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

Description

TECHNICAL FIELD

[0001] The present invention relates to 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, a cell composition and a pharmaceutical composition comprising such immune effector cell and a method of producing such immune effector cell. Furthermore, the present invention relates to a particle 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 a complex comprising such particles. Further, the present invention relates to a method of using the immune effector cell, the particle or the complex for treating a subject having a disease, disorder or condition associated with the expression or elevated expression of an antigen. BACKGROUND

[0002] With the success of CD19 CAR-T cells in the treatment of hematological malignancies, there are currently hundreds of ongoing clinical trials testing engineered T cells as cancer therapies, and the number of suitable patients for T cell-based treatment modalities is expected to increase substantially. However, current T cell manufacturing technologies struggle to meet the growing demand for CAR-T cells and related engineered T cell immunotherapies, especially since current manufacturing methods rely on time- and cost-intensive production protocols (Rafiq et al., Nat. Rev. Clin. Oncol. 2020 Mar; 17(3): 147-167).

[0003] Standard production protocols for engineered T cells are mainly performed in a manual or semi-automated fashion, which limits production volume and increases product variability and costs. This approach typically involves the collection of a patient’s leukapheresis material and shipping to a production facility, T cell stimulation and transduction (usually by viral vectors encoding antigen receptors), expansion and cryopreservation of the T cell product under good manufacturing practice (GMP) conditions, and shipping back to the original patient. Thus, improving speed, scalability, and cost-effectiveness while maintaining or improving the biosafety and therapeutic efficacy of the product are major 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 generally rely on the use of gamma- or lentiviruses with high transduction efficiency to introduce the CAR nucleic acid sequence into the patient’s T cells. Further developments of these viral transduction methods have shortened the manufacturing time (Ghassemi et al., Nat. Biomed. Eng. 2022 Feb; 6(2): 118-128). However, viral vectors have limited gene payload capacity, often require a T cell activation step (which is detrimental to their persistence and anti-tumor efficacy), are costly to produce under GMP conditions, and face a complex regulatory environment. Therefore, the development of alternative genetic engineering modalities has been the focus 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 advantages over traditional viral transduction in terms of safety, reproducibility, and speed (Irving et al., Hum. Gene Ther. 2021 Oct; 32(19-20): 1044-1058).

[0005] However, the drawback of the transposon-based approaches used so far is that they have a low integration efficiency, or, if the transposase is expressed for a long time, there is a risk that sequences that have been integrated are excised again. The efficiency is even lower for resting cells, such as unactivated T cells.

[0006] Therefore, there is an urgent need for improved methods for producing sufficient amounts of immune effector cells in vivo and / or in vitro, preferably in a safe and efficient manner. This need is particularly prevalent for immune effector cells that require stable expression of a receptor.

[0007] The present invention meets this need. A particular advantage of the present invention is that it provides for shorter manufacturing times, less quality testing, and / or lower costs for the immune effector cells. In particular, no additional activation step is required. SUMMARY

[0008] 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 are further useful in methods of treating a subject. The immune effector cells of the present disclosure are characterized by (i) the second nucleotide sequence not being integrated into a genomic nucleic acid molecule of the immune effector cell, and / or (ii) the activator molecule being transiently expressed. If the second nucleic acid is not integrated into a genomic nucleic acid molecule (which would be equally passed on to daughter cells during cell division), the activator molecule is only expressed for a limited time.

[0009] Transient expression allows activator molecules to exist for a desired period of time before disappearing. Transient expression can be achieved by preventing the second nucleic acid from integrating into the genomic nucleic acid molecule, but it can also be achieved in other ways, such as by regulating the induction of expression of the second nucleic acid molecule.

[0010] One aspect of this disclosure is an immune effector cell comprising a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface antigen receptor, and the 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 the genomic nucleic acid molecule of the immune effector cell, and / or (ii) the activator molecule is transiently expressed.

[0011] Unbound by theoretical constraints, providing first antigen receptors and activator molecules to immune effector cells offers several advantages: additional activation steps can be eliminated from the production method or integrated into it. Furthermore, activation, particularly with second antigen receptors, can contribute to overall transfection efficiency, as it helps the first nucleotide sequence overcome the nuclear envelope and enter the nucleus for amplification, especially through integration via systems such as transposon-based systems. Moreover, the elimination of the need for other cells to express the activator molecule increases regulatory acceptance. Because the second nucleic acid is not integrated and / or the activator molecule is transiently expressed, the activation signal disappears when it is no longer needed, increasing the safety of the employed protocol.

[0012] In one implementation, the immune effector cells can be isolated. The immune effector cells can be present in vitro (e.g., in cell cultures or frozen samples) 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 the stable and long-term expression of the encoded first cell surface antigen receptor.

[0014] In one embodiment, the first nucleotide sequence may be integrated into the genomic nucleic acid molecule of an immune effector cell. Preferably, the genomic nucleic acid molecule may be a chromosome, an episome, 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 poly-A-based retrotransposon system. According to this embodiment, the first nucleotide sequence can be contained within a suitable transposon element.

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

[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 with transposase activity may be Sleeping Beauty, PiggyBac, Frog, Prince, Himall, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmar1, or a functionally equivalent variant thereof with transposase activity. Preferably, the molecule with transposase activity is Sleeping Beauty transposase SB100X.

[0019] In one implementation, the third nucleic acid molecule does not integrate into the genomic nucleic acid molecule of the immune effector cell, and / or the molecule encoded by the third nucleic acid molecule can be transiently expressed. The third nucleic acid molecule is characterized by its unsuitability for the stable, long-term expression of molecules with transposase activity. This lack of theoretical constraint helps prevent the removal of the integrated first nucleotide sequence.

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

[0021] In one embodiment, the activator molecule encoded by the second nucleotide sequence enables immune effector cells to be activated, expanded, differentiated, and / or proliferated. Preferably, the activator molecule can be a non-coding RNA or a protein.

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

[0023] In one implementation, the activator molecule may be a cytokine.

[0024] In one embodiment, the activator molecule may 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 may further comprise a fourth nucleic acid molecule containing a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor, and / or the immune effector cell may further comprise a fifth nucleic acid molecule containing a fifth nucleotide sequence encoding a binding target of a second cell surface antigen receptor. In this embodiment, either the fourth or fifth nucleic acid molecule may contain a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface antigen receptor.

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

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

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

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

[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 its epitope. 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 preferably 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 blood cells, preferably another type of immune effector cell. Preferably, the blood cells can be T cells, NK cells, dendritic cells, macrophages, or B cells.

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

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

[0034] In one embodiment, the RNA may include a 5' cap structure. Preferably, the 5' cap structure is a naturally occurring cap or cap analogue. The 5' cap structure may be one of the following: cap0, cap1, cap2, cap3, cap4, ARCA (anti-reverse cap analogue), modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deazo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. Preferably, the 5' cap structure may be cap0, which is m7G(5')ppp(5') or it may be cap1, which is m7G(5')ppp(5')(N1 2'-OMe In this embodiment, N1 can be selected from A, C, G, or U. cap1 may also include a second nucleotide N2, which is the proximal cap end A, G, C, or U at position +2, and is denoted as m7G(5')ppp(5')(N1) 2'-OMe )pN2. In one implementation, the structure of the 5' end is m7G(5')ppp(5')(A 2'-OMe )pGpApApU.

[0035] In one embodiment, the immune effector cells may 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 implementation, the second nucleic acid may not be inherited by the daughter cells of the immune effector cells in the same manner as chromosomal inheritance, and / or the second nucleic acid may be diluted compared to the total number of cells in each generation of daughter cells of the immune effector cells, and / or, after one round of cell division, the amount of the second nucleic acid molecule in each daughter cell may be less than the amount in the parent cells.

[0037] Another embodiment may be an immune effector cell comprising (i) a DNA molecule containing 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] Another embodiment may 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 in an appendage 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] Another implementation 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 implementation, the immune effector cells may be CD8+ cytotoxic T cells.

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

[0042] In one implementation, immune effector cells may not contain DNA nucleotide sequences encoding activator molecules.

[0043] In one embodiment, the cell surface expression of endogenous T cell receptors on immune effector cells is reduced to a level that prevents graft-versus-host activity of the immune effector cells when they are administered to a subject from a different origin. Preferably, the immune effector cells do not express their endogenous T cell receptors on their cell surface.

[0044] In one embodiment, the cell surface expression of the endogenous HLA complex of the immune effector cells is reduced to a level that prevents host antigraft activity in a subject who has been administered the immune effector cells, wherein the subject administered the immune effector cells is different from the subject from whom the immune effector cells originated. Preferably, the immune effector cells do not express their endogenous HLA complex on their cell surface.

[0045] On the other hand, there is a cell composition comprising the immune effector cells disclosed herein. The cell composition may also comprise a cryopreservative.

[0046] On the other hand, it is a pharmaceutical composition comprising the immune effector cells or cell compositions of the present disclosure, and a pharmaceutically acceptable carrier.

[0047] In one embodiment, a method of using the immune effector cells, cell compositions, or pharmaceutical compositions of this disclosure for treating a subject suffering from a disease, condition, or ailment associated with the expression or elevated expression of a binding target of a first cell surface antigen receptor, wherein the method includes administering the immune effector cells, cell compositions, or pharmaceutical compositions to the subject. Preferably, the disease, condition, or ailment may be cancer, and more preferably, the cancer is a solid tumor.

[0048] In one embodiment, the immune effector cells, cell compositions, or pharmaceutical compositions used in this disclosure may be an infection, preferably a viral infection.

[0049] In one embodiment, the immune effector cells, cell compositions, or pharmaceutical compositions used in this disclosure are autologous or allogeneic to the subject to which the immune effector cells, cell compositions, or pharmaceutical compositions are administered.

[0050] All implementation schemes applicable to immune effector cells are equally applicable to the particles, complexes, and methods disclosed herein.

[0051] On the other hand, a particle comprises (i) a first nucleic acid molecule containing a first nucleotide sequence encoding a first cell surface antigen receptor, the first nucleotide sequence being contained within a transposon element; and (ii) a second nucleic acid molecule containing a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not contained within a transposon element. Preferably, the particle further comprises a third nucleic acid molecule containing a third nucleotide sequence encoding a molecule (preferably a transposase) having transposase activity, wherein the third nucleotide sequence is not contained within a transposon element. Preferably, the first nucleic acid molecule or episome is a DNA microcircle or a linear DNA molecule.

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

[0053] In embodiments of the particles disclosed herein, the transposon element in the first nucleic acid may be derived from a DNA-based transposon system, a virus-based transposon system, or a poly-A-based retrotransposon system.

[0054] In one embodiment of the particles disclosed herein, the third nucleic acid molecule may be DNA or RNA. Preferably, the third nucleic acid molecule is RNA, more preferably mRNA.

[0055] In one embodiment of the particles disclosed herein, the molecule having transposase activity may be SleepingBeauty, PiggyBac, Frog, Prince, Himall, 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 particles disclosed herein, the second nucleic acid molecule may be DNA or RNA. Preferably, the second nucleic acid molecule is RNA, more preferably mRNA.

[0057] In one embodiment of the particles disclosed herein, the first cell surface expresses an antigen receptor that can bind to an antigen of a tumor-associated antigen or an infectious agent, or its epitope.

[0058] In one embodiment of the particles disclosed herein, the first cell surface expressing an antigen receptor may be a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0059] In one embodiment of the particles disclosed herein, the activator molecule enables immune effector cells to activate, expand, differentiate, and / or proliferate. The activator molecule may be a non-coding RNA or a protein.

[0060] In one embodiment of the particles disclosed herein, the activator molecule may bind to the extracellular portion of a first cell surface expressing an antigen receptor. Preferably, the activator molecule is an antigen targeted by the first cell surface expressing an antigen receptor.

[0061] In one embodiment of the particles disclosed herein, the activator molecule may be a cytokine.

[0062] In one embodiment of the particles disclosed herein, the activator molecule may 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 may further comprise a fourth nucleic acid molecule containing a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor, wherein the fourth nucleotide sequence is not contained within a transposon element; or the particle may further comprise a fifth nucleic acid molecule containing a fifth nucleotide sequence encoding a binding target of a second cell surface antigen receptor, wherein the fifth nucleotide sequence is not contained within a transposon element. Preferably, both the fourth and fifth nucleic acid molecules contain a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface antigen receptor.

[0064] In one embodiment of the particles disclosed herein, the fourth and / or fifth nucleic acid molecules may be DNA or RNA. Preferably, the fourth and / or fifth nucleic acid molecules are RNA, more preferably mRNA.

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

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

[0067] In one embodiment, the particles may comprise polyalkylimide or lipids. Preferably, the particles comprise lipids, and more preferably lipids having cationic head groups and / or pH-responsive lipids and / or PEGylated lipids.

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

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

[0070] In one embodiment, the particles may be lipid nanoparticles (LNP), lipid complexes (lipoplex, LPX), polyplexes (PLX), or lipid polyplexes (LPLX) particles.

[0071] In one embodiment, the particles may further contain at least one phosphatidylserine.

[0072] In one embodiment, the particles may be nanoparticles, wherein:

[0073] (i) The number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or

[0074] (ii) The nanoparticles have a neutral or net negative charge, and / or

[0075] (iii) The zeta potential of the nanoparticles is 0 or lower. Optionally or additionally, the charge ratio of positive to negative charge in the nanoparticles may be 1.4:1 or lower.

[0076] In one embodiment, the particles may comprise a polyalkylene imide, preferably wherein (a) the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkylene imide to the number of phosphorus atoms (P) in the first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule is 2.0-15.0, preferably 6.0-12.0; or (b) the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkylene imide to the number of phosphorus atoms (P) in the first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule is at least about 48, optionally about 48-300, about 60-200, or about 80-150, or preferably wherein the ionic strength of the composition is 50 mM or less, preferably wherein the concentration of the monovalent cation is 25 mM or less, and the concentration of the divalent cation is 20 μM or less.

[0077] In one embodiment, the particles may be polymeric composite particles.

[0078] In one embodiment, the particle may include a hydrophobic portion having a covalently bonded portion therewith, preferably wherein the hydrophobic portion having the covalently bonded portion and the particle are non-covalently associated with each other. The hydrophobic portion having the covalently bonded portion may be a component of the particle. Preferably, the hydrophobic portion having the covalently bonded portion comprises a polymer.

[0079] In one embodiment, the hydrophobic portion having a binding portion covalently linked thereto may comprise a compound of formula I.

[0080] L-X1-P-X2-B(I)

[0081] in

[0082] P contains polymers;

[0083] L includes a hydrophobic portion attached to a first end of the polymer;

[0084] B includes a binding portion attached to the second end of the polymer;

[0085] X1 either does not exist or is the first connection part; and

[0086] X2 is absent or is a second linking portion, preferably X1 contains a carbonyl group and / or preferably X2 contains a reaction product of a maleimide group and a thiol or cysteine ​​group of a compound containing the linking portion.

[0087] In one embodiment, the hydrophobic portion may be a lipid or may be contained within a lipid.

[0088] In one embodiment, the polymer can provide stealth properties, extended cyclic half-life, and / or reduced nonspecific protein binding or cell adhesion.

[0089] In one embodiment, the polymer may comprise polyethylene glycol (PEG).

[0090] In one embodiment of the particles disclosed herein, the hydrophobic portion having a binding portion covalently linked thereto may comprise a compound of formula II.

[0091]

[0092] Wherein B contains a binding portion, preferably B contains a portion containing the structure -N-peptide-C(O)-NH2.

[0093] In one embodiment, the binding portion covalently linked to the hydrophobic portion may comprise an antibody or an antibody derivative.

