charge. Alternatively, the polar portion can have both a formal positive and a formal negative charge and can be a zwitterion or inner salt. For purposes of this disclosure, an amphiphilic compound can be, but is not limited to, one or more of natural or non-natural lipids and lipid-like compounds.

[0493] The terms "lipid-like material," "lipid-like compound," or "lipid-like molecule" refer to substances, particularly amphiphiles, that are structurally and / or functionally related to lipids but are not strictly considered lipids. For example, this term includes compounds that can form amphiphilic layers due to their presence in vesicles, multilamellar / unilamellar liposomes, or membranes within an aqueous environment, and includes surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, this term includes molecules containing hydrophilic and hydrophobic moieties with different structural configurations that may or may not resemble lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSLs) and synthetic function-spacer-sterol constructs (FSSs), as well as artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Conventional surfactant monomers, containing only a single linear alkyl chain and a hydrophilic head group, are generally conical in shape. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble. Lipids also have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—but lipids differ from surfactants in the shape of the monomer, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" should be interpreted to encompass both lipids and lipid-like materials, unless otherwise indicated herein or clearly contradicted by context.

[0494] Generally, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used synonymously with fat, but fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as steroids, i.e., cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0495] Fatty acids, or fatty acid residues, are a diverse group of molecules made up of hydrocarbon chains terminating in a carboxylic acid group. This arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chains, typically 4 to 24 carbons long, can be saturated or unsaturated and may be attached to functional groups, including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, they can be either cis or trans geometric isomers, which greatly influence the molecular configuration. Cis double bonds cause bending of the fatty acid chain, and the greater the number of cis double bonds in the chain, the greater the effect. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0496] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. An additional subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0497] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by ester bonds to two fatty acid-derived "tails" and a phosphate ester bond to a single "head" group. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0498] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The predominant phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues linked via glycosidic bonds to a sphingoid base. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0499] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0500] Glycolipids describe compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0501] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes as well as iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They encompass a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and possess great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0502] According to the present disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0503] Cationic lipids / cationically ionizable lipids In some embodiments, the DNA and / or RNA compositions and formulations and nucleic acid particles described herein comprise at least one cationic lipid or cationically ionizable lipid as a particle-forming agent. The cationic lipids or cationically ionizable lipids contemplated for use herein include any cationic lipids or cationically ionizable lipids (including lipid-like materials) that can electrostatically bind to nucleic acids. In some embodiments, the cationic lipids or cationically ionizable lipids contemplated for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0504] As used herein, "cationic lipid" refers to a lipid or lipid-like material that has a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl chain, or more acyl chains, and the head group of the lipid typically carries a positive charge.

[0505] In some embodiments, cationic lipid only has net positive charge at certain pH, particularly acidic pH, but at different, preferably higher pH such as physiological pH, it does not have net positive charge, preferably has no charge, i.e., is neutral.This ionizable behavior is believed to improve efficacy by helping endosomal escape and reducing toxicity, compared with particles that remain cationic at physiological pH.

[0506] As used herein, " cationically ionizable lipid " refers to lipid or lipid-like material that has net positive charge or is neutral, i.e., is not permanently cationic.Therefore, depending on the pH of the composition in which the cationically ionizable lipid is dissolved, the cationically ionizable lipid is either positively charged or neutral.For the purpose of this disclosure, the cationically ionizable lipid is covered by the term "cationic lipid", unless otherwise contradicted by the context.

[0507] In some embodiments, cationic lipids or cationically ionizable lipids comprise a head group that contains at least one nitrogen atom (N) that is positively charged or protonatable, e.g., under physiological conditions.

[0508] Examples of cationic lipids or cationically ionizable lipids include, but are not limited to, N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammoniumpropane (DDAB); Pan (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium 1,2-Dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-Dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), Dioctadecylamidoglycylspermine (DOGS), 3-dimethyl Amino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DM A), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide nium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxybenzoyl) N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(tetradecyloxyethyl)propan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), (Dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl)ethyl] ... Decylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoylethylamino)-ethyl]-amino}-ethylamino]propionamide (Lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

[0509] In some embodiments, the cationic lipid or cationically ionizable lipid is DOTMA. In some embodiments, the cationic lipid or cationically ionizable lipid is DODMA.

[0510] DOTMA is a cationic lipid with a quaternary amine head group. The structure of DOTMA can be represented as follows: TIFF2026504516000022.tif19170

[0511] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows: TIFF2026504516000023.tif13170

[0512] In some embodiments, the cationic lipid or cationically ionizable lipid may comprise from about 10 mol% to about 95 mol%, from about 20 mol% to about 95 mol%, from about 20 mol% to about 90 mol%, from about 30 mol% to about 90 mol%, from about 40 mol% to about 90 mol%, or from about 40 mol% to about 80 mol% of the total lipid present in the particle.

[0513] Additional fats The DNA and / or RNA compositions and formulations and DNA and / or RNA particles described herein may also contain lipids (including lipid-like materials) other than cationic lipids or cationically ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic lipids or non-cationically ionizable lipids or lipid-like materials). Collectively, anionic lipids and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. In addition to cationic lipids or cationically ionizable lipids, the addition of other hydrophobic moieties, such as cholesterol and lipids, can optimize the formulation of DNA and / or RNA particles to improve particle stability and nucleic acid delivery effectiveness.

[0514] One or more additional lipids may or may not affect the overall charge of DNA and / or RNA particles.In some embodiments, one or more additional lipids are non-cationic lipids or lipid-like materials.Non-cationic lipids can include, for example, one or more anionic lipids and / or neutral lipids.As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH.As used herein, "neutral lipid" refers to any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at a selected pH.

[0515] In some embodiments, the DNA and / or RNA compositions and formulations and DNA and / or RNA particles described herein comprise a cationic lipid or a cationically ionizable lipid and one or more additional lipids.

[0516] Without wishing to be bound by theory, the amount of cationic lipid or cationically ionizable lipid, relative to the amount of one or more additional lipids, can affect important properties of the DNA and / or RNA particles, such as the charge of the DNA and / or RNA, particle size, stability, tissue selectivity, and biological activity. Thus, in some embodiments, the molar ratio of cationic lipid or cationically ionizable lipid to one or more additional lipids is about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1.

[0517] In some embodiments, the one or more additional lipids included in the DNA and / or RNA compositions and formulations and DNA and / or RNA particles described herein comprise one or more of neutral lipids, steroids, and combinations thereof.

[0518] In some embodiments, the one or more additional lipids comprise a neutral lipid that is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin.Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (D BPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine ( DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and even phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0519] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0520] Thus, in some embodiments, the DNA and / or RNA compositions and formulations and DNA and / or RNA particles described herein comprise (1) a cationic lipid or cationically ionizable lipid and a phospholipid such as DSPC or DOPE, or (2) a cationic lipid or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

[0521] In some embodiments, the DNA and / or RNA particles described herein (particularly particles comprising mRNA) comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0522] DSPC is a neutral phospholipid. The structure of DSPC can be represented as follows: TIFF2026504516000024.tif29170

[0523] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows: TIFF2026504516000025.tif24170

[0524] The structure of cholesterol can be represented as follows: TIFF...

Claims

1. 1. An immune effector cell comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the immune effector cell, and / or (ii) the activator molecule is transiently expressed.

2. The immune effector cell of claim 1 , wherein the immune effector cell is isolated.

3. The immune effector cell of claim 1 or claim 2, wherein the first nucleic acid molecule is DNA or RNA.

4. The immune effector cell of claim 1 , wherein the first nucleotide sequence is integrated into a genomic nucleic acid molecule of the immune effector cell.

5. The immune effector cell of claim 1 , wherein the genomic nucleic acid molecule is a chromosome, an episome, or a non-viral episome.

6. 6. The immune effector cell of claim 4 or claim 5, wherein the first nucleotide sequence is integrated into the genomic nucleic acid molecule via a DNA-based transposon system, a virus-based retrotransposon system, or a polyA-based retrotransposon system.

7. The immune effector cell of claim 1 , wherein the first nucleotide sequence is contained within a transposable element.

8. The immune effector cell of any one of claims 1 to 7, wherein the immune effector cell further comprises a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity.

9. The immune effector cell of claim 8 , wherein the third nucleic acid molecule is DNA or RNA.

10. The immune effector cell of claim 8 or claim 9, wherein the third nucleic acid molecule is mRNA.

11. The immune effector cell according to any one of claims 8 to 10, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof having transposase activity.

12. The immune effector cell of any one of claims 8 to 12, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.

13. 13. The immune effector cell of any one of claims 8 to 12, wherein (i) the third nucleic acid molecule is not integrated into a genomic nucleic acid molecule of the immune effector cell, and / or (ii) the encoded molecule having transposase activity is transiently expressed.

14. 14. The immune effector cell of claim 1, wherein the first cell surface-expressed antigen receptor is stably expressed.

15. 15. The immune effector cell of any one of claims 1 to 14, wherein the activator molecule enables activation, expansion, differentiation and / or proliferation of the immune effector cell.