[0094] In one embodiment, the particles are compounded with nucleic acid molecules and / or the particles encapsulate nucleic acid molecules.

[0095] On the other hand, it is a pharmaceutical composition comprising the particles described herein, and a pharmaceutically acceptable carrier.

[0096] In one embodiment, the particulate or pharmaceutical composition of this disclosure can be used in a method of treating a subject with a disease, condition, or ailment associated with the expression or elevated expression of a binding target of a first cell surface antigen receptor, wherein the method includes administering the particulate or pharmaceutical composition to the subject, preferably the disease, condition, or ailment being cancer, wherein the cancer is preferably a solid tumor.

[0097] In one embodiment, the particles or pharmaceutical composition may be used for purposes according to this disclosure, wherein the disease, symptom, or condition may be an infection, preferably wherein the infection is a viral infection.

[0098] Another aspect of this disclosure is a complex comprising:

[0099] (a) the particles of this disclosure, wherein the particles comprise a hydrophobic portion having a binding portion covalently linked thereto, and (b) a compound comprising (i) a portion binding to the binding portion covalently linked to the hydrophobic portion, and (ii) a portion targeting a cell surface antigen, preferably wherein the portion binding to the binding portion covalently linked to the hydrophobic portion comprises an antibody or antibody derivative, preferably wherein the binding portion covalently linked to the hydrophobic portion comprises a peptide containing an ALFA-tag; and the portion binding to the binding portion covalently linked to the hydrophobic portion comprises an antibody or antibody derivative, preferably a nanobody containing a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY. Preferably, (i) the portion binding to the binding portion covalently linked to the hydrophobic portion and (ii) the portion targeting the cell surface antigen are interconnected.

[0100] In one implementation, the compound in (b) may comprise a peptide or polypeptide.

[0101] In one implementation, the portion targeting the cell surface antigen may contain an antibody or an antibody derivative.

[0102] In one embodiment, the cell surface antigen may be characteristic of immune effector cells. Preferably, the cell surface antigen comprises CD4 and / or CD8 and / or CD3.

[0103] In one embodiment, the complex disclosed herein can be used to treat a subject suffering from a disease, condition, or ailment associated with the expression or elevated expression of a binding target of a first cell surface antigen receptor.

[0104] On the other hand, there is a method for generating immune effector cells expressing a first antigen receptor on the cell surface, the method comprising contacting the immune effector cells with: (i) a first nucleic acid molecule containing a first nucleotide sequence encoding the first cell surface antigen receptor, and (ii) a second nucleic acid molecule containing a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not contained within a transposon element, and wherein the first cell surface 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.

[0105] In one embodiment, the method may further include integrating a first nucleotide sequence into the genomic nucleic acid molecule of an immune effector cell. Preferably, the first nucleotide sequence is contained within a transposon element.

[0106] In one embodiment, the method may further include contacting immune effector cells with a third nucleic acid molecule, the third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule (preferably a transposase) having transposase activity, 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. The third nucleic acid molecule is characterized in that it is not suitable for the stable, long-term expression of molecules having transposase activity.

[0107] In one embodiment, the method may further include contacting a fourth nucleic acid molecule and / or a fifth nucleic acid molecule on immune effector cells, said fourth and / or fifth nucleic acid molecules comprising a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor and / or a binding target of a second cell surface antigen receptor. Preferably, both the fourth and fifth nucleic acid molecules comprise a fourth nucleotide sequence encoding a binding target of a first cell surface antigen receptor and a fifth nucleotide sequence encoding a binding target of a second cell surface antigen receptor. The fourth and / or fifth nucleic acid molecules may be DNA or RNA. Preferably, the fourth and / or fifth nucleic acid molecules are RNA, more preferably mRNA.

[0108] In one implementation, the first cell surface expressing an antigen receptor and / or the second cell surface expressing an antigen receptor may be a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0109] In one embodiment of the method for generating immune effector cells, the binding target of the second cell surface antigen receptor is expressed on or by cells different from those expressing the binding target of the first cell surface antigen receptor. The binding target of the first cell surface antigen receptor may be an antigen of a tumor-associated antigen or an infectious agent, or its epitope.

[0110] In one embodiment of the method for generating immune effector cells, the second nucleic acid molecule is DNA or RNA. Preferably, the second nucleic acid molecule is RNA, more preferably mRNA.

[0111] In one embodiment of the method for generating immune effector cells, the RNA contains a 5' cap structure, preferably a naturally occurring cap or cap analogue.

[0112] In one embodiment of the method for generating 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, and more preferably cytotoxic T cells.

[0113] In one embodiment of the method for generating immune effector cells, the second nucleic acid is not inherited to the daughter cells of the immune effector cells in the same manner as chromosomal inheritance, and / or, in each generation of daughter cells of the immune effector cells, the second nucleic acid may be diluted, and / or, after one round of cell division, the amount of the second nucleic acid molecule in each daughter cell is less than the amount in the parent cells.

[0114] In one embodiment, the method includes contacting immune effector cells with particles or complexes of the present disclosure. Preferably, the contact is performed in vitro. The method may further include, after contacting the nucleic acid molecules with the immune effector cells, contacting the immune effector cells with a second cell surface-expressing antigen receptor binding target or a cell expressing the binding target.

[0115] In one embodiment, the method may be a method for generating immune effector cells expressing two antigen receptors on their cell surfaces, the method comprising, under in vitro or ex vivo conditions, contacting the immune effector cells with: (i) a DNA molecule containing a first nucleotide sequence encoding a first cell surface antigen receptor, the first nucleotide sequence being contained within a transposon element; (ii) an RNA molecule containing a second nucleotide sequence encoding a second cell surface antigen receptor, the second nucleotide sequence not contained within a transposon element; and (iii) an RNA molecule containing a third nucleotide sequence encoding a transposase, the third nucleotide sequence not contained within a transposon element, wherein the extracellular domains of the first and second cell surface antigen receptors bind to different targets, preferably wherein the binding target of the first cell surface antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface antigen receptor is expressed on the surface of blood cells. Preferably, the method further comprises contacting the immune effector cells with the binding target of the second cell surface antigen receptor or with cells expressing the binding target of the second cell surface antigen receptor.

[0116] In one embodiment, the method may be a method of generating immune effector cells expressing two antigen receptors on their cell surfaces, the method comprising contacting the immune effector cells with a particle comprising (i) a DNA molecule containing a first nucleotide sequence encoding a first cell surface antigen receptor, the first nucleotide sequence being contained within a transposon element; (ii) an mRNA molecule containing a second nucleotide sequence encoding a second cell surface antigen receptor; and (iii) an mRNA molecule containing a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell surface antigen receptors bind to different targets, preferably wherein the binding target of the first cell surface antigen receptor is a tumor or tumor-associated antigen, and the binding target of the second cell surface antigen receptor is expressed on the surface of blood cells. Preferably, the contact is in vivo.

[0117] Another aspect of this disclosure is a method for treating a subject suffering from a disease, condition, or illness associated with the expression or elevated expression of an antigen, the method comprising administering to the subject a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface antigen receptor, and the 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 antigen receptor is the antigen associated with the disease, condition, or illness, wherein (i) the second nucleotide sequence is not integrated into the genomic nucleic acid molecule of the subject's cells or contained in an appendage present in the subject's cells, and / or (ii) the activator molecule is transiently expressed in the subject. Preferably, the nucleic acid molecule is in a lipid-containing particle.

[0118] In one embodiment, the method may be a method of treating a subject suffering from a disease, condition, or illness associated with the expression or elevated expression of an antigen, the method comprising administering to the subject a particle comprising (i) a DNA molecule containing a first nucleotide sequence encoding a first cell surface antigen receptor, the first nucleotide sequence being contained within a transposon element, and wherein the binding target of the first cell surface antigen receptor is the antigen associated with the disease, condition, or illness; (ii) an mRNA molecule containing a second nucleotide sequence encoding a second cell surface antigen receptor; and (iii) an mRNA molecule containing a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first cell surface antigen receptor and the second cell surface antigen receptor bind to different targets.

[0119] In one embodiment, the method may be a method of treating a subject suffering from a disease, condition, or illness associated with the expression or elevated expression of an antigen, the method comprising administering to the subject an immune effector cell of the present disclosure, a cell composition of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the binding target of a first cell surface expressing an antigen receptor is the antigen associated with the disease, condition, or illness.

[0120] In one embodiment, the method may be a method of treating a subject suffering from a disease, condition, or illness associated with the expression or elevated expression of an antigen, the method comprising administering to the subject the particles of the present disclosure or the pharmaceutical composition of the present disclosure, wherein the binding target of a first cell surface antigen receptor is the antigen associated with the disease, condition, or illness.

[0121] In one embodiment, the method may be a method of treating a subject suffering from a disease, condition, or illness associated with the expression or elevated expression of an antigen, the method comprising administering to the subject the complex disclosed herein, wherein the binding target of a first cell surface antigen receptor is the antigen associated with the disease, condition, or illness.

[0122] In one embodiment of the method for treating a subject, the antigen associated with the disease, symptom, or condition may be a tumor-associated antigen. Preferably, the method is a method for treating or preventing cancer in the subject.

[0123] In one embodiment of the method for treating a subject, the antigen associated with the disease, symptom, or condition comprises an antigen of an infectious agent. Preferably, the infectious agent is a virus.

[0124] In one embodiment of the method for treating a subject, the method is a means of treating or preventing infection in the subject. Detailed Implementation

[0125] While the invention is described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can 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 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.

[0126] Preferably, the definitions of the terms used herein are as in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G. Leuenberger, B. Nagel, and H. As described in Eds., Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

[0127] Unless otherwise stated, the present invention will be practiced using conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, as illustrated in the literature in the field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0128] The elements of the invention will be described below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to the explicitly described embodiments. This specification should be understood to disclose and cover embodiments combining the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described in this application should be considered as disclosed in this specification.

[0129] The term “about” means approximately or close to, and in the context of the numerical values ​​or ranges shown herein, it preferably means + / - 10% of the enumerated or claimed numerical values ​​or ranges.

[0130] The terms “a,” “an,” and “this,” as well as similar designations, used in the context of describing this invention (particularly in the context of the claims) should be understood to cover both singular and plural forms, unless otherwise specified herein or clearly contradicted by the context. The enumeration of ranges of values ​​herein is merely a method of abbreviating the terminology for individually referring to each distinct value falling within the range. Each individual value is incorporated into this specification as it is individually enumerated herein, unless otherwise specified herein or clearly contradicted by the context elsewhere. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is merely for the purpose of better illustrating the invention and does not constitute a limitation on the scope of the otherwise claimed invention. No language in this specification should be construed as indicating any unclaimed element essential to the implementation of this invention.

[0131] Unless otherwise expressly stated, in the context of this document, the term "comprising" is used to indicate that, in addition to the list members introduced by "comprising," there may optionally be other members. However, as a particular embodiment of the invention, the term "comprising" is considered to cover the possibility that no other members exist; that is, for the purposes of this embodiment, "comprising" is understood to mean "consisting of."

[0132] The relative quantity index of a component characterized by a generic term refers to the total amount of all specific variants or members covered by the generic term. If a component defined by a generic term is specified to exist in a specific relative quantity, and if the component is further characterized as a specific variant or member covered by the generic term, it means that no other variants or members covered by the generic term may exist such that the total relative quantity of the components covered by the generic term exceeds the specified relative quantity; more preferably, no other variants or members covered by the generic term exist at all.

[0133] Several documents are referenced throughout the main text of this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer's instructions, user manuals, etc.), whether mentioned above or below, is incorporated herein in its entirety. Nothing herein should be construed as an admission that the invention was not entitled to prior disclosure.

[0134] As used herein, terms such as “reduction” or “inhibition” mean a reduction in the overall level, 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 “inhibition” or similar phrases include complete or substantially complete inhibition, i.e., a reduction to 0 or substantially to 0.

[0135] Terms such as “increase” or “enhance” preferably refer to an increase or enhancement of about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

[0136] The term "net charge" refers to the electrical charge on an entire object (such as a compound or particle).

[0137] Ions with an overall net positive charge are cations, while ions with an overall net negative charge are anions. Therefore, according to the present invention, anions are ions with more electrons than protons and thus a net negative charge; cations are ions with fewer electrons than protons and thus a net positive charge.

[0138] With respect to a given compound or particle, the terms “charged,” “net charge,” “negatively charged,” or “positively charged” refer to the net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0139] The term "nucleic acid" in this invention also includes the chemical derivatization of nucleic acids on nucleotide bases, sugars, or phosphates, 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, preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared, and chemically synthesized molecules. According to the invention, nucleic acids can exist in the form of single-stranded or double-stranded molecules and linear or covalently closed circular molecules.

[0140] According to the present invention, a "nucleic acid sequence" refers to a nucleotide sequence in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to the entire nucleic acid molecule (such as a single strand of the entire nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0141] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinant RNA, such as modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, such as addition to the ends or interior of the RNA, for example at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs, particularly analogs of naturally occurring RNA.

[0142] According to the present invention, RNA can be single-stranded or double-stranded. In some embodiments of the invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule to which no complementary nucleic acid molecule (usually no complementary RNA molecule) binds. Single-stranded RNA may contain self-complementary sequences that enable a portion of the RNA to fold back and form secondary structural motifs, including but not limited to base pairs, stems, stem loops, and protrusions. Single-stranded RNA can exist as a negative strand [(-) strand] or a positive strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. The genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can be directly used 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 bind together to form double-stranded RNA ("double-stranded RNA").

[0143] The term "stability" in RNA refers to the "half-life" of RNA. "Half-life" refers to the time required to eliminate half the activity, amount, or quantity of a molecule. In the context of this invention, the half-life of RNA is an indicator of the stability of said RNA. The half-life of RNA may affect the "expression duration" of RNA. RNA with a long half-life can be expected to be expressed over an extended period.

[0144] The term "translation efficiency" refers to the amount of translational product that an RNA molecule provides within a given time period.

[0145] Regarding nucleic acid sequences, a "fragment" refers to a portion of a nucleic acid sequence, specifically a sequence representing a shortened nucleic acid sequence at the 5'- and / or 3'- ends. Preferably, the nucleic acid sequence fragment contains at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In this invention, fragments of RNA molecules that retain RNA stability and / or translation efficiency are preferred.

[0146] Regarding amino acid sequences (peptides or proteins), a "fragment" refers to a portion of the amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame (OPF) lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated OPF lacking the 5' end, provided the truncated OPF contains a start codon used to initiate translation. A fragment of the amino acid sequence comprises, for example, 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.

[0147] According to the present invention, the term "variant" relating to, for example, nucleic acid and amino acid sequences includes any variant, particularly mutants, viral strain variants, splicing variants, conformations, alloforms, allele variants, interspecific variants, and species homologs, especially those that are naturally occurring. Allele variants involve alterations in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies many allele variants of a given gene. Regarding nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein the degenerate nucleic acids of the present invention differ in their codon sequences from a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence that has a different species origin than a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence that has a different viral origin than a given nucleic acid or amino acid sequence.

[0148] Compared to a reference nucleic acid, nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions, and / or insertions. Deletions involve removing one or more nucleotides from the reference nucleic acid. Addition variants involve the fusion of one or more nucleotides at their 5'- and / or 3'-termini, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. In the case of substitutions, at least one nucleotide in the sequence is removed, and at least one other nucleotide is inserted at its position (e.g., transversion and transition). Mutations include baseless sites, crosslinked sites, and chemically altered or modified bases. Insertions involve adding at least one nucleotide to the reference nucleic acid.