16. The immune effector cell of claim 1 , wherein the activator molecule is a non-coding RNA or a protein.

17. 17. The immune effector cell of any one of claims 1 to 16, wherein the activator molecule binds to the extracellular portion of the first cell surface-expressed antigen receptor.

18. 17. The immune effector cell of claim 1, wherein the activator molecule is a cytokine.

19. 17. The immune effector cell of claim 1, wherein the activator molecule is a second cell surface-expressed antigen receptor, and the extracellular portions of the first and second cell surface-expressed antigen receptors do not bind to the same binding target.

20. 20. The immune effector cell of claim 19, wherein the immune effector cell further comprises a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of the first cell surface-expressed antigen receptor.

21. 21. The immune effector cell of claim 19 or claim 20, wherein the immune effector cell further comprises a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor.

22. 22. The immune effector cell of claim 20 or 21, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises both a fourth nucleotide sequence encoding a binding target of a first cell surface-expressed antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor.

23. 23. The immune effector cell of any one of claims 20 to 22, wherein (i) the fourth and / or fifth nucleic acid molecule is not integrated into a genomic nucleic acid molecule of the immune effector cell, and / or (ii) the encoded binding target of the fourth and / or fifth nucleic acid molecule is transiently expressed.

24. 24. The immune effector cell of any one of claims 20 to 23, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.

25. 25. The immune effector cell of any one of claims 20 to 24, wherein the fourth and / or fifth nucleic acid molecule is mRNA.

26. 26. The immune effector cell of any one of claims 1 to 25, wherein the first cell surface-expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

27. 27. The immune effector cell of any one of claims 19 to 26, wherein the second cell surface-expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

28. 28. The immune effector cell of any one of claims 19 to 27, wherein the binding target of the second cell surface-expressed antigen receptor is expressed on or from a different cell than the cell expressing the binding target of the first cell surface-expressed antigen receptor.

29. 29. The immune effector cell of any one of claims 1 to 28, wherein the binding target of the first cell surface-expressed antigen receptor is a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

30. 31. The immune effector cell of any one of claims 19 to 30, wherein the binding target of the second cell surface-expressed antigen receptor is a cell surface-expressed or soluble protein, or an epitope thereof.

31. The immune effector cell of claim 30 , wherein the cell surface-expressed protein is a glycoprotein or a cell surface-expressed cytokine.

32. 32. The immune effector cell of claim 30 or claim 31, wherein the cell surface expressed protein is a cluster of differentiation (CD) protein.

33. The immune effector cell of claim 30 , wherein the soluble protein is a soluble cytokine.

34. 35. The immune effector cell of any one of claims 19 to 34, wherein the binding target of the second cell surface-expressed antigen receptor is a cell surface protein expressed on a blood cell, the blood cell preferably being another immune effector cell.

35. 35. The immune effector cell of claim 34, wherein the blood cell is a T cell, NK cell, dendritic cell, macrophage, or B cell.

36. 36. The immune effector cell of claim 34 or claim 35, wherein the cell surface protein is CD19.

37. The immune effector cell of claim 30, wherein the cell surface protein is CLDN18.

2.

38. 38. The immune effector cell of any one of claims 1 to 37, wherein the second nucleic acid molecule is DNA or RNA.

39. 39. The immune effector cell of any one of claims 1 to 38, wherein the second nucleic acid molecule is mRNA.

40. 40. The immune effector cell of claim 3, 9, 10, 24, 25, 38 or 39, wherein the RNA or mRNA comprises ribonucleobases other than A, C, G and U.

41. 41. The immune effector cell of claim 40, wherein the ribonucleobase is pseudouridine, preferably 1-methyl-pseudouridine.

42. 40. The immune effector cell of claim 3, 9, 10, 24, 25, 38, or 39, wherein the RNA comprises a 5' cap structure.

43. The immune effector cell of claim 42, wherein the 5' cap structure is a naturally occurring cap.

44. The immune effector cell of claim 42, wherein the 5' cap structure is a cap analog.

45. 45. The immune effector cell of claim 43 or claim 44, wherein the 5' cap structure is one of cap 0, cap 1, cap 2, cap 3, cap 4, ARCA (Anti-Reverse Cap Analogs), modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

46. The 5' cap structure is ｍ７ The immune effector cell of claim 45, wherein cap 0 is G(5')ppp(5').

47. The 5' cap structure is ｍ７ G(5')ppp(5')(N １ ２’－ＯＭｅ 46. ​​The immune effector cell of claim 45, wherein the Cap1 is CAP1.

48. N １ is selected from A, C, G, or U.

49. Cap 1 is a cap-proximal A, G, C, or U at position +2; ｍ７ G(5')ppp(5')(N １ ２’－ＯＭｅ ) pN ２ The second nucleotide N is represented as ２ 49. The immune effector cell of claim 47 or 48, further comprising:

50. 50. The immune effector cell of any one of claims 1 to 49, wherein the immune effector cell is a T cell, a B cell, a dendritic cell, or an NK cell.

51. 51. The immune effector cell of any one of claims 1 to 50, wherein the immune effector cell is a CD8+ and / or CD4+ T cell.

52. 52. The immune effector cell of any one of claims 1 to 51, wherein the immune effector cell is a cytotoxic T cell.

53. 53. The immune effector cell of any one of claims 1 to 52, wherein the second nucleic acid is not inherited by progeny cells of the immune effector cell in the same way that chromosomes are inherited, and / or the second nucleic acid is diluted compared to the total number of cells in each generation of progeny cells of the immune effector cell, and / or after one cell division, the amount of the second nucleic acid molecule is less in each daughter cell compared to the amount in the parent cell.

54. An immune effector cell comprising: (i) a DNA molecule comprising a transposable element, the transposable element comprising a nucleotide sequence encoding a first T cell receptor or chimeric antigen receptor that binds to a tumor-associated antigen; (ii) an mRNA molecule encoding a second T cell receptor or chimeric antigen receptor that binds to a target different from the tumor-associated antigen; and (iii) an mRNA molecule encoding a transposase.

55. An immune effector cell comprising: (i) a nucleotide sequence encoding a first T cell receptor or chimeric antigen receptor that binds to a tumor-associated antigen, the nucleotide sequence being integrated into the genome of the immune effector cell or contained within an episome present in the immune effector cell; and (ii) a nucleic acid molecule encoding a second T cell receptor or chimeric antigen receptor that binds to a target different from the tumor-associated antigen, the nucleic acid molecule not being integrated into the genomic nucleic acid molecule of the immune effector cell.

56. An immune effector cell comprising: (i) a DNA sequence encoding a first chimeric antigen receptor that binds to a tumor-associated antigen, the DNA sequence being integrated into the genome of the immune effector cell; and (ii) an mRNA molecule encoding a second chimeric antigen receptor that binds to a target different from the tumor-associated antigen.

57. 57. The immune effector cell of any one of claims 54 to 56, wherein the immune effector cell is a CD8+ cytotoxic T cell.

58. 58. The immune effector cell of any one of claims 54 to 57, wherein the immune effector cell is activated by binding of a second T cell receptor or a chimeric antigen receptor to its target.

59. 59. The immune effector cell of any one of claims 1 to 58, wherein the immune effector cell does not comprise a DNA nucleotide sequence encoding an activator molecule.

60. 60. The immune effector cell of any one of claims 1 to 59, wherein the cell surface expression of an endogenous T cell receptor has been reduced to a level that prevents graft-versus-host activity of the immune effector cell when the immune effector cell is administered to a subject different from the subject from which the immune effector cell was derived.

61. 61. The immune effector cell of any one of claims 1 to 60, wherein the immune effector cell does not express its endogenous T cell receptor on its cell surface.

62. 62. The immune effector cell of any one of claims 1 to 61, wherein the immune effector cell has reduced cell surface expression of endogenous HLA complexes to a level that prevents host-versus-graft activity in a subject to which the immune effector cell is administered, wherein the subject to which the immune effector cell is administered is different from the subject from which the immune effector cell was derived.

63. 63. The immune effector cell of any one of claims 1 to 62, wherein the immune effector cell does not express its endogenous HLA complex on its cell surface.

64. 64. A cell composition comprising an immune effector cell according to any one of claims 1 to 63.

65. 65. The cell composition of claim 64, further comprising a cryopreservative.

66. 66. A pharmaceutical composition comprising an immune effector cell described in any one of claims 1 to 63 or a cell composition described in claim 64 or claim 65, and a pharmaceutically acceptable carrier.

67. An immune effector cell described in any one of claims 1 to 63, a cell composition described in claim 64 or claim 65, or a pharmaceutical composition described in claim 66, for use in a method for treating a subject having a disease, disorder, or condition associated with expression or increased expression of a binding target of a first cell surface-expressed antigen receptor, the method comprising administering the immune effector cell, cell composition, or pharmaceutical composition to the subject.

68. 68. The immune effector cell, cell composition, or pharmaceutical composition for use according to claim 67, wherein the disease, disorder, or condition is cancer.

69. 69. The immune effector cell, cell composition, or pharmaceutical composition for use according to claim 68, wherein the cancer is a solid cancer.

70. 68. The immune effector cell, cell composition, or pharmaceutical composition for use according to claim 67, wherein the disease, disorder, or condition is an infectious disease.