[0149] According to the present invention, a "nucleotide alteration" can refer to a single or multiple nucleotide deletion, addition, mutation, substitution, and / or insertion compared to a reference nucleic acid. In some embodiments, a "nucleotide alteration" is selected from a single nucleotide deletion, single nucleotide addition, single nucleotide mutation, single nucleotide substitution, and / or single nucleotide insertion compared to a reference nucleic acid. According to the present invention, a nucleic acid variant may include one or more nucleotide alterations compared to a reference nucleic acid.

[0150] Variants of a specific nucleic acid sequence preferably have at least one functional property of the specific sequence, and are preferably functionally equivalent to the specific sequence, for example, nucleic acid sequences that exhibit the same or similar properties as the specific nucleic acid sequence.

[0151] As described below, some embodiments of the present invention are characterized in particular by nucleic acid sequences homologous to other nucleic acid sequences. These homologous sequences are variants of other nucleic acid sequences.

[0152] 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 is at least 70%, preferably at least 75%, more 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 given for regions 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 a preferred embodiment, the degree of identity is given for the entire length of the reference nucleic acid sequence.

[0153] "Sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of identical amino acids or nucleotides between the sequences.

[0154] The term "% identical" specifically refers to the percentage of identical nucleotides in the optimal alignment between two sequences to be compared. This percentage is purely statistical, and the differences between the two sequences can be randomly distributed across the entire length of the sequences. Furthermore, the sequences to be compared may contain additions or deletions compared to a reference sequence to achieve the optimal alignment. The comparison of two sequences is typically performed after the optimal alignment by comparing the sequences against specific segments or "comparison windows" to identify corresponding sequences in local regions. The best alignment for comparison can be performed manually or by means of the local homology algorithm of Smithand Waterman, 1981, Ads App. Math. 2:482, the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85:2444, or by means of computer programs using the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0155] The percentage similarity is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared, and multiplying the result by 100.

[0156] For example, the BLAST program “BLAST2 sequence” available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi can be used.

[0157] If two sequences are complementary to each other, the nucleic acid is “capable of hybridization” or “hybridizes” with another nucleic acid. If two sequences are capable of forming a stable double helix with each other, the nucleic acid is “complementary” with another nucleic acid. According to the invention, hybridization is preferably performed under conditions that allow specific hybridization between multiple nucleotides (strict conditions). Strict 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*, FMAusubel et al., Editors, John Wiley & Sons, Inc., New York, and refer to, for example, hybridization at 65°C in a hybridization buffer (3.5x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride / 0.15M sodium citrate, pH 7. After hybridization, the membranes with transferred DNA were washed, for example, in 2×SSC at room temperature, and then in 0.1-0.5×SSC / 0.1×SDS at temperatures up to 68°C.

[0158] Percentage complementarity refers to the percentage of consecutive 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, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfect complementarity" or "complete complementarity" means that all consecutive residues in the nucleic acid sequence will bind to the same number of consecutive residues in the second nucleic acid sequence via hydrogen bonds. Preferably, the complementarity of 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 complementarity of the present invention is 100%.

[0159] The term "derivative" encompasses any chemical derivatization of nucleic acids on nucleotide bases, sugars, or phosphates. The term "derivative" also includes nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs. Preferably, derivatization of nucleic acids increases their stability.

[0160] "A nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, the sequence is a variant of a specific sequence that, when it replaces the specific sequence in an RNA molecule, preserves the stability and / or translation efficiency of the RNA.

[0161] "nt" is an abbreviation for nucleotide; or for nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0162] According to the present invention, the term "codon" refers to a base triplet that encodes a nucleic acid and specifies which amino acid will be added next during the protein synthesis process of the ribosome.

[0163] The terms “transcription” and “transcribing” refer to the process by which RNA polymerase reads a nucleic acid molecule having a specific nucleic acid sequence (“nucleic acid template”) to enable the RNA polymerase to produce a single-stranded RNA molecule. During transcription, genetic information in the nucleic acid template is transcribed. The nucleic acid template can be DNA; however, for example, in the case of transcription using an alphavirus nucleic acid template, the template is usually RNA. Subsequently, the transcribed RNA can be translated into a protein. According to the 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 generate the transcript. These cloning vectors are generally designated as transcription vectors and are covered within the term “vector” according to the invention. The cloning vector is preferably a plasmid. According to the invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0164] Single-stranded nucleic acid molecules produced during transcription usually have a nucleic acid sequence that serves as a template complementary sequence.

[0165] According to the present invention, the terms "template" or "nucleic acid template" or "template nucleic acid" generally refer to a nucleic acid sequence that can be replicated or transcribed.

[0166] "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to the nucleic acid sequence of the transcription product (which, where appropriate, is part of a complete RNA molecule) that serves as a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0167] According to the present invention, the "3' end of a nucleic acid" refers to the end having a free hydroxyl group. In illustrations of double-stranded nucleic acids, particularly DNA, the 3' end is always on the right. According to the present invention, the "5' end of a nucleic acid" refers to the end having a free phosphate group. In illustrations of double-stranded nucleic acids, particularly DNA, the 5' end is always on the left.

[0168] 5' terminal 5'--P-NNNNNNN-OH-3' 3' terminal

[0169] 3'-HO-NNNNNNN-P--5'

[0170] "Upstream" describes the relative positioning of the first element of a nucleic acid molecule with respect to the second element of the same molecule, where both elements are contained within the same molecule, and the first element is closer to the 5' end of the molecule than the second element. The second element is then referred to as "downstream" of the first element. An element located "upstream" of the second element can be synonymously referred to as being located at the "5'" of the second element. For double-stranded nucleic acid molecules, similar "upstream" and "downstream" indications are given relative to the (+) strand.

[0171] According to the present invention, "functional linkage" or "functionally connected" refers to a connection within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, then the promoter and the coding sequence are functionally linked. Functionally linked nucleic acids are typically adjacent to each other (and may be separated by other nucleic acid sequences where appropriate), and in a particular embodiment, are transcribed by RNA polymerase to yield a single RNA molecule (co-transcription).

[0172] In a particular embodiment, according to the present invention, a nucleic acid is functionally linked to an expression control sequence, which may be homologous to or heterologous to the nucleic acid.

[0173] According to the present invention, the term "expression control sequence" includes a promoter, a ribosome-binding sequence, and other control elements that control the transcription of a gene or the translation of derived RNA. In specific embodiments of the invention, the expression control sequence can be modulated. The precise structure of the expression control sequence may vary depending on the species or cell type, but generally includes 5'-non-transcriptional sequences and 5'- and 3'-non-translational sequences, respectively, involved in initiating transcription and translation. More specifically, the 5'-non-transcriptional expression control sequence includes a promoter region that encompasses the promoter sequence for transcriptional control of genes used for functional linkage. The expression control sequence may also include enhancer sequences or upstream activator sequences. Expression control sequences of DNA molecules typically include 5'-non-transcriptional sequences and 5'- and 3'-non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, etc. Expression control sequences of alphavirus RNA may include subgenomic promoters and / or one or more conserved sequence elements. Specific expression control sequences of the present invention are subgenomic promoters of alphaviruses, as described herein.

[0174] The nucleic acid sequences specified herein, particularly transcribed and coding nucleic acid sequences, can be combined with any expression control sequences (particularly promoters) that may be homologous or heterologous to the nucleic acid sequences. The term “homological” means that the nucleic acid sequence is also functionally linked to the expression control sequence in its native state, while the term “heterologous” means that the nucleic acid sequence is not functionally linked to the expression control sequence in its native state.

[0175] If a transcribed nucleic acid sequence (particularly a nucleic acid sequence encoding a peptide or protein) is covalently linked to an expression control sequence, thereby allowing the transcription or expression of the transcribed nucleic acid sequence (particularly a coding nucleic acid sequence) to be controlled or influenced by the expression control sequence, then they are "functionally" linked to each other. If the nucleic acid sequence is translated into a functional peptide or protein, inducing the expression control sequence, which is functionally linked to the coding sequence, will result in the transcription of said coding sequence without causing a frameshift in the coding sequence or preventing the coding sequence from being translated into the desired peptide or protein.

[0176] 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 a recognition and binding site for RNA polymerase. A promoter region may include additional recognition or binding sites for other factors involved in regulating the transcription of the gene. Promoters can control the transcription of prokaryotic or eukaryotic genes. Promoters can be "inducible," initiating transcription in response to an inducer, or "constitutive," if transcription is not controlled by an inducer. In the absence of an inducer, inducible promoters are expressed only at a very low level or not at all. In the presence of an inducer, the gene is "switch on," or the transcriptional level increases. This is typically mediated by the binding of a specific transcription factor. Specific promoters of this invention are subgenomic promoters, such as the subgenomic promoters of alphaviruses as described herein. Other specific promoters are positive or negative genomic promoters, such as the positive or negative genomic promoters of alphaviruses.

[0177] The term "core promoter" refers to the nucleic acid sequence contained within a promoter. The core promoter is typically the smallest part of the promoter required for the proper initiation of transcription. A core promoter usually includes a transcription start site and an RNA polymerase binding site.

[0178] "Polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of polymeric molecules from monomeric building blocks. "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of RNA molecules from ribonucleotide building blocks. "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of DNA molecules from deoxyribonucleotide building blocks. For both DNA and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, DNA polymerases synthesize DNA molecules based on a template nucleic acid, which is usually a DNA molecule. Typically, RNA polymerases synthesize RNA molecules based on a template nucleic acid, which is either a DNA molecule (in this case, the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in this case, the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0179] 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 and (+) strand of genomic RNA leads to RNA replication. Therefore, RNA-dependent RNA polymerase is synonymously called "RNA replicase" or simply "replicaase." In nature, all RNA viruses, except retroviruses, typically encode RNA-dependent RNA polymerases. A typical example of a virus encoding RNA-dependent RNA polymerase is alphavirus.

[0180] According to the present invention, "RNA replication" generally refers to the synthesis of an RNA molecule based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule may, for example, be identical to or complementary to the template RNA molecule. Generally, RNA replication can occur through the synthesis of DNA intermediates, or directly through RNA-dependent RNA polymerase (RdRP)-mediated RNA-dependent RNA replication. In the case of alphavirus, RNA replication does not occur through DNA intermediates, but is mediated by RNA-dependent RNA polymerase (RdRP): the template RNA strand (first RNA strand) – or a portion thereof – serves as the template for the synthesis of the second RNA strand, which is complementary to the first RNA strand or a portion thereof. The second RNA strand – or a portion thereof – may optionally serve as the template for the synthesis of the third RNA strand, which is 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. Therefore, RNA-dependent RNA polymerase can directly synthesize the complementary RNA strand of the template, as well as indirectly synthesize the same RNA strand (through a complementary intermediate strand).

[0181] According to the present invention, the term "template RNA" refers to RNA that can be transcribed or replicated by RNA-dependent RNA polymerase.

[0182] 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 performing one or more intercellular or intracellular functions. More specifically, the term refers to a nucleic acid portion (typically DNA; however, in the case of RNA viruses, RNA) containing nucleic acids encoding specific proteins or functional or structural RNA molecules.

[0183] As used herein, "isolated molecule" refers to a molecule that is substantially free of other molecules (such as other cellular material). According to the invention, the term "isolated nucleic acid" means that the nucleic acid is: (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced by clonal recombination, (iii) purified, for example by cleavage and gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Isolated nucleic acids are nucleic acids that can be manipulated using recombinant technologies.

[0184] The term "vector" is used herein in its broadest sense, including any intermediate vector for nucleic acids, such as those enabling the nucleic acid to be introduced into prokaryotic and / or eukaryotic host cells and, where appropriate, integrated into the genome. Such vectors preferably replicate and / or are expressed in cells. Vectors include plasmids, phages, viral genomes, and fragments thereof.

[0185] In the context of this invention, the term "recombinant" means "prepared by genetic engineering". Preferably, in the context of this invention, the "recombinant object" such as recombinant cells is not naturally occurring.

[0186] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature," including known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0187] According to the present invention, the term "expression" is used in its broadest sense, including the production of RNA and / or proteins. It also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable. Regarding RNA, the term "expression" or "translation" refers to the process in which an amino acid sequence is directed by a chain encoding RNA (e.g., messenger RNA) in cellular ribosomes to produce a peptide or protein.

[0188] According to the present invention, the term "mRNA" means "messenger RNA" and refers to transcripts that are typically generated using a DNA template and encode peptides or proteins. Typically, mRNA contains a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be generated from a DNA template by in vitro transcription. In vitro transcription methods 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 stabilization modifications and capping.

[0189] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to a continuous or discontinuous sequence of adenosine residues, typically located at the 3' end of an RNA molecule. A continuous sequence is characterized by a series of adenosine residues. Continuous poly(A) sequences are typical in nature. Although eukaryotic DNA generally does not encode poly(A) sequences, DNA-encoded poly(A) sequences are covered in this invention because they are linked to the free 3' end of RNA during eukaryotic transcription in the cell nucleus by template-independent RNA polymerases post-transcriptionally.

[0190] According to the present invention, with respect to nucleic acid molecules, the term "primary structure" refers to the linear sequence of nucleotide monomers.

[0191] According to the present invention, the term "secondary structure" refers to a two-dimensional representation of a nucleic acid molecule reflecting base pairing; for example, in the case of single-stranded RNA molecules, particularly intramolecular base pairing. Although each RNA molecule has only one polynucleotide chain, the molecule is typically characterized by regions of (intramolecular) base pairs. According to the present invention, the term "secondary structure" includes structural motifs, including but not limited to base pairs, stems, stem-loops, protrusions, and loops (such as inner loops and multi-branched loops). The secondary structure of a nucleic acid molecule can be represented by a two-dimensional diagram (planar diagram) showing base pairing (for more detailed information on the secondary structure of RNA molecules, see Auber et al., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structures of certain RNA molecules are relevant to the content of this invention.

[0192] According to the present invention, the secondary structure of nucleic acid molecules, particularly the secondary structure of 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, "secondary structure" specifically refers to the secondary structure determined by the prediction for nucleic acid molecules. MFOLD structure prediction can also be used to perform or confirm the prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0193] According to the present invention, a "base pair" is a divalent structural motif in which two nucleotide bases are linked together by hydrogen bonds between donor and acceptor sites. Complementary bases A:U and G:C form a stable base pair through hydrogen bonds between donor and acceptor sites; the A:U and G:C base pair is called a Watson-Crick base pair. Weaker base pairs (called swing base pairs) are formed by the bases G and U (G:U). The base pairs A:U and G:C are called classical base pairs. Other base pairs, such as G:U (frequently found in RNA) and other rare base pairs (e.g., A:C; U:U), are called non-classical base pairs.

[0194] According to the present invention, "nucleotide pairing" refers to two nucleotides that are linked together, and their bases thereby form a base pair (classical or non-classical base pair, preferably classical base pair, and most preferably Watson-Crick base pair).