71. 71. The immune effector cell, cell composition, or pharmaceutical composition for use according to claim 70, wherein the infectious disease is a viral infection.

72. 72. The immune effector cell, cell composition, or pharmaceutical composition for use according to any one of claims 67 to 71, wherein the immune effector cell is autologous or heterologous to the subject to which the immune effector cell, cell composition, or pharmaceutical composition is administered.

73. A particle comprising: (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is contained within a transposable element; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not contained within a transposable element.

74. 74. The particle of claim 73, further comprising a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, wherein the third nucleotide sequence is not contained within a transposable element.

75. A particle comprising: (i) a DNA episome, preferably a non-viral episome, comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleic acid molecule, when present in a cell, provides for transient expression of the activator molecule.

76. 75. The particle of claim 73 or claim 74, wherein the first nucleic acid molecule is DNA or RNA.

77. 77. The particle of any one of claims 73 to 76, wherein the first nucleic acid molecule or episome is a DNA minicircle or a linear DNA molecule.

78. 78. The particle of any one of claims 73 to 77, wherein the transposable element in the first nucleic acid is derived from a DNA-based transposon system, a viral-based transposon system, or a polyA-based retrotransposon system.

79. 75. The particle of claim 74, wherein the third nucleic acid molecule is DNA or RNA.

80. 79. The particle of claim 78, wherein the third nucleic acid molecule is mRNA.

81. 81. The particle of any one of claims 74 or 76 to 80, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof having transposase / transposition activity.

82. 82. A particle described in any one of claims 74 or 76 to 81, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.

83. 83. The particle of any one of claims 73 to 82, wherein the second nucleic acid molecule is RNA.

84. 84. The particle of any one of claims 73 to 83, wherein the second nucleic acid molecule is mRNA.

85. 85. The particle of any one of claims 73 to 84, wherein the first cell surface expressed antigen receptor binds to a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

86. 86. The particle of any one of claims 73 to 85, wherein the first cell surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

87. 87. The particle of any one of claims 73 to 86, wherein the activator molecule enables activation, expansion, differentiation and / or proliferation of immune effector cells.

88. 88. The particle of any one of claims 73 to 87, wherein the activator molecule is a non-coding RNA or a protein.

89. 89. The particle of any one of claims 73 to 88, wherein the activator molecule binds to an extracellular portion of the first cell surface-expressed antigen receptor.

90. 89. The particle of any one of claims 73 to 88, wherein the activator molecule is a cytokine.

91. 89. The particle of any one of claims 73 to 88, wherein the activator molecule is a second cell surface-expressed antigen receptor, and wherein the extracellular portions of the first and second cell surface-expressed antigen receptors do not bind to the same binding target.

92. 92. The particle of claim 91, wherein the particle further comprises a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of the first cell surface-expressed antigen receptor, wherein the fourth nucleotide sequence is not contained within a transposable element.

93. 93. The particle of claim 91 or claim 92, wherein the particle further comprises a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor, wherein the fifth nucleotide sequence is not contained within a transposable element.

94. 94. The particle of claim 92 or claim 93, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises both a fourth nucleotide sequence encoding a binding target of a first cell surface-expressed antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor.

95. 95. A particle according to any one of claims 92 to 94, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.

96. 96. A particle described in any one of claims 92 to 95, wherein the fourth and / or fifth nucleic acid molecule is mRNA.

97. 97. The particle of any one of claims 91 to 96, wherein the second cell surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

98. 73. A particle comprising the first, second and third nucleic acid molecules of any one of claims 1 to 72.

99. 99. The particle of claim 98, further comprising a fourth and / or fifth nucleic acid molecule of any one of claims 20 to 72.

100. 73. A particle comprising the first, second, third and fourth nucleic acid molecules of any one of claims 19 to 72.

101. 101. The particle of claim 100, further comprising a fifth nucleic acid molecule of any one of claims 19 to 72.

102. 102. The particle of any one of claims 73 to 101, wherein the particle comprises a polyalkyleneimine or a lipid.

103. 103. A particle according to any one of claims 73 to 102, wherein the particle comprises a lipid, preferably a lipid with a cationic head group.

104. 104. The particle of any one of claims 73 to 103, wherein the particle comprises a pH-responsive lipid.

105. 105. The particle of any one of claims 73 to 104, wherein the particle comprises a PEGylated lipid.

106. 106. The particle of any one of claims 73 to 105, wherein the particle is a lipid particle, a polymer particle, or a mixture thereof.

107. 107. The particle of any one of claims 73 to 106, wherein the particle is a nanoparticle.

108. 108. The particle of any one of claims 73 to 107, wherein the particle is a lipid nanoparticle (LNP), lipoplex (LPX), polyplex (PLX), or lipopolyplex (LPLX) particle.

109. 109. The particle of any one of claims 73 to 108, wherein the particle further comprises at least one phosphatidylserine.

110. the particles are nanoparticles, (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the zeta potential of the nanoparticles is less than or equal to 0; 111. A particle according to any one of claims 73 to 110.

111. 103. The particle of any one of claims 73 to 102, wherein the particle comprises a polyalkyleneimine.

112. 112. The particle of claim 111, wherein: (a) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) is 2.0 to 15.0, preferably 6.0 to 12.0; or (b) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) is at least about 48, optionally about 48 to 300, about 60 to 200, or about 80 to 150.

113. 112. The particle of claim 111, wherein the ionic strength of the composition is 50 mM or less, preferably the concentration of monovalent cations is 25 mM or less and the concentration of divalent cations is 20 μM or less.

114. 114. The particle of any one of claims 111 to 113, wherein the particle is a polyplex particle.

115. 115. The particle of any one of claims 73 to 114, wherein the particle comprises a hydrophobic portion having a covalently attached binding portion.

116. 116. The particle of claim 115, wherein the hydrophobic moiety and the particle having the covalently bound binding moiety are non-covalently bound to each other.

117. 117. A particle according to claim 115 or claim 116, wherein the hydrophobic moiety having the covalently attached binding moiety is an integral part of the particle.

118. 118. The particle of any one of claims 115 to 117, wherein the hydrophobic portion having a covalently attached binding moiety comprises a polymer.

119. The hydrophobic moiety having a covalently attached binding moiety is represented by Formula I L-X1-P-X2-B (I) [In the formula, P comprises a polymer; L comprises a hydrophobic moiety attached to a first end of the polymer; B comprises a linking moiety attached to the second end of the polymer; X1 is absent or a first linking moiety; X2 is absent or a second linking moiety.

119. The particle of any one of claims 115 to 118, comprising a compound of formula:

120. 120. The particle of claim 119, wherein X1 comprises a carbonyl group.

121. 121. The particle of claim 119 or claim 120, wherein X2 comprises the reaction product of a maleimide group and a thiol or cysteine ​​group of a compound comprising a binding moiety.

122. 122. A particle according to any one of claims 115 to 121, wherein the hydrophobic moiety is a lipid or is contained in a lipid.

123. 123. The particle of any one of claims 118 to 122, wherein the polymer provides stealth properties, extends circulating half-life, and / or reduces non-specific protein binding or cell adhesion.

124. 124. The particle of any one of claims 118 to 123, wherein the polymer comprises polyethylene glycol (PEG).

125. The hydrophobic moiety having a covalently attached binding moiety is represented by Formula II wherein B comprises a bond moiety.

125. The particle of any one of claims 115 to 124, comprising a compound of the formula:

126. B is a peptide having the structure -N-peptide-C(O)-NH ２ 126. The particle of claim 125, comprising a moiety comprising:

127. 127. The particle of any one of claims 115 to 126, wherein the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or antibody derivative.

128. 128. A particle according to any one of claims 73 to 127, wherein the particle is complexed with and / or encapsulates a nucleic acid molecule.

129. 129. A pharmaceutical composition comprising a particle according to any one of claims 73 to 128 and a pharmaceutically acceptable carrier.

130. A particle described in any one of claims 73 to 128 or a pharmaceutical composition described in claim 129 for use in a method of treating a subject having a disease, disorder or condition associated with expression or up-regulation of a binding target of a first cell surface-expressed antigen receptor, the method comprising administering the particle or pharmaceutical composition to the subject.

131. 131. The particle or pharmaceutical composition for use according to claim 130, wherein the disease, disorder or condition is cancer.

132. 132. The particle for use of claim 131, wherein the cancer is a solid cancer.

133. 131. The particle or pharmaceutical composition for use according to claim 130, wherein the disease, disorder or condition is an infectious disease.

134. 134. The particle for use according to claim 133, wherein the infectious disease is a viral infection.

135. 129. A conjugate comprising: (a) a particle of any one of claims 73-128, wherein the particle comprises a hydrophobic moiety having a covalently attached binding moiety; and (b) a compound comprising: (i) a moiety that binds to the binding moiety covalently attached to the hydrophobic moiety, and (ii) a moiety that targets a cell surface antigen.

136. 136. The conjugate of claim 135, wherein the moiety that binds to the binding moiety covalently bound to the hydrophobic moiety comprises an antibody or antibody derivative.