[0195] According to the present invention, the terms "stem-loop" or "hairpin" or "hairpin loop" can be used interchangeably to refer 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 consists of a continuous nucleic acid sequence containing a stem and a (terminal) loop, also known as a hairpin loop, where the stem is formed by two adjacent, fully or partially complementary sequence elements; it is separated by a short sequence (e.g., 3-10 nucleotides) to form a stem-loop structure. Two adjacent, fully or partially complementary sequences can be defined, for example, stem-loop elements stem 1 and stem 2. When these two adjacent, fully or partially anticomplementary sequences, such as stem-loop elements stem 1 and stem 2, form a base pair with each other, a stem-loop is formed, resulting in a double-stranded nucleic acid sequence containing an unpaired loop at its end formed by a short sequence located between stem-loop elements stem 1 and stem 2. Therefore, the stem-loop comprises two stems (stem 1 and stem 2) that form base pairs with each other at the bivalent structural level of the nucleic acid molecule, while at the primary structural level, they are separated by short sequences not belonging to stem 1 or stem 2. For illustration, the two-dimensional representation of the stem-loop resembles a lollipop-shaped structure. The formation of the stem-loop structure requires the presence of sequences that can fold back to form paired double strands; these paired double strands are formed by stem 1 and stem 2. The stability of the paired stem-loop element is generally determined by the length, the number of nucleotides in stem 1 that can form base pairs (preferably classical base pairs, more preferably Watson-Crick base pairs) with nucleotides in stem 2, relative to the number of nucleotides in stem 1 that cannot form such base pairs with nucleotides in stem 2 (mismatches or bulges). According to the invention, the optimal loop length is 3-10 nucleotides, more preferably 4-7 nucleotides, such as 4, 5, 6, or 7 nucleotides. If a given nucleic acid sequence is characterized by a stem-loop, the corresponding complementary nucleic acid sequence is generally also characterized by a stem-loop. Stem-loops are typically formed from single-stranded RNA molecules. For example, there are several stem-loops in the 5' replication recognition sequence of alphavirus genomic RNA.

[0196] According to the present invention, regarding a specific secondary structure (e.g., stem-loop) of a nucleic acid molecule, "disruption" or "disrupt" means that the specific secondary structure is absent or altered. Typically, a secondary structure may be disrupted as a result of a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop may be disrupted due to a change in one or more nucleotides forming the stem, making nucleotide pairing impossible.

[0197] According to the present invention, the term "tertiary structure" refers to the three-dimensional structure of a nucleic acid molecule as defined by atomic coordinates.

[0198] According to the present invention, nucleic acids such as RNA, for example rRNA, can encode peptides or proteins. Therefore, transcribed nucleic acid sequences or their transcripts can contain open reading frames (ORFs) encoding peptides or proteins.

[0199] According to the present invention, the term "nucleic acid encoding a peptide or protein" means that, if present in a suitable environment (preferably intracellular), the nucleic acid can direct the assembly of amino acids during translation to produce a peptide or protein. Preferably, the encoding RNA according to the present invention is capable of interacting with cellular translation mechanisms, such that the encoding RNA can be translated to produce a peptide or protein.

[0200] According to the present invention, the term "peptide" includes oligopeptides and polypeptides, referring to a substance comprising 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and at most preferably 50, preferably 100 or preferably 150 consecutive amino acids linked together by peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having at least about 151 amino acids, but the terms "peptide" and "protein" are generally used synonymously herein.

[0201] According to the present invention, the terms "peptide" and "protein" include substances that contain not only amino acid components but also non-amino acid components (such as sugar and phosphate structures), and also substances that contain bonds such as ester bonds, thioether bonds or disulfide bonds.

[0202] According to the present invention, the terms "initiation codon" and "startcodon" are synonymous with the codon (base triplet) in an RNA molecule that is likely the first codon translated by the ribosome. This codon typically encodes the amino acid methionine in eukaryotes and a modified methionine in prokaryotes. The most common start codon in both eukaryotes and prokaryotes is AUG. Unless specifically stated herein that the start codon being referred to is not AUG, the term "start codon" for the purposes of the RNA molecule refers to the codon AUG. According to the present invention, the term "start codon" is also used to refer to the corresponding base triplet in deoxyribonucleic acid, i.e., the base triplet encoding the RNA start codon. If the start codon of messenger RNA is AUG, then the base triplet encoding AUG is ATG. According to the present invention, the term "start codon" preferably refers to a functional start codon, i.e., a start codon that is used or will be used by the ribosome as a codon to begin translation. RNA molecules may contain AUG codons that are not used by ribosomes to initiate translation, for example, because these codons are too close to the cap. The term "functional start codon" does not cover these codons.

[0203] Specific and / or preferred variations of the various features of the invention are provided below. The invention also contemplates particularly preferred embodiments that are generated by combining two or more specific and / or preferred variations described by two or more features of the invention.

[0204] "Separated" means altered or separated from its natural state. For example, cells, nucleic acids, or peptides naturally present in living animals are not "separated," while the same cells, nucleic acids, or peptides that are partially or completely separated from substances coexisting in their natural state are "separated." Preferably, the separated cells, nucleic acids, or peptides are in a purified or substantially purified state. The separated cells or cell populations preferably do not coexist with different types of cells; for example, separated T cells do not coexist with other blood cells such as dendritic cells. Preferably, the separated cells coexist only with cells of the same genetic type.

[0205] The term "syngenetic" is used to describe cells that have the same genetic information as another cell or cell group.

[0206] The term "autologous" is used to describe anything that originates from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organ derived from the same subject. The advantage of such methods is that they overcome the immune barrier, which would otherwise lead to rejection.

[0207] As used in this article, the term "alien" is used to describe anything that originates from different individuals of the same species. Two or more individuals are called alliens when their genes differ at one or more loci.

[0208] The term "homogeneous" is used to describe anything that originates from individuals or tissues with the same genotype, i.e., identical twins or animals of the same inbred lineage, or their tissues.

[0209] The term "heterologous" is used to describe something composed of multiple different elements. For example, transplanting bone marrow from one individual to another constitutes a heterologous transplant. Heterologous genes are genes derived from sources other than the recipient.

[0210] In the context of this invention, the term "recombinant" means "prepared through genetic engineering." Preferably, the "recombinant object" in the context of this invention, such as recombinant cells, is not naturally occurring.

[0211] As used in this article, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring.

[0212] The term "lentivirus" as used in this article refers to the genus *Lentinvirus* within the family Retroviridae. Lentivirals are unique among retroviruses, capable of infecting non-dividing cells; they can deliver substantial genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral vectors provide a means to achieve significant levels of gene transfer in vivo.

[0213] As used herein, the term "specific binding" refers to a molecule, such as an antibody or CAR, that recognizes a specific antigen but does not significantly recognize or bind to other molecules in the sample or the subject's body. For example, an antibody that specifically binds to an antigen of one species may also bind to that antigen of one or more other species. However, this cross-species reactivity itself does not change the antibody's specific classification. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, this cross-reactivity itself does not change the antibody's specific classification. In some cases, "specific binding" or "specifically binding" can be used to describe the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that this interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than generally recognizing and binding to proteins. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled "A") will reduce the amount of labeled A bound to the antibody.

[0214] As used in this article, the term "microloop" refers to supercoiled DNA molecular vectors that lack bacterial origins of replication and antibiotic resistance genes. They are primarily composed of eukaryotic expression cassettes (see, for example, F. Jia et al. Nature methods, Vol. 7, no. 3, pp. 197-199, March 2010).

[0215] Immune effector cells

[0216] Cells used in connection with this invention and into which nucleic acids (DNA and / or RNA) can be introduced are immune effector cells, such as cells with lytic potential, particularly lymphoid cells, preferably T cells, especially 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 triggers the destruction of the target cell. For example, cytotoxic T cells trigger the destruction of the target cell by one or both of the following: First, upon activation, the T cell releases cytotoxins such as perforin, granzyme, and granzyme-lysin. Perforin and granzyme-lysin create pores in the target cell, granzyme enters the cell and triggers a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death). Second, apoptosis can be induced by Fas-Fas ligand interactions between the T cell and the target cell. Cells used in connection with this invention are preferably autologous cells, although allogeneic or allogeneic cells can be used.

[0217] In the context of this invention, the terms "immune effector cells" or "immune reactive cells" refer to cells that perform effector functions during an immune response.

[0218] In the context of this invention, the term "effective function" includes any function mediated by components 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 the suppression of tumor development, including the inhibition of tumor dissemination and metastasis. Preferably, in the context of this invention, effector functions are T cell-mediated effector functions. Such functions are mediated by helper T cells (CD4+). + In the case of T cells, this includes the release of cytokines and / or CD8. + Activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, this includes the elimination of cells (i.e., cells characterized by expressing a certain antigen) (e.g., cell lysis mediated by apoptosis or perforin), the production of cytokines (such as IFN-γ and TNF-α), and specific cytolytic killing of target cells expressing antigens.

[0219] In one embodiment, "immune effector cells" are capable of binding antigens (such as antigens presented on cells or expressed on the cell surface in the context of MHC) 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 this invention, "immune effector cells" are T cells, preferably CD4+. + and / or CD8 +T cells. According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, particularly helper T cells or cytolytic T cells) upon appropriate stimulation. Immune effector cells contain CD34. + Hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. T cell precursors differentiate into cytolytic T cells upon exposure to antigens, similar to clonal selection in the immune system.

[0220] Preferably, "immune effector cells" can recognize antigens with a degree of specificity, particularly when the antigen is presented in the context of MHC or is present on the surface of diseased cells (such as cancer cells). Preferably, this recognition enables the cells recognizing the antigen to be responsive or reactive. If the cells are helper T cells (CD4+)... + T cells), this responsiveness or reactivity may involve the release of cytokines and / or CD8+. + Activation of lymphocytes (CTLs) and / or B cells. If the cells are CTLs, this responsiveness or reactivity may involve the elimination of cells (i.e., cells characterized by expressing a certain antigen) (e.g., through apoptosis or perforin-mediated cell lysis). According to the invention, the responsiveness of CTLs may include sustained calcium ion flow, 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. The responsiveness of CTLs can also be determined using artificial reporter systems that can accurately indicate the responsiveness of CTLs. Such CTLs that recognize antigens and are responsive or reactive are also referred to herein as “antigen-responsive CTLs”.

[0221] In one embodiment, the immune effector cell is an immune effector cell expressing a CAR. In another embodiment, the immune effector cell is an immune effector cell expressing a TCR.

[0222] The immune effector cells used according to the present invention may express endogenous antigen receptors, such as T cell receptors or B cell receptors, or may lack the expression of endogenous antigen receptors.

[0223] "Lymphoid cells" are cells or precursor cells of such cells that, optionally after appropriate modification (e.g., after transferring antigen receptors such as TCRs or CARs), are capable of generating an immune response (e.g., a cellular immune response), 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 antigen receptors on their cell surface. In one embodiment, the lymphoid cells lack endogenous expression of T cell receptors.

[0224] The terms "T cell" and "T lymphocyte" are used interchangeably in this article and include helper T cells (CD4+). + T cells) and cytotoxic T cells (CTL, CD8) + T cells, including cytotoxic T cells, are also referred to as T cells. "Antigen-specific T cells" or similar terms refer to T cells that recognize antigens targeted by T cells and preferably perform T cell effector functions. A T cell is considered specific to an antigen if it can kill target cells expressing that antigen. T cell specificity can be assessed using any of a variety of standard techniques, such as chromium release assays or proliferation assays. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0225] T cells belong to a group of white blood cells called 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 a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the main organ responsible for T cell maturation. Several different subsets of T cells have been identified, each with different functions.

[0226] Helper T cells assist other white blood cells in the immune process, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4 cells. + T cells are activated because they express the CD4 glycoprotein on their surface. Helper T cells are activated when they are presented with MHC class II peptide antigens expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in active immune responses.

[0227] Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. These cells are also known as CD8 cells. + T cells are formed because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to MHC class I-associated antigens that are present on the surface of almost every cell in the body.

[0228] Regulatory T cells, or Tregs, are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and typically inhibit or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0229] As used herein, the term "naive T cell" refers to a mature T cell that, unlike activated or memory T cells, has not yet encountered its corresponding antigens in the periphery. Naive T cells are typically characterized by surface expression of L-selectin (CD62L), lack of activation markers CD25, CD44, or CD69, and lack of the memory CD45RO isoform.

[0230] As used herein, the term "memory T cells" refers to a subset or population of T cells that have previously encountered and responded to their corresponding antigens. Upon a second encounter with the antigen, memory T cells can proliferate to initiate a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells can be CD4+ cells. + or CD8 + And it is usually expressed as CD45RO.

[0231] According to the present invention, the term "T cell" also includes cells that can mature into T cells under appropriate stimulation.

[0232] Most T cells possess a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor consists of two separate polypeptide chains, derived from the independent T cell receptor α and β (TCRα and TCRβ) genes, and are called the α-TCR and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells that possess a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is much rarer than αβ T cells (comprising 2% of all T cells).

[0233] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells, derived from these stem cells, colonize the thymus and proliferate through cell division to produce a large number of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as double-negative (CD4+). - CD8 - ) cells. As they develop, they transform into double-positive thymocytes (CD4+) cells. + CD8 + ), and eventually matures into a single positive (CD4+) . + CD8 - or CD4 - CD8 + Thymocytes, which are then released from the thymus into the peripheral tissues.

[0234] T cells can typically be prepared in vitro or ex vivo using standard procedures. For example, commercially available cell isolation systems can be used to isolate T cells from the bone marrow, peripheral blood, or portions of bone marrow or peripheral blood of mammals (such as patients). Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures. Samples containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0235] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined as those expressing CD56 or CD16 and lacking T cell receptors. As provided herein, NK cells can also differentiate from stem cells or progenitor cells.

[0236] Nucleic acid

[0237] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant-produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVTRNA) or synthetic RNA. According to the invention, the polynucleotides are preferably isolated.

[0238] Nucleic acids can be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage vectors (such as λ phage), viral vectors (such as retrovirus, adenovirus, or baculovirus vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). Vectors include expression vectors and cloning vectors. Expression vectors contain plasmids and viral vectors and typically contain the desired coding sequence and the appropriate DNA sequence required to express the operable linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are typically used to engineer and amplify a desired DNA fragment and may lack the functional sequence required to express the desired DNA fragment.

[0239] In one embodiment of all aspects of the invention, nucleic acids (such as nucleic acids encoding antigen receptors or nucleic acids encoding immune effector cell activator molecules) are expressed in immune effector cells and provide antigen receptors or activator molecules.

[0240] The nucleic acids of the present invention can be introduced into immune effector cells in a variety of ways, such as by the particles or complexes of the present disclosure or any combination comprising one or both of the particles and complexes, or by electroporation, or by a virus-based system, or by any particles capable of introducing nucleic acids into cells, particularly lipid- or polymer-based particles. In some embodiments, the nucleic acids are introduced into or taken up by cells, wherein the cells may be present in a subject (e.g., a patient). Thus, according to the present invention, cells incorporating the nucleic acids described herein may be present in vitro or in vivo, for example, the cells may form part of an organ, tissue, and / or organism of a patient.

[0241] In some embodiments, the first nucleic acid molecule is a DNA or RNA molecule introduced into the cell. In other embodiments, the first nucleic acid molecule is a DNA molecule with an integrated first nucleotide sequence, which was previously present in the cell but did not contain the first nucleotide sequence. In this embodiment, the first nucleotide sequence has been previously introduced into the cell via another first nucleic acid molecule (which may be DNA or RNA), from which the first nucleotide sequence is retrieved and integrated into a DNA molecule (e.g., genomic DNA) already present in the cell. The DNA molecule with the integrated first nucleotide sequence becomes the first nucleic acid molecule after integration.

[0242] In some implementations, the first nucleic acid is an RNA molecule that is integrated into the genome of immune effector cells via a retrotransposon-based system (preferably a virus-based or poly-A-based retrotransposon system).

[0243] The term "genome," "genomic DNA," or "genomic nucleic acid molecule" refers to any kind of DNA molecule that can be replicated and equally distributed from the mother cell to daughter cells. Genomic DNA includes chromosomal DNA and extrachromosomal DNA, such as episomes, preferably nonviral episomes.

[0244] The term "epitaphone" should be understood as a DNA molecule that is not integrated into the eukaryotic genome but still exists as part of the eukaryotic genome. Epiitaphones achieve this by replicating along with the rest of the genome and then being equally distributed to each daughter cell, just like chromosomes.

[0245] In some embodiments, the second nucleic acid molecule does not integrate into the genomic nucleic acid molecule of the immune effector cell, and in particular, is not contained in the appendage present on the immune effector cell. Preferably, the second nucleic acid is an RNA molecule, more preferably mRNA, and more preferably modified RNA or mRNA. In some embodiments, the RNA molecule may be degraded or lost during cell division.

[0246] In another embodiment, the second nucleic acid molecule is a DNA molecule (preferably a plasmid), which is preferably degraded or epigenetically silenced in immune effector cells, or cannot be equally transmitted during cell division.

[0247] In some implementations, the second nucleic acid is expressed transiently.

[0248] The term "transient expression" should be understood as meaning that a nucleic acid or transcript is expressed only for a limited time. Preferably, this means that the nucleic acid encoding the transcript, non-coding RNA, or protein is lost from the cell due to degradation or uneven distribution of nucleic acids during cell division, and in particular, the nucleic acid does not replicate in the cell to replenish any lost nucleic acid. Transiently expressed nucleic acids are preferably not part of the cell's genome. For example, a DNA plasmid encoding a specific transcript and lacking a eukaryotic origin of replication (preferably lacking a mammalian (preferably human) origin of replication) is only transiently expressed because, although it expresses the encoded transcript, the plasmid cannot be replenished after degradation due to the absence of an origin of replication and cannot replicate before cell division; the plasmid is lost. For example, a protein is considered transiently expressed when it is expressed only for a limited time (e.g., because the nucleic acid encoding the protein is lost and not replicated). Another example of transiently expressed nucleic acids is mRNA molecules introduced into the cell (e.g., via electroporation).

[0249] In some implementations, the third nucleic acid molecule is DNA or RNA. In cases where the first nucleotide sequence needs to be integrated into the genome of an immune effector cell, the third nucleic acid molecule provides the necessary enzyme (e.g., transposase, reverse transcriptase, or integrase) for integrating the first nucleotide sequence into the genomic nucleic acid molecule. Providing a means of integrating the first nucleotide sequence via a separate nucleic acid has a particular advantage: it helps prevent the loss of the integrated first nucleotide sequence, since the means for integration (which may also remove the integrated nucleotide sequence) is only available for a limited time. The third nucleic acid molecule can be one or more nucleic acid molecules. If integration requires more than one enzyme, providing separate nucleic acids encoding the required enzymes helps provide further flexibility (e.g., in terms of the amount of enzyme produced in the cell or the combination of different enzymes).

[0250] In some embodiments, the third nucleic acid molecule does not integrate into the genomic nucleic acid molecule of the immune effector cell, and in particular, is not contained in the appendage present in the immune effector cell. Preferably, the third nucleic acid is an RNA molecule, more preferably mRNA, and more preferably modified RNA or mRNA. In some embodiments, the RNA molecule may be degraded or lost during cell division.

[0251] In another embodiment, the third nucleic acid molecule is a DNA molecule, preferably a plasmid, which is preferably not transmitted during cell division, or is degraded or epigenetically silenced in immune effector cells.

[0252] In another implementation, the enzymes required for integration can also be provided in other forms than those encoded by nucleic acid molecules.

[0253] In some embodiments, the immune effector cells or particles of this disclosure comprise a fourth nucleic acid molecule and / or a fifth nucleic acid molecule. In some embodiments, the fourth and / or fifth nucleic acid molecule is one or more nucleic acid molecules.

[0254] In some implementations, the fourth nucleic acid molecule encodes one or more antigens that bind to the first cell surface antigen receptor.

[0255] In some implementations, the fifth nucleic acid molecule encodes one or more antigens that bind to the second cell surface antigen receptor.

[0256] In some embodiments, the fourth nucleic acid molecule does not integrate into the genomic nucleic acid molecule of the immune effector cell, and in particular, is not contained in the appendage present in the immune effector cell. Preferably, the fourth nucleic acid is an RNA molecule, more preferably mRNA, and more preferably modified RNA or mRNA. In some embodiments, the RNA molecule may be degraded or lost during cell division.

[0257] In another embodiment, the fourth nucleic acid molecule is a DNA molecule (preferably a plasmid), which is preferably not transmitted during cell division, or is degraded or epigenetically silenced in immune effector cells.

[0258] In some embodiments, the fifth nucleic acid molecule does not integrate into the genomic nucleic acid molecules of immune effector cells, and in particular, is not contained in appendages present on immune effector cells. Preferably, the fifth nucleic acid is an RNA molecule, more preferably mRNA, and more preferably modified RNA or mRNA. In some embodiments, the RNA molecule may be degraded or lost during cell division.

[0259] In another embodiment, the fifth nucleic acid molecule is a DNA molecule (preferably a plasmid), which is preferably not transmitted during cell division, or is degraded or epigenetically silenced in immune effector cells.

[0260] In some implementations, at least one or all of the first, second, third, fourth, and fifth nucleic acid molecules are modified RNA molecules.

[0261] Modified RNA

[0262] In some embodiments, the RNA or RNA molecule described herein is modified RNA. In some embodiments, the modified RNA contains at least one functional analogue of A, C, G and / or U.

[0263] 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.

[0264] In this context, the modified RNA molecules defined herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. Backbone modifications relevant to this disclosure are modifications that chemically modify the phosphate group of the nucleotide backbone contained in the RNA molecule as defined herein. Sugar modifications relevant to this disclosure are chemical modifications of the sugar group of the nucleotides in the RNA molecule as defined herein. Furthermore, base modifications relevant to this disclosure are chemical modifications of the base portion of the nucleotides in the RNA molecule. In this context, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and / or translation.

[0265] Sugar Modification: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be modified at the sugar moiety. For example, the 2' hydroxyl (OH) can be modified or replaced by a number of different "oxygen" or "deoxy" substituents. Examples of "oxygen"-2' hydroxyl modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or glycosyl); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked to the 4' carbon of the same ribose via, for example, a methylene bridge; and amino (-O-amino, in which the amino, e.g., NRR, can be alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, diethylamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, an amino group (e.g., NH₂; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group may be linked to a glycosyl group via a linker containing one or more atoms of C, N, and O. The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Therefore, modified RNA molecules may include nucleotides containing, for example, arabinose as a glycosyl group.

[0266] Backbone Modification: The phosphate backbone can be further modified in modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein. The phosphate groups of the backbone can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can include complete replacement of unmodified phosphate groups with the modified phosphate groups described herein. Examples of modified phosphate groups include, but are not limited to, thiophosphates, selenophosphates, boranophosphates, boranophosphate esters, hydrophosphonates, phosphoramide esters, alkyl or aryl phosphonates, and phosphate triesters. In dithiophosphates, both non-linked oxygen atoms are replaced with sulfur. The phosphate linker can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramide ester), sulfur (bridged thiophosphate), and carbon (bridged methylene phosphonate).

[0267] 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. In some embodiments, major groove chemical modifications may include amino, thiol, alkyl, or halogen groups.

[0268] In specific embodiments of this disclosure, the nucleotide analog / modification is selected from base modifications, preferably from 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-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-iodino-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 nucleoside-5'-triphosphate, 7-deazo-adenosine-5'-triphosphate, 7-deazoguanosine- 5'-Triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-nucleoside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudo-uridine-5'-triphosphate or puromycin-5'-triphosphate, and xanthoside-5'-triphosphate. Nucleotides for base modification may be particularly preferred, selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazoguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudo-uridine-5'-triphosphate.In some embodiments, the modified nucleosides include pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-2-thiouridine, 1-taurate methyluridine The uridine derivatives include 4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 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 analogue replacing uridine is N1-methyl-pseudouridine (m1Ψ).

[0269] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrole-cytidine, pyrrole-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio- -1-Methyl-1-denitro-pseudoisocytidine, 1-Methyl-1-denitro-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.

[0270] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaa-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N... 6-Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methyl-thio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine. In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0271] In some embodiments, the nucleotide can be modified on the major groove face, and may include replacing the hydrogen on C-5 of uracil with a methyl or halogen group. In specific embodiments, the modified nucleoside is 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine, 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)-pseuuridine.

[0272] In a further embodiment, the modified RNA may include nucleoside modifications selected from the following: 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, Nl-methyl-pseudo-uridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5 α-Methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deazo-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deazo-adenosine.

[0273] In some preferred embodiments, the RNA contains a modified nucleoside in place of at least one (e.g., each) uridine.

[0274] As used herein, the term "uracil" describes one of the nucleobases that can appear in RNA nucleic acids. The structure of uracil is:

[0275]

[0276] As used in this article, the term "uridine" describes one of the nucleosides that can appear in RNA. The structure of uridine is:

[0277]

[0278] UTP (uridine 5'-triphosphate) has the following structure:

[0279]

[0280] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0281]

[0282] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, in which uracil is linked to a pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0283] Another exemplary modified nucleoside is N1-methyl-pseuuridine (m1Ψ), which has the following structure:

[0284]

[0285] N1-methyl-pseudo-UTP has the following structure:

[0286]

[0287] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure:

[0288]

[0289] In some preferred embodiments, one or more uridines in the RNA described herein are replaced with modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0290] In some preferred embodiments, the RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, the RNA comprises a modified nucleoside replacing each uridine.

[0291] In some preferred embodiments, the modified nucleoside is 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 more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0292] In some preferred embodiments, the nucleoside that replaces the uridine modification in one or more (e.g., all) RNAs can be any one or more of the following: 3-methyluridine (m 3 U), 5-methoxyuridine (mo) 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thiouridine (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-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyluridine (cm) 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm) 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) 5 U), 5-methoxycarbonylmethyl-uridine (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm) 5 s 2 U), 5-methylaminomethyluridine (mnm) 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm) 5 se 2 U), 5-carbamoylmethyluridine (ncm) 5 U), 5-Carboxymethylaminomethyluridine (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm) 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauric acid methyl-uridine (τm) 5 U), 1-taurine methyl-pseuuridine, 5-taurine methyl-2-thio-uridine (τm) 5 s 2 U), 1-Tauratemethyl-4-thio-pseudouridine), 5-Methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuuridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D) 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 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-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m) 5 Um), 2′-O-methyl-pseuuridine (ψm), 2-thio-2′-O-methyl-uridine (s) 2 Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm)5 Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm) 5 Um), 3,2′-O-dimethyluridine (m) 3 Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm) 5 Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

[0293] In one embodiment, the RNA comprises other modified nucleosides, or further modified nucleosides, such as modified cytidines, as described above. For example, in one embodiment, 5-methylcytidine in the RNA partially or completely (preferably completely) replaces cytidine. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine, and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0294] Cap

[0295] In some embodiments, the RNA or RNA molecule 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 use of codons for the coding sequence or open reading frame may be optimized.

[0296] "RNA containing a 5'-cap," "RNA with a 5'-cap," "RNA modified with a 5'-cap," or "capped RNA" refers to RNA containing 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 said 5'-cap, wherein said 5'-cap is co-transcribed into the resulting RNA strand, or RNA can be generated, for example, by in vitro transcription, and the 5'-cap can be transcribed and ligated to RNA using a capping enzyme (e.g., a capping enzyme of vaccinia virus). In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked via a phosphodiester bond to the 5' position of the next base ("the second base") of the RNA molecule.

[0297] In this disclosure, naturally occurring caps are typically selected from unmethylated cap dinucleotides (G(5')ppp(5')N; also known as GpppN) and methylated cap dinucleotides ((m 7 G(5')ppp(5')N; also known as m 7 GpppN). m 7 GpppN (where N is G) is represented by the following formula:

[0298]

[0299] The capped RNA disclosed herein can be prepared in vitro and is therefore independent of the capping mechanism in the host cell. Co-transcriptional capping is achieved through all four ribonucleoside triphosphates or their functional analogues, along with a capping reagent (such as m...). 7 G(5')ppp(5')G(also known as m) 7 In the presence of GpppG), in vitro transcription of DNA templates using bacterial or bacteriophage nucleic acid polymerases is performed. The nucleic acid polymerase operates via m... 7 The 3'-OH of the GpppG guanosine moiety initiates transcription by nucleophilically attacking the α-phosphate of the next template nucleoside triphosphate (pppN), producing the intermediate m. 7 GpppGpN (where N is the second base in the RNA molecule).

[0300] In a preferred embodiment of this disclosure, the RNA molecule comprises a 5'-cap analog. Cap analogs were originally described as facilitating the large-scale synthesis of RNA transcripts via in vitro transcription.

[0301] For messenger RNA, several cap analogs (also known as synthetic caps) have been generally described to date, and all of them can be used in the context of this disclosure. Ideally, cap analogs associated with higher translation efficiency and / or increased in vivo and / or increased in vitro degradation resistance should be selected.

[0302] Preferably, the cap analog used can only be incorporated into the RNA chain in one direction. Pasquinelli et al. (1995, RNA J.1:957-967) demonstrated that during in vitro transcription, phage RNA polymerase initiates transcription using a 7-methylguanosine unit, thereby approximately 40-50% of the capped transcripts have an inverted cap dinucleotide (i.e., the initial reaction product is Gpppm). 7 RNA with a reversed cap is not functional in translating nucleic acid sequences into proteins, compared to RNA with a correctly oriented cap. Therefore, it is desirable to incorporate the cap in the correct direction, i.e., to produce RNA with a cap that substantially corresponds to m... 7RNAs with structures such as GpppGpN, etc., have been shown to inhibit reverse integration of cap dinucleotides by substituting 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 analogues" produces higher yields than that synthesized in the presence of conventional 5'-cap dinucleotides. 7 In the presence of GpppG, in vitro transcribed RNA is translated more efficiently. For this purpose, a cap analogue in which the 3'-OH group of a methylated guanosine unit is replaced by OCH3 has been described, for example by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analogue (ARCA)). ARCA is a suitable cap dinucleotide according to this disclosure.

[0303]

[0304] In one embodiment, the cap has the effect of making the RNA bearing the cap substantially difficult to uncap. This is important because, generally, the amount of protein produced by synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase containing a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α and β phosphate groups of the triphosphate bridge. In this disclosure, a cap that is insensitive or less sensitive to this type of cleavage can be selected. For this purpose, a suitable cap analogue may be selected from a cap dinucleotide according to formula (I):

[0305]

[0306] Where R 1 Selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted ynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl.

[0307] R 2 and R 3 Independently selected from H, halogen, OH and optionally substituted alkoxy groups, or R 2 and R 3 Together they form OXO, where X is selected from optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3), and

[0308] C(CH3)2, or R 2With R 2 The hydrogen atoms at position 4' of the connected ring combine to form -O-CH2- or -CH2-O-.

[0309] R 5 Selected from S, Se and BH3,

[0310] R 4 and R 6 It is independently selected from O, S, Se and BH3.

[0311] n is 1, 2, or 3.

[0312] R 1 R 2 R3, R 4 R 5 R 6 Preferred embodiments are disclosed in WO2011 / 015347 A1, and can be selected accordingly in this disclosure.

[0313] For example, in one embodiment, the RNA disclosed herein comprises a phosphate thioester-cap-analyte. A phosphate thioester-cap-analyte is a specific cap-analyte in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom, i.e., R in formula (I). 4 R 5 or R 6 One is the S-thiophosphate-cap analogue, described by Kowalska et al., 2008, RNA, 14:1119-1131, as a solution to the undesirable uncapping process and thus increase the stability of RNA in vivo. Specifically, replacing the oxygen atom with a sulfur atom on the β-phosphate group of the 5'-cap results in stability against Dcp2. In the preferred embodiment of this disclosure, R in formula (I) 5 It is S; R 4 and R 6 It is O.

[0314] In another embodiment, the RNA molecule of this disclosure comprises a phosphate thioester-cap-analyte, wherein the phosphate thioester modification of the RNA 5'-cap is combined with an "anti-reverse cap analog" (ARCA) modification. The corresponding ARCA-phosphate thioester-cap-analytes are described in WO2008 / 157688 A2, and they can all be used in the RNA of this disclosure. In this embodiment, R in formula (I) 2 or R 3 At least one of them is not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3Another preferred form is OH. In a preferred embodiment, the oxygen atom at the β-phosphate group is replaced by a sulfur atom (so that R in formula (I) is substituted with OH). 5 It is S; and R 4 and R 6 (Yes, it is O). It is believed that thiophosphate modification of ARCA ensures that the α, β, and γ thiophosphate groups are precisely positioned within the active site of cap-binding proteins during translation and uncapping mechanisms. At least some of these analogues exhibit fundamental resistance to pyrophosphatases Dcp1 / Dcp2. Thiophosphate-modified ARCAs are described to have a significantly higher affinity for eIF4E than their counterparts lacking the thiophosphate group.

[0315] The corresponding cap, namely m2, is particularly preferred in this disclosure. 7,2’-O Gpp s pG, referred to as β-S-ARCA (WO2008 / 157688A2; Kuhn et al., 2010, Gene Ther. 17:961-971). Therefore, in one embodiment of this disclosure, the RNA of this disclosure is modified with β-S-ARCA. β-S-ARCA is represented by the following structure:

[0316]

[0317] Typically, replacing an oxygen atom with a sulfur atom at the bridging phosphoric acid site yields diastereomers of thiophosphates, named D1 and D2 based on their elution patterns in HPLC. In short, "D1 diastereomer of β-S-ARCA" or "β-S-ARCA(D1)" is a diastereomer of β-S-ARCA that elutes first from the HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)). The determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0318] In a first particularly preferred embodiment of this disclosure, the RNA of this disclosure is modified with the β-S-ARCA(D2) diastereomer. The two diastereomers of β-S-ARCA have different sensitivities to nucleases. Studies have shown that RNA carrying the D2 diastereomer of β-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 with the β-S-ARCA(D1) 5'-cap exhibits intermediate sensitivity to Dcp2 cleavage (71% cleavage). Further studies have shown that increased stability against Dcp2 cleavage is associated with increased protein expression in mammalian cells. In particular, studies have shown that RNA carrying the β-S-ARCA(D2) cap is translated more efficiently in mammalian cells than RNA carrying the β-S-ARCA(D1) cap. Therefore, in one embodiment of this disclosure, the RNA of this disclosure is modified with a cap analog according to formula (I), characterized by the inclusion of the substituent R in formula (I). 5 The stereochemical configuration at the P atom corresponds to the P atom of the D2 diastereomer of β-S-ARCA. β Stereochemical configuration at the atom. In this embodiment, R in formula (I) 5 It is S; and R 4 and R 6 It is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred component is OH.

[0319] In a second particularly preferred embodiment, the RNA of this disclosure is modified with a β-S-ARCA(D1) diastereomer. This embodiment is particularly suitable for transferring capped RNA into immature antigen-presenting cells, such as for vaccination purposes. It has been shown that, after transferring separately capped RNA into immature antigen-presenting cells, the β-S-ARCA(D1) diastereomer is particularly suitable for increasing RNA stability, improving RNA translation efficiency, prolonging RNA translation, increasing total protein expression of RNA, and / or increasing the immune response against antigens or antigenic peptides encoded by said RNA (Kuhn et al., 2010, Gene Ther. 17:961-971). Therefore, in an alternative embodiment of this disclosure, the RNA of this disclosure is modified with a capped analog according to formula (I), characterized in that formula (I) contains the substituent R. 5The stereochemical configuration at the P atom corresponds to the P atom of the D1 diastereomer of β-S-ARCA. β Stereochemical configuration at the atom. Corresponding cap analogs and embodiments thereof are described in WO 2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther. 17:961-971. Any cap analog described in WO2011 / 015347 A1 may be used in this disclosure, containing the substituent R. 5 The stereochemical configuration at the P atom corresponds to the P atom of the D1 diastereomer of β-S-ARCA. β The stereochemical configuration of the atom. Preferably, R in formula (I) 5 It is S; and R 4 and R 6 It is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred component is OH.

[0320] In one embodiment, the RNA of this disclosure is modified with a 5'-cap structure according to formula (I), wherein any phosphate group is replaced by a boranophosphate group or a phosphoroselenoate group. Such caps exhibit increased stability both in vitro and in vivo. Optionally, the corresponding compound has a 2'-O- or 3'-O-alkyl group (wherein the alkyl group is preferably methyl); the corresponding cap analogs are referred to as BH3-ARCA or Se-ARCA. Compounds particularly suitable for capping mRNA include β-BH3-ARCA and β-Se-ARCA, as described in WO 2009 / 149253A2. For these compounds, the preferred formula (I) contains a substituent R. 5 The stereochemical configuration at the P atom corresponds to the P atom of the D1 diastereomer of β-S-ARCA. β Stereochemical configuration at the atom.

[0321] In some embodiments, the RNA may include a cap, cap0 (methylation of the first nucleobase, e.g., methylation of the first nucleobase), which may be appropriate in the context of this disclosure. m7 GpppN), cap1 ( m7 Additional methylation of the ribose of adjacent nucleotides of GpppN), cap2 (in m7 Additional methylation of the ribose of the second nucleotide downstream of GpppN), cap3 (inm7 Additional methylation of the ribose of the third nucleotide downstream of GpppN), cap4 (in m7 Additional methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphate thioester modified ARCA, such as β-S-ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0322] In some implementations, the RNA contains 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, for example, Ramanathan A et al. Figure 1 and Decroly E et al. Figure 1 .

[0323] In some embodiments, Cap0 contains a guanosine nucleoside methylated at the 7-position of guanine ( m7 G). In some implementations, Cap0 is linked to RNA via a 5'-to-5'-triphosphate bond, and is also referred to herein as... m7 Gppp or m7 G(5')ppp(5').

[0324] In some embodiments, Cap1 contains a guanosine nucleoside methylated at the 7-position of guanine ( m7 G or 7m G) and the first nucleotide methylated at 2'O in RNA (G) 2 'OMeN1 or N12'OMe or N1 2'OMe In some implementations, Cap1 is linked to RNA via a 5'-to-5'-triphosphate bond; in some implementations, Cap1 may be represented as... 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.

[0325] In some implementation schemes, m7 G(5')ppp(5')(N1 2'OMeCap1 contains a second nucleotide, and N2 is the proximal cap A, G, C, or U at position +2. In some embodiments, such Cap1 is represented as ( 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.

[0326] In some implementations, Cap1 is or includes m7 G(5')ppp(5')(A1 2'OMe pG2, where A1 is the proximal end A of the cap at position +1, G2 is the proximal end G of the cap at position +2, and has the following structure:

[0327]

[0328] In some implementations, Cap1 is or includes m7 G(5')ppp(5')(A1 2'OMe pU2, where A1 is the proximal end A of the cap at position +1, and U2 is the proximal end U of the cap at position +2, and has the following structure:

[0329]

[0330] In some implementations, Cap1 is or includes m7 G(5')ppp(5')(G1 2'OMe pG2, where G1 is the proximal end of the cap at position +1, G2 is the proximal end of the cap at position +2, and has the following structure:

[0331]

[0332] In some embodiments, Cap1 contains a guanosine nucleoside methylated at the 7-position of guanine ( m7 G) and one or more additional modifications (e.g., methylation on the ribose) and the first nucleotide with 2'O methylation in the RNA. In some embodiments, Cap1 comprises a guanosine nucleotide with 7-methylation at the guanine position and 3'O methylation at the ribose (m7G3'OMe or 7m G 3'OMe ); and the first nucleotide (N1) methylated at 2'O in RNA. 2'OMe In some implementations, Cap1 is linked to RNA via a 5'-to-5'-triphosphate bond, and is also referred to herein as (m7G3'OMe)ppp(2'OMeN1) or ( m7 G 3'OMe (5')ppp(5')(2'OMe N1). 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.

[0333] In some implementation schemes, ( m7 G 3’OMe (5')ppp(5')(N1 2'OMe Cap1 contains a second nucleotide, which is the cap proximal nucleotide at position 2, and is 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.

[0334] In some implementations, Cap1 is or includes ( m7 G 3'OMe (5')ppp(5')(A1 2'OMe pG2, where A1 is the proximal end A of the cap at position +1, G2 is the proximal end G of the cap at position +2, and has the following structure:

[0335]

[0336] In some implementations, Cap1 is or includes ( m7 G 3'OMe (5')ppp(5')(G1 2'OMe pG2, where G1 is the proximal end of the cap at position +1, G2 is the proximal end of the cap at position +2, and has the following structure:

[0337]

[0338] In some embodiments, the second nucleotide in Cap1 may include one or more modifications, such as methylation. In some embodiments, Cap1 containing a second nucleotide including 2'O methylation is a Cap2 structure.

[0339] In some embodiments, the RNA polynucleotide containing Cap1 exhibits increased translation efficiency, increased translation rate, and / or increased expression of the encoded payload relative to a suitable reference control. In some embodiments, it contains ( m7 G 3'OMe (5')ppp(5')(A1 2'OMeThe RNA polynucleotide of Cap1 in pG2 (where A1 is the cap proximal nucleotide at position +1 and G2 is the cap proximal nucleotide at position +2) relative to the RNA polynucleotide containing ( m7 G 3'OMe (5')ppp(5')(G1 2'OMe The Cap1 RNA polynucleotide of pG2 (where G1 is the cap proximal nucleotide at position 1 and G2 is the cap proximal nucleotide at position 2) has increased translation efficiency. In some embodiments, the increase in translation efficiency can be assessed by administering the RNA polynucleotide to cells or organisms.

[0340] In some implementations, the cap analogue used in RNA polynucleotides is m7 G 3'OMe Gppp(m1 2’-OMe ApG (sometimes also called m2) 7,3'-OMe G(5')ppp(5')m 2’-OMe ApG or ( m7 G 3'OMe (5')ppp(5')(A 2'OMe pG), which has the following structure:

[0341]

[0342] The following is an example Cap1 RNA, which contains RNA and m2. 7,3`OMe G(5')ppp(5')m 2’-OMe ApG.

[0343]

[0344] Here is another example of Cap1 RNA:

[0345]

[0346] UTR

[0347] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (such as mRNA). Untranslated regions (UTRs) can be located at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR).

[0348] If a 3'-UTR is present, it is located at the 3' end of the gene, downstream of the stop codon in the protein-coding region; however, the term "3'-UTR" preferably does not include the poly(A) tail. Therefore, the 3'-UTR is located upstream of the poly(A) tail (if present), for example, directly adjacent to the poly(A) tail.

[0349] If a 5'-UTR exists, it is located at the 5' end of the gene, upstream of the start codon of the protein-coding region. A 5'-UTR is located downstream of the 5'-cap, for example, directly adjacent to it.

[0350] According to this disclosure, 5'- and / or 3'-untranslated regions can be linked to open reading frames (OPFs) to associate these regions with OPFs, thereby improving the stability and / or translation efficiency of RNA containing the OPFs.

[0351] In some embodiments, the RNA molecule disclosed herein contains a 5'-UTR and / or a 3'-UTR.

[0352] UTRs are related to RNA stability and translation efficiency. Both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs), in addition to structural modifications concerning the 5'-cap and / or 3' poly(A)-tail as described herein. Sequence elements within the UTR are generally considered to affect translation efficiency (primarily the 5'-UTR) and RNA stability (primarily the 3'-UTR). The presence of a 5'-UTR that functions to improve RNA translation efficiency and / or stability is preferred. Independently or additionally, the presence of a 3'-UTR that functions to improve the translation efficiency and / or stability of the RNA molecule is also preferred.

[0353] Regarding the first nucleic acid sequence (e.g., UTR), the terms "functions to improve translation efficiency" and / or "functions to improve stability" indicate that the first nucleic acid sequence is able to modify the translation efficiency and / or stability of the second nucleic acid sequence in a co-transcription with the second nucleic acid sequence, thereby improving the translation efficiency and / or stability compared to the translation efficiency and / or stability of the second nucleic acid sequence without the first nucleic acid sequence.

[0354] The 5'-UTR of this disclosure may contain any combination of more than one nucleic acid sequence, optionally separated by an adapter. The 3'-UTR of this disclosure may contain any combination of more than one nucleic acid sequence, optionally separated by an adapter.

[0355] The term "connector" in this disclosure refers to a nucleic acid sequence added between two nucleic acid sequences to link the two nucleic acid sequences. There are no particular limitations on the adapter sequence.

[0356] 3'-UTRs typically range in length from 200 to 2000 nucleotides, for example, 500 to 1500 nucleotides. The 3'-untranslated regions (UTRs) of immunoglobulin mRNAs are relatively short (less than about 300 nucleotides), while those of other genes are relatively long. For example, the 3'-UTR of tPA is about 800 nucleotides long, that of factor VIII is about 1800 nucleotides long, and that of erythropoietin is about 560 nucleotides long. The 3'-UTR of mammalian mRNAs often contains a homologous region called the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) linker signal, typically located 10–30 bases upstream of the poly(A) linker site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures, which act as a barrier against ribonucleases or interact with proteins known to increase RNA stability, such as RNA-binding proteins.

[0357] The human β-globin 3'-UTR, particularly two consecutive identical copies of the human β-globin 3'-UTR, contributes to high transcript stability and translation efficiency (Holtkamp et al., 2006, Blood 108:4009-4017). Therefore, in one embodiment, the RNA molecule of this disclosure comprises two consecutive identical copies of the human β-globin 3'-UTR. Thus, it comprises, in the 5'→3' direction: (a) an optionally present 5'-UTR; (b) an open reading frame; and (c) a 3'-UTR; said 3'-UTR comprising two consecutive identical copies of the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0358] In one embodiment, the RNA molecule disclosed herein contains a 3'-UTR that functions to improve translation efficiency and / or stability, but is not the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0359] In one embodiment, the RNA molecule disclosed herein contains a 5'-UTR that functions to improve translation efficiency and / or stability.

[0360] Poly(A) sequence

[0361] In some embodiments, the RNA molecule disclosed herein contains a 3'-poly(A) sequence.

[0362] According to this disclosure, in one embodiment, the poly(A) sequence comprises at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and preferably up to 500, preferably up to 400, preferably up to 300, preferably up to 200, particularly up to 150 A nucleotides, particularly about 120 A nucleotides, or substantially composed of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and... Preferably, it consists of up to 500, up to 400, up to 300, up to 200, and especially up to 150 A nucleotides, particularly about 120 A nucleotides, or is composed of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and preferably up to 500, up to 400, preferably at least 300, preferably at least 200, and especially up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consistently composed of" means that the majority of the nucleotides in the poly(A) sequence, typically at least 50%, preferably at least 75% (by number of nucleotides in the "poly(A) sequence"), are A nucleotides (adenosine), but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine), G nucleotides (guanosine), or C nucleotides (cytidine). In this context, "composed of" means that all nucleotides in the poly(A) sequence are A nucleotides, i.e., 100% (by number of nucleotides in the poly(A) sequence). The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0363] In fact, the 3'-poly(A) sequence of about 120 A nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells and on protein levels translated from an open reading frame located upstream (5') of the 3'-poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0364] This disclosure provides a 3'-poly(A) sequence linked to a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand during RNA transcription (i.e., during the preparation of in vitro transcribed RNA). The DNA sequence (coding strand) encoding the poly(A) sequence is called a poly(A) box.

[0365] In some embodiments of this disclosure, the 3'-poly(A) box present in the coding strand of the DNA template molecule is essentially composed of dA nucleotides, but interrupted by a random sequence having an equal distribution of four nucleotides (dA, dC, dG, and dT). The length of such a random sequence can be 5-50 nucleotides, preferably 10-30, more preferably 10-20 nucleotides. Such a box is disclosed in WO 2016 / 005004A1. Any poly(A) box disclosed in WO 2016 / 005004A1 can be used in this disclosure. A poly(A) box that is essentially composed of dA nucleotides but interrupted by a random sequence having an equal distribution of four nucleotides (dA, dC, dG, and dT) and having a length of, for example, 5-50 nucleotides, exhibits constant proliferation of plasmid DNA at the DNA level in *E. coli*, and remains associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level.

[0366] Therefore, in some embodiments of this disclosure, the 3'-poly(A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U) with equal distribution. Such a random sequence can be 5-50 nucleotides long, preferably 10-30, and more preferably 10-20 nucleotides long.

[0367] Codon usage

[0368] Generally, the degeneracy of the genetic code allows certain codons (base triplets encoding amino acids) in an RNA sequence to be substituted by other codons (base triplets) while maintaining the same coding capacity (the substituted codon encodes the same amino acid as the substituted codon). In some embodiments of this disclosure, at least one codon in an open reading frame (OPF) of the RNA molecule differs from the corresponding codon in the corresponding ORF of the species from which the ORF originated. In this embodiment, the coding sequence of the ORF is referred to as "adpatted" or "modified." The coding sequence of the ORF contained in the RNA molecule can be adjusted.

[0369] For example, when the coding sequence of an open reading frame is modified, commonly used codons can be selected: WO 2009 / 024567A1 describes modifications to the coding sequence of nucleic acid molecules, including replacing rare codons with more commonly used ones. Since the frequency of codon usage depends on the host cell or host organism, this type of modification is suitable for making the nucleic acid sequence appropriately expressed in a specific host cell or host organism. Generally, more commonly used codons are translated more efficiently in the host cell or host organism, although it is not always necessary to modify all codons in the open reading frame.

[0370] For example, when the coding sequence of an open reading frame is adjusted, the content of G (guanosine monophosphate) or C (cytidine monophosphate) residues can be altered by selecting codons with the highest GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames have been reported to have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther. 23:1457-1465).

[0371] Antibody

[0372] The term "immunoglobulin" refers to a class of structure-associated glycoproteins composed of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains linked together by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Chapter 7 of Basic Immunology (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). In short, each heavy chain typically contains a heavy chain variable region (abbreviated as V in this paper). H or VH) and heavy chain constant region (abbreviated as C in this article) H (or CH). The heavy chain constant region typically contains three domains: CH1, CH2, and CH3. The hinge region is the area between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bonds in the hinge region are the part where the two heavy chains interact in the IgG molecule. Each light chain typically contains a light chain variable region (abbreviated as V in this article). L or VL) and light chain constant region (abbreviated as C in this article) L (or CL). The light chain constant region typically contains a single domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or highly variable regions, which are highly variable in the form of a ring defined by the sequence and / or structure), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol). 196 ,901-917(1987)).

[0373] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof capable of binding (preferably specifically binding) an antigen. In some embodiments, binding occurs under typical physiological conditions and has a considerably long half-life, 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 longer, about 48 hours or longer, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant functionally defined time period (such as a time sufficient to induce, promote, enhance, and / or modulate the physiological response associated with antibody binding to an antigen). Variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. As used herein, the terms "antigen-binding region," "binding region," or "binding domain" refer to a region or domain that interacts with the antigen and typically include VH and VL regions. When the term "antibody" is used herein, it includes not only monospecific antibodies but also multispecific antibodies, which contain multiple (e.g., two or more, e.g., three or more) distinct antigen-binding regions. The constant regions of an antibody (Ab) 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 (e.g., the first component C1q in the classical complement activation pathway). As stated above, unless otherwise stated or clearly contradicted by the context, as used herein, the term "antibody" includes fragments of an antibody that are antigen-binding fragments (i.e., retain the ability to specifically bind antigens) and antibody derivatives (i.e., constructs derived from antibodies). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments covered by the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of 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 disulfide bonds in the hinge region; (iii) Fd fragments, which consist essentially of VH and CH1 domains; (iv) Fv fragments, which consist essentially of the VL and VH domains of an antibody single arm; and (v) dAb fragments (Ward et al., Nature). 341 ,544-546(1989)), which is basically composed of VH domains, also known as domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21(11):484-90); vi) Cameloid or nanobody molecules (Revets et al; Expert Opin Biol Ther. Jan; 5(1):111-24) and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by synthetic linkers using recombination methods to create single protein chains, where the VL and VH regions pair to form monovalent molecules (called single-chain antibodies or single-chain Fv (scFv)), see, for example, Bird et al., Science. 242 ,423-426(1988) and Huston et al.,PNAS USA 85 ,5879-5883 (1988)). Unless otherwise stated or the context clearly indicates otherwise, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included in the meaning of antibody, they are common and independent features of this disclosure, exhibiting distinct biological properties and utilities. These and other antibody fragments useful in the context of this disclosure, as well as bispecific forms of such fragments, are further discussed herein. It should also be understood that, unless otherwise stated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides (such as chimeric antibodies and humanized antibodies), and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind antigens and are provided by any known technique (such as enzymatic digestion, peptide synthesis, and recombinant techniques).

[0374] The phrase "single-chain Fv" or "scFv" refers to an antibody in which the variable domains (VH and VL) of the heavy and light chains of a conventional double-chain antibody are linked together to form a single chain. Optionally, a linker (usually a peptide) is inserted between the two chains to enable proper folding and the creation of an active binding site.

[0375] Single-domain antibodies, also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. In some embodiments, single-domain antibodies are variable domains (V) of heavy chain antibodies. H These are called VHH fragments. Like intact antibodies, single-domain antibodies can selectively bind to specific antigens. The first single-domain antibody was engineered from a heavy-chain antibody found in camels. Cartilaginous fish also have heavy-chain antibodies (IgNAR, "immunoglobulin neoantigen receptor"), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimer variable domain of common human or mouse immunoglobulin G (IgG) into monomers. Although most current research on single-domain antibodies is based on heavy-chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes.

[0376] Antibodies can be of any isotype. As used herein, the term "isotype" refers to a class of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region gene. When a particular isotype (e.g., IgG1) is mentioned herein, the term is not limited to a particular isotype sequence (e.g., a specific IgG1 sequence), but is used to indicate that the antibody is sequence-closer to that isotype (e.g., IgG1) rather than 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.

[0377] In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, κ or IgG1, λ 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).

[0378] As used herein, the term "monoclonal antibody" refers to an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity and having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomal nonhuman animals (such as transgenic mice) having a genome containing human heavy chain and light chain transgenes, fused to immortalized cells.

[0379] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., from rodents) and the constant region is derived from a different species (e.g., humans). Chimeric monoclonal antibodies are developed for therapeutic applications to reduce the immunogenicity of the antibody. In the context of chimeric antibodies, the term "variable region" or "variable domain" refers to the region containing the CDR and framework regions of the immunoglobulin heavy and light chains. Chimeric antibodies can be generated using standard DNA techniques described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies can be genetically engineered recombinant antibodies or enzyme-engineered recombinant antibodies. The generation of chimeric antibodies is within the knowledge of those skilled in the art; therefore, the generation of chimeric antibodies can be performed by methods other than those described herein.

[0380] As used herein, the term "humanized antibody" refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain, wherein the nonhuman variable domain is modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that co-form the antigen-binding site onto the homologous human receptor frame region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues of the parent antibody (i.e., the nonhuman antibody) with human frame regions (reversion mutations). Structural homology modeling can help identify amino acid residues in the frame regions that are important for antibody binding properties. Therefore, a humanized antibody may contain a nonhuman CDR sequence, primarily a human frame region, optionally containing one or more amino acid reversion mutations to nonhuman amino acids, and a fully human constant region. Optionally, additional amino acid modifications (not necessarily reversion mutations) may be applied to obtain humanized antibodies with preferred properties (such as affinity and biochemical properties).

[0381] 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 through random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies for which CDR sequences derived from another mammalian species (such as mice or rats) have been grafted onto human frame sequences. Human monoclonal antibodies can be generated using a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization is preferred, other techniques for generating monoclonal antibodies can also be used in principle, such as viral or tumorigenic transformation of B-lymphocytes or phage display using human antibody gene libraries. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. The generation of hybridomas in mice is a well-established method. Immunization protocols and techniques for isolating spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known. Thus, human monoclonal antibodies can be produced, for example, using transgenic or transchromosomal mice or rats carrying a portion of the human immune system rather than a mouse or rat system. Thus, in some embodiments, human antibodies are obtained from transgenic animals (e.g., mice or rats) carrying human germline immunoglobulin sequences rather than animal immunoglobulin sequences. In such embodiments, the antibody is derived from the human germline immunoglobulin sequence introduced into the animal, but the final antibody sequence is the result of further modification of the human germline immunoglobulin sequence through somatic hypermutation and affinity maturation via an endogenous animal antibody mechanism, see, for example, Mendez et al. 1997 Nat Genet. 15(2):146-56.

[0382] When used herein, unless contradicted by context, the terms “Fab arm,” “binding arm,” or “arm” include a heavy-light chain pair and are used interchangeably with “half-molecule” herein.

[0383] When used in the context of antibodies, the term "full-length" means that the antibody is not a fragment, but contains all the domains of a particular isotype that are commonly found in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0384] When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region consisting of two Fc sequences of the immunoglobulin heavy chain, wherein the Fc sequences contain at least a hinge region, a CH2 domain, and a CH3 domain.

[0385] This disclosure also envisions antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies described herein. The VL, VH, or CDR functional variants used in the context of the antibody still allow 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 antibodies may be associated with higher affinity, selectivity, and / or specificity compared to the parent antibody.

[0386] These functional variants typically retain significant sequence identity with the parent antibody.

[0387] Exemplary variants include those that differ primarily from the VH and / or VL and / or CDR regions of the parent antibody sequence through conserved substitutions; for example, up to 10 substitutions (such as 9, 8, 7, 6, 5, 4, 3, 2, or 1) in the variant are conserved amino acid residue substitutions.

[0388] Functional variants of the antibody sequences described herein (such as VL or VH regions), or antibody sequences that are homologous or identical to the antibody sequences described herein (such as VL or VH regions), preferably contain modifications or variations in the non-CDR sequence, while the CDR sequence preferably remains unchanged.

[0389] Unless contradicted by the context, as used herein, the term "specificity" is intended to mean the following: If two antibodies bind to the same antigen and the same epitope, they have "the same specificity".

[0390] The antibodies or fragments available in this article may compete with the specific antibodies or fragments described herein.

[0391] The terms "competite" and "competition" can refer to the competition between a primary antibody and a secondary antibody for the same antigen. Those skilled in the art are well aware of how to test the competition between antibodies for binding to a target antigen. An example of such a method is the so-called cross-competition assay, which can be performed, for example, by ELISA or by flow cytometry. Alternatively, biomembrane interference methods can be used to determine the competition.

[0392] Antibodies that competitively bind to a target antigen may bind to different epitopes on the antigen, wherein said epitopes are so close to each other that a first antibody binding to one epitope prevents a second antibody from binding to the other epitope. However, in other cases, two different antibodies may bind to the same epitope on the antigen and will compete for binding in a competitive binding assay. Such antibodies binding to the same epitope are considered herein to have the same specificity. Therefore, in some embodiments, antibodies binding to the same epitope are considered to bind to the same amino acid on the target molecule. The binding of the same epitope on the target antigen can be determined by standard alanine scanning assays or antibody-antigen crystallization assays known to those skilled in the art. Preferably, antibodies or binding domains binding to different epitopes do not compete with each other for binding to their respective epitopes.

[0393] Naturally occurring antibodies are typically monospecific, meaning they bind to a single antigen. This document describes conjugates, such as docking compounds, that bind to, for example, a primary target and different epitopes on a linker compound. Such conjugates are at least bispecific or multispecific, such as trispecific, tetraspecific, etc. Therefore, conjugates can comprise two or more antibodies or fragments thereof as described herein. In particular, the conjugates described herein can be artificial proteins composed of two different antibodies, a fragment of one antibody and a different antibody, and fragments of two different antibodies (the fragments of the two different antibodies forming two binding domains).

[0394] According to this disclosure, bispecific binders, particularly bispecific proteins such as bispecific antibodies, are molecules with two different binding specificities and therefore can bind to two epitopes. Specifically, as used herein, the term "bispecific antibody" refers to an antibody containing two antigen-binding sites, a first binding site having an affinity for a first epitope, and a second binding site having a binding affinity for a second epitope different from the first epitope.

[0395] As used in this article, the term "bispecific" refers to a substance having two distinct antigen-binding regions that bind to different epitopes.

[0396] "Multispecific binders" are molecules that have two or more different binding specificities.

[0397] Many different forms and uses of bispecific antibodies are known in the art and were reviewed by Kontermann in Drug Discov Today, 2015 Jul; 20(7):838-47 and MAbs, 2012 Mar-Apr; 4(2):182-97.

[0398] The bispecific binders disclosed herein are not limited to any particular bispecific form or method of production thereof.

[0399] Examples of bispecific antibody molecules that may be used in this paper include (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody specific for two different epitopes, for example, two scFvs tandemly linked by an additional peptide linker; and (iii) a dual variable domain antibody (DVD-Ig) wherein each light and heavy chain contains two variable domains tandemly linked by a short peptide linker (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM (iv) Chemically linked bispecific (Fab')2 fragments; (v) Tandab, which is a fusion of two single-chain diabody antibodies, thereby producing a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibody, which is a combination of scFv and diabody, thereby producing a multivalent molecule; (vii) So-called "dock and lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) So-called scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (ix) diabody antibodies.

[0400] The term "bispecific antibody" includes biantibodies. Biantibodies are bivalent bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but a linker is used that is too short for the two domains on the same chain to pair, thus forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, for example, 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 preferably refer to an antibody construct consisting of at least two antibody variable regions linked in a single polypeptide chain, which lacks the constant regions and / or Fc portions present in intact immunoglobulins. For example, a bispecific single-chain antibody can be a construct having a total of two antibody variable regions (e.g., two VH regions), each capable of specifically binding to a single epitope, and linked to each other by short polypeptide spacers, such that the two antibody variable regions and their inserted spacers exist as a single, continuous polypeptide chain. Another example of a bispecific single-chain antibody can be a single polypeptide chain with three antibody variable regions. Here, two antibody variable regions, such as one VH and one VL, can constitute an scFv, wherein the two antibody variable regions are linked to each other by synthetic polypeptide linkers, which are typically genetically engineered to have minimal immunogenicity while maintaining maximum anti-proteolytic activity. This scFv can specifically bind to a particular epitope and link to another antibody variable region, such as the VH region, which can bind to an epitope different from the one bound to the scFv. Another example of a bispecific single-chain antibody can be a single polypeptide chain having four antibody variable regions. Here, the first two antibody variable regions, such as the VH and VL regions, can form an scFv capable of binding to one epitope, while the second VH and VL regions can form a second scFv capable of binding to another epitope. Within a single continuous polypeptide chain, the individual antibody variable regions having a specificity can advantageously be separated by synthetic peptide linkers, while the corresponding scFvs can advantageously be separated by short polypeptide spacers as described above. According to some embodiments, the first binding domain of the bispecific antibody comprises an 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 an antibody variable domain, preferably a VHH domain.According to some embodiments, the second binding domain of the bispecific antibody comprises two antibody variable domains, preferably scFv, i.e., VH-VL or VL-VH. Therefore, in its 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 or two VHH domains. According to some embodiments, the first and second binding domains of the bispecific antibody each comprise one antibody variable domain, preferably a VHH domain. According to 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 binder 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.

[0401] In some embodiments, the bispecific molecule comprises two Fab regions, each targeting a different epitope. In some embodiments, the molecule disclosed herein is an antigen-binding fragment (Fab)2 complex. The Fab2 complex consists of two Fab fragments, one containing an Fv domain specific to one epitope, namely the VH and VL domains, and the other containing an Fv domain specific to another epitope. Each Fab fragment may consist of two single strands, a VL-CL module and a VH-CH module. Alternatively, each individual Fab fragment may be arranged in a single strand, preferably VL-CL-CH-VH, and the variable and constant domains may be linked by peptide linkers.

[0402] In some embodiments, the binders of this disclosure include various types of divalent and trivalent single-chain variable fragments (scFvs) that mimic the variable domains of two antibodies in a fusion protein. Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be designed by linking two scFvs. This can be achieved by generating a single peptide chain having two VH and two VL regions, thereby producing tandem scFvs. This disclosure also includes multispecific molecules containing more than two scFv binding domains.

[0403] Another possibility is to create scFvs with a linker peptide that is too short for the two variable regions to fold together (about five amino acids), thus forcing the scFv to dimerize. This type is called a biantibody. Shorter linkers (one or two amino acids) lead to the formation of trimers, known as tribosomes. Tetrabosomes have also been produced. They have even higher affinity for their targets than biantibodies.

[0404] A particularly preferred example of a bispecific antibody fragment is a biantibody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small, bivalent, and bispecific antibody fragment. A biantibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) linked by a peptide linker on the same polypeptide chain (VH-VL). This linker is so short that the two domains on the same chain cannot pair. This forces pairing with the complementary domain of the other chain and promotes the assembly of a dimer molecule with two functional antigen-binding sites.

[0405] In some embodiments, the bispecific or multispecific molecules of this disclosure comprise variable domains (VH, VL) and constant domains (C) of an immunoglobulin. In some embodiments, the bispecific molecule is a minibody, preferably comprising two single VH-VL-C chains interconnected by a constant domain (C) of each chain. According to this aspect, the corresponding variable heavy chain region (VH), the corresponding variable light chain region (VL), and the constant domain (C) are arranged from the N-terminus to the C-terminus in the order VH(epitope 1)-VL(epitope 1)-(C) and VH(epitope 2)-VL(epitope 2)-C, wherein C is preferably a CH3 domain, epitope 1 refers to the first epitope, and epitope 2 refers to the second epitope. The pairing of constant domains leads to the formation of the minibody.

[0406] According to another aspect, the bispecific binder of this disclosure is in the form of a bispecific single-chain antibody construct, wherein the construct comprises or consists of at least two binding domains. In some embodiments, each binding domain comprises a variable region (“VH region”) from the antibody heavy chain, wherein 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 interconnected by short polypeptide spacers. Each binding domain may additionally comprise a variable region (“VL region”) from the antibody light chain, wherein the VH and VL regions within each of the first and second binding domains are interconnected by polypeptide linkers that are long enough to allow the VH and VL regions of the first binding domain and the VH and VL regions of the second binding domain to pair with each other.

[0407] In some embodiments, the conjugates described herein comprise antibodies, such as full-length antibodies, that include a first binding domain. In some embodiments, the conjugates described herein comprise antibody fragments, such as scFv or VHH, that include a second binding domain, said antibody fragment being covalently linked to an antibody including the first binding domain. In some embodiments, the conjugates comprise antibody fragments, such as scFv or VHH, that are covalently linked to the N-terminus or C-terminus of the light or heavy chain of an antibody.

[0408] In some embodiments, the binding portion described herein, for example, the binding portion contained in a docking compound that binds to a primary target, includes DARPin. In some embodiments, the binding portion directs the particle to immune effector cells, particularly T cells such as CD8 cells. + T cells.

[0409] The term "DARPin" refers to a designed ankyrin repeat protein. DARPin is based on naturally occurring ankyrin repeat proteins but contains one or more amino acid mutations that can affect, for example, its binding affinity to target molecules and its cell surface expression. DARPin preferably comprises 2-3 ankyrin repeat modules flanked by N- and C-capped repeats. Each ankyrin repeat module comprises approximately 33 amino acid residues.

[0410] In 1987, ankyrin repeats were discovered by comparing the sequences of four such proteins in *Saccharomyces cerevisiae*, *Drosophila melanogaster*, and *Caenorhabditis elegans*. Breeden and Nasmyth reported multiple copies of repeat units of approximately 33 residues in the sequences swi6p, cddOp, notch, and lin-12 (Breeden et al., *Nature* 329, 651–654 (1987)). Subsequently, 24 copies of this repeat unit were found in ankyrin proteins, leading to the naming of this repeat unit as ankyrin repeats (Lux et al., *Nature* 344, 36–42 (1990)). Later, this repeat unit was found in hundreds of proteins in various organisms and viruses (Bork, *Proteins* 17(4), 363–74 (1993)). These proteins are located in the nucleus, cytoplasm, or extracellular space. This is consistent with the fact that the ankyrin repeat domains of these proteins are independent of disulfide bridges and therefore independent of the oxidative state of the environment. The number of repeat units in each protein ranges from two to more than twenty. The tertiary structure of the ankyrin repeat unit shares a characteristic fold (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6(1999)), which consists of a β hairpin, followed by two antiparallel α-helices and a loop connecting the end of the repeat unit to the next repeat unit. The domains constructed from the ankyrin repeat units are formed by stacking the repeat units into extended and curved structures. Proteins containing ankyrin repeat domains often contain additional domains. While the latter domains have variable functions, the function of the ankyrin repeat domain is usually to bind other proteins. When analyzing the repeat units of these proteins, target-interacting residues are mainly found in the exposed portions of the β hairpin and the first α-helix. Thus, these target-interacting residues form a large contact surface on the ankyrin repeat domain. This contact surface is exposed on a framework constructed from stacked α-helices 1, α-helices 2 and rings.

[0411] DARPins that bind to a specific target can be identified by screening combinatorial libraries of DARPins and selecting those with the desired target-binding properties. Such screening methods have been described, for example, in Muench et al., Molecular Therapy, 16(4), 686-693, 2011. For example, target-specific DARPins can be selected from a wide variety of libraries using ribosome display or phage display methods.

[0412] The term "repetitive protein" refers to a (poly)peptide / protein comprising one or more repeating domains. In one embodiment, the repeating protein comprises up to four repeating domains. In one embodiment, the repeating protein comprises up to three repeating domains. In one embodiment, the repeating protein comprises up to two repeating domains. In the most preferred embodiment, the repeating protein comprises one repeating domain.

[0413] The domains of a repeating protein can be directly interconnected or interconnected via (poly)peptide linkers. The term "(poly)peptide linker" refers to an amino acid sequence capable of connecting two protein domains. Such linkers include, for example, variable-length glycine-serine linkers, and are known to those skilled in the art.

[0414] The term "repetitive domain" refers to a protein domain comprising two or more consecutive repeating units (modules). In one embodiment, the repeating units are structural units having the same or similar folding structures, and are preferably closely stacked to preferably produce a supercoiled structure with a joint hydrophobic core.

[0415] The term "structural unit" refers to a locally ordered portion of a (poly)peptide, formed by three-dimensional interactions between two or more adjacent segments of secondary structures on the (poly)peptide chain. Such structural units contain structural motifs.

[0416] 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 art. Individually, the structural units may not achieve a definite three-dimensional arrangement; however, their continuous arrangement as repeating modules in a repeating structural domain leads to mutual stability of adjacent units, thereby generating a superhelical structure.

[0417] The term "repetitive module" refers to the repeating amino acid sequence of a repeating protein, which is derived from the repeating units of naturally occurring proteins. Each repeating module contained in a repeating domain is derived from one or more repeating units of a naturally occurring family of repeating proteins (such as ankyrin repeating proteins).

[0418] The term "repeating module group" refers to the total number of repeating modules present in a repeating structural domain. Such a "repeating module group" in a repeating structural domain contains two or more consecutive repeating modules, and may contain two or more copies of repeating modules of only one type, or may contain two or more different types of modules, each with one or more copies. For example, a repeating module group may contain three repeating modules, which can be consecutively represented from N to C as repeating module 1, repeating module 2, and repeating module 3.

[0419] Each repeating structural domain can have the same number of repeating modules, or the number of repeating modules in each repeating structural domain can be different.

[0420] Preferably, the repeating modules included in the group are homologous repeating modules. In the context of this disclosure, the term "homologous repeating module" refers to a repeating module in which more than 70% of the framework residues are homologous. Preferably, more than 80% of the framework residues of the repeating module are homologous. Most preferably, more than 90% of the framework residues of the repeating module are homologous. Computer programs for determining the percentage of homology between peptides, such as Fasta, Blast, or Gap, are known to those skilled in the art.

[0421] The term "repetitive unit" refers to an amino acid sequence that comprises one or more naturally occurring protein sequence motifs, wherein the "repetitive unit" exists in multiple copies and exhibits a well-defined folding topology common to all said motifs. Such repeatable units contain framework residues and interacting residues.

[0422] An example of such repeating units is the ankyrin repeating unit. Naturally occurring proteins containing two or more of these repeating units are called "naturally occurring repeating proteins." The amino acid sequences of the individual repeating units of a repeating protein can have numerous mutations, substitutions, additions, and / or deletions compared to each other, but they still largely retain the general pattern or motif of the repeating unit.

[0423] The term "repetitive sequence motif" or "repetitive common sequence" refers to an amino acid sequence derived from one or more repeating units. Such repetitive sequence motifs include framework residue positions and target-interacting residue positions. The framework residue positions correspond to the positions of framework residues within the repeating unit. The target-interacting residue positions correspond to the positions of target-interacting residues within the repeating unit. Such repetitive sequence motifs can include fixed positions and random positions. The term "fixed position" refers to an amino acid position within the repetitive sequence motif, where the position is specified as a particular amino acid. Typically, such fixed positions correspond to the positions of framework residues.

[0424] The term "random position" refers to an amino acid position in a repeating sequence motif, where two or more amino acids are allowed at that position. Typically, this random position corresponds to the position of target-interacting residues. However, some positions of framework residues can also be random.

[0425] The term "fold topology" refers to the tertiary structure of the repeating unit. Fold topology can be determined by amino acid sequence fragments that form at least a portion of an α-helix or β-sheet, or amino acid sequence fragments that form a linear polypeptide or ring, or any combination of α-helices, β-sheets, and / or linear polypeptides / rings.

[0426] The term "continuous" refers to an arrangement in which the modules are connected in series.

[0427] Repeated proteins contain at least two, typically six or more, ten or more, or twenty or more repeating units, but generally about two to six repeating units. Most repeating proteins are structural and / or adhesion proteins, found in prokaryotes and eukaryotes, including vertebrates and invertebrates.

[0428] In most cases, the repeating units will exhibit high sequence identity (identical amino acid residues at corresponding positions) or sequence similarity (different amino acid residues but with similar physicochemical properties), and some amino acid residues may be key residues that are highly conserved in different repeating units and are found in naturally occurring proteins.

[0429] However, as long as a common folding topology is maintained, a high degree of sequence variability is possible between different repeating units found in naturally occurring proteins through amino acid insertions and / or deletions and / or substitutions.

[0430] The term "framework residue" refers to an amino acid residue of a repeating unit, or a corresponding amino acid residue of a repeating module, that contributes to folding topology, i.e., to the folding of the repeating unit (or module) or to interaction with adjacent units (or modules). This contribution can be an interaction with other residues in the repeating unit (module), or an effect on the scaffolding of a polypeptide in an α-helix or β-sheet, or an effect on amino acid sequence fragments that form linear polypeptides or loops.

[0431] The term "target-interacting residue" refers to an amino acid residue of a repeating unit, or a corresponding amino acid residue of a repeating module, that contributes to the interaction with a target substance. This contribution can be a direct interaction with the target substance, or an effect on other directly interacting residues, such as by stabilizing the conformation of the (poly)peptide of the repeating unit (module) to allow or enhance the interaction of the directly interacting residue with the target.

[0432] A “target” can be a single molecule, such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule, including any part of such a single molecule, or a complex of two or more such molecules. Targets can be particularly molecules on immune effector cells, especially CD8.

[0433] In one embodiment, the repeat modules are directly linked. In the context of this invention, the term "directly linked" means that the repeat modules are arranged as direct repeats in the repeat protein without any spacer amino acid sequences.

[0434] In another embodiment, the repeating modules are linked via (poly)peptide linkers. Therefore, the repeating modules can be indirectly linked via (poly)peptide linkers that serve as spacer sequences separating the individual modules. The "spacer sequence" can be any amino acid sequence that allows the individual modules to be linked without interfering with the folding topology or stacking of the modules. Preferably, the spacer sequence is a short (poly)peptide linker of fewer than 10, more preferably, fewer than 5 amino acid residues.

[0435] In one embodiment, the repeating protein further comprises an N-terminal capping module and / or a C-terminal capping module having an amino acid sequence different from any of the repeating modules. The term "capping module" refers to a polypeptide fused to an N-terminal or C-terminal repeating module of a repeating domain, wherein the capping module forms a tight tertiary interaction with the repeating module, thereby providing a cap that protects the hydrophobic core of the repeating module from solvent influence on the side not in contact with the continuous repeating modules.

[0436] The N-terminal capping module and / or C-terminal capping module may be or may be derived from capping units or other domains adjacent to the repeating unit found in naturally occurring repeating proteins.

[0437] The term "capped unit" refers to a naturally occurring folded (poly)peptide that defines a specific structural unit fused to a repeating unit at either an N-terminus or a C-terminus, wherein the (poly)peptide forms a tight tertiary interaction with the repeating unit, thereby providing a cap that protects the hydrophobic core of the repeating unit from solvent influence on one side. Such capped units may have sequence similarity to the repeating sequence motif.

[0438] antigen receptor

[0439] The immune effector cells of this 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 a processed product thereof, particularly when the antigen or a processed product thereof is present in or presented by a target cell. The immune effector cells can be modified (e.g., in vitro / ex vivo or in a subject to be treated) to express the antigen receptor. In one embodiment, the modification to express the antigen receptor occurs in vitro / ex vivo. Subsequently, the modified cells can be administered to a patient. In one embodiment, the modification to express the antigen receptor occurs in vivo. The cells may be endogenous cells of the patient or may have already been administered to the patient.

[0440] In one embodiment of all aspects of the invention, the antigen receptor is expressed on the cell surface.

[0441] Immune effector cells, particularly T cells, have a first cell surface antigen receptor. In some embodiments, immune effector cells, particularly T cells, have a first cell surface antigen receptor and a second cell surface antigen receptor. The terms "cell surface antigen receptor" and "antigen receptor" are used synonymously throughout this application.

[0442] In one embodiment, the antigen receptor of this disclosure may also bind to cell surface or soluble cytokines. In this embodiment, the antigen receptor is a cytokine receptor.

[0443] T cell receptors