137. The conjugate of claim 136, wherein the binding moiety covalently bound to the hydrophobic moiety comprises a peptide comprising an ALFA-tag; and the binding moiety covalently bound to the hydrophobic moiety comprises an antibody or antibody derivative comprising a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

138. 138. The conjugate of any one of claims 135 to 137, wherein (i) a moiety that binds to a binding moiety covalently bound to a hydrophobic moiety, and (ii) a moiety that targets a cell surface antigen are linked to each other.

139. 139. The conjugate of any one of claims 135 to 138, wherein the compound in (b) comprises a peptide or polypeptide.

140. 140. The conjugate of any one of claims 135 to 139, wherein the cell surface antigen targeting moiety comprises an antibody or antibody derivative.

141. 141. The conjugate of any one of claims 135 to 140, wherein the cell surface antigen is characteristic of an immune effector cell.

142. 142. The conjugate of any one of claims 135 to 141, wherein the cell surface antigen comprises CD4 and / or CD8.

143. 143. The conjugate of any one of claims 135 to 142, wherein the cell surface antigen comprises CD3.

144. 144. A conjugate according to any one of claims 135 to 143 for use in treating a subject having a disease, disorder or condition associated with expression or up-regulation of a binding target of a first cell surface-expressed antigen receptor.

145. 1. A method for producing an immune effector cell that expresses a first antigen receptor on its cell surface, comprising contacting the immune effector cell with (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding the first cell surface-expressed antigen receptor, and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not contained within a transposable element, and wherein the first cell surface-expressed antigen receptor is stably expressed in the cell and the activator molecule is transiently expressed in the cell.

146. 146. The method of claim 145, wherein the first nucleic acid molecule is DNA or RNA.

147. 147. The method of claim 145 or claim 146, wherein the method further comprises incorporating the first nucleotide sequence into a genomic nucleic acid molecule of an immune effector cell.

148. 148. The method of any one of claims 145 to 147, wherein the first nucleotide sequence is contained within a transposable element.

149. 149. The method of any one of claims 145 to 148, wherein the method further comprises contacting the immune effector cell with a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, wherein the third nucleotide sequence is not contained within a transposable element.

150. 150. The method of claim 149, wherein the third nucleic acid molecule is DNA or RNA.

151. 151. The method of claim 149 or claim 150, wherein the third nucleic acid molecule is mRNA.

152. 152. The method of any one of claims 149 to 151, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof having transposase / transposition activity.

153. 152. A method according to any one of claims 149 to 151, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.

154. 147. The method of claim 145 or claim 146, wherein the first nucleic acid is episomal.

155. 155. The method of claim 154, wherein the episome is a non-viral episome.

156. 156. The method of any one of claims 145 to 155, wherein the activator molecule enables activation, expansion, differentiation and / or proliferation of immune effector cells.

157. 157. The method of any one of claims 145 to 156, wherein the activator molecule is a non-coding RNA or a protein.

158. 158. The method of any one of claims 145 to 157, wherein the activator molecule binds to the extracellular portion of the first cell surface expressed antigen receptor.

159. 159. The method of any one of claims 145 to 158, wherein the activator molecule is a cytokine.

160. 160. The method of any one of claims 145 to 159, wherein the activator molecule is a second cell surface expressed antigen receptor, wherein the extracellular portions of the first and second cell surface expressed antigen receptors do not bind to the same binding target.

161. 161. The method of claim 160, wherein the method further comprises contacting the immune effector cell with a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding a binding target of the first cell surface-expressed antigen receptor.

162. 162. The method of claim 160 or claim 161, further comprising contacting the immune effector cell with a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor.

163. 163. The method of claim 161 or claim 162, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises both a fourth nucleotide sequence encoding a binding target of a first cell surface-expressed antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface-expressed antigen receptor.

164. 164. The method of any one of claims 161 to 163, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.

165. 165. The method of any one of claims 161 to 164, wherein the fourth and / or fifth nucleic acid molecule is mRNA.

166. 166. The method of any one of claims 145 to 165, wherein the first cell surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

167. 167. The method of any one of claims 160 to 166, wherein the first cell surface expressed antigen receptor and / or the second cell surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

168. 168. The method of any one of claims 145 to 167, wherein the binding target of the second cell surface-expressed antigen receptor is expressed on or from a different cell than the cell expressing the binding target of the first cell surface-expressed antigen receptor.

169. 169. The method of any one of claims 145 to 168, wherein the binding target of the first cell surface-expressed antigen receptor is a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.

170. 170. The method of any one of claims 145 to 169, wherein the second nucleic acid molecule is DNA or RNA.

171. 171. The method of any one of claims 145 to 170, wherein the second nucleic acid molecule is mRNA.

172. 172. The method of claim 146, 150, 151, 164, 165, 170 or 171, wherein the RNA or mRNA comprises ribonucleobases other than A, C, G and U.

173. 173. The method of claim 172, wherein the ribonucleobase is pseudouridine, preferably 1-methyl-pseudouridine.

174. 172. The method of claim 146, 150, 151, 164, 165, 170 or 171, wherein the RNA comprises a 5' cap structure.

175. 175. The method of claim 174, wherein the 5' cap structure is a naturally occurring cap.

176. 175. The method of claim 174, wherein the 5' cap structure is a cap analog.

177. 177. The method of claim 175 or claim 176, wherein the 5' cap structure is one of cap 0, cap 1, cap 2, cap 3, cap 4, ARCA (Anti-Reverse Cap Analogs), modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

178. 178. The method of any one of claims 145 to 177, wherein the immune effector cell is a T cell, a B cell, a dendritic cell, or an NK cell.

179. 179. The method of any one of claims 145 to 178, wherein the immune effector cells are CD8+ and / or CD4+ T cells.

180. 179. The method of any one of claims 145 to 179, wherein the immune effector cell is a cytotoxic T cell.

181. 181. The method of any one of claims 145-180, wherein the second nucleic acid is not inherited by progeny cells of the immune effector cell in the same way that chromosomes are inherited, and / or the second nucleic acid is diluted in each generation of progeny cells of the immune effector cell, and / or after one cell division, the amount of the second nucleic acid molecule is less in each daughter cell compared to the amount in the parent cell.

182. 143. A method for producing immune effector cells that express a first antigen receptor on the cell surface, the method comprising contacting immune effector cells with a particle described in any one of claims 73 to 134 or a complex described in any one of claims 135 to 144.

183. 183. The method of any one of claims 145 to 182, wherein the contacting occurs in vitro.

184. 184. The method of any one of claims 160 to 183, further comprising the step of contacting the immune effector cell with a binding target, or a cell expressing a binding target, of a second cell surface-expressed antigen receptor after contacting the nucleic acid molecule with the immune effector cell.

185. 1. A method for producing immune effector cells that express two antigen receptors on their cell surface, the method comprising contacting immune effector cells in vitro or ex vivo with: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is contained in a transposable element; (ii) an RNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor, wherein the second nucleotide sequence is not contained in a transposable element; and (iii) an RNA molecule comprising a third nucleotide sequence encoding a transposase, wherein the third nucleotide sequence is not contained in a transposable element; wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets, preferably, the binding target of the first cell surface-expressed antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface-expressed antigen receptor is expressed on the surface of a blood cell.

186. 186. The method of claim 185, further comprising contacting the immune effector cell with a binding target of a second cell surface-expressed antigen receptor or with a cell expressing a binding target of a second cell surface-expressed antigen receptor.

187. A method for producing immune effector cells that express two antigen receptors on their cell surface, comprising contacting immune effector cells with particles comprising: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is comprised in a transposable element; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets, preferably, the binding target of the first cell surface-expressed antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface-expressed antigen receptor is expressed on the surface of a blood cell.

188. 188. The method of claim 187, wherein the contacting is performed in vivo.

189. 1. A method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition, and wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of a cell of the subject or is contained within an episome present in the cell of the subject, and / or (ii) the activator molecule is transiently expressed in the subject.

190. 190. The method of claim 189, wherein the nucleic acid molecule is within a particle comprising a lipid.

191. A method for treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject particles comprising: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, wherein the first nucleotide sequence is contained in a transposable element, and a binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell surface-expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell surface-expressed antigen receptors bind to different targets.

192. 67. A method for treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject an immune effector cell described in any one of claims 1 to 63, a cell composition described in claim 64 or claim 65, or a pharmaceutical composition described in claim 66, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

193. 130. A method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a particle described in any one of claims 73 to 128 or a pharmaceutical composition described in claim 129, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

194. 145. A method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, comprising administering to the subject a conjugate of any one of claims 135 to 144, wherein the binding target of the first cell surface-expressed antigen receptor is an antigen associated with the disease, disorder, or condition.

195. 195. The method of any one of claims 189 to 194, wherein the antigen associated with the disease, disorder or condition is a tumor-associated antigen.

196. 196. The method of any one of claims 189 to 195, which is a method for treating or preventing cancer in a subject.

197. 195. The method of any one of claims 189 to 194, wherein the antigen associated with the disease, disorder or condition comprises an antigen of an infectious agent.

198. 198. The method of claim 197, wherein the infectious agent is a virus.

199. 200. The method of any one of claims 189 to 194, 197 or 198, which is a method for treating or preventing an infection in a subject.

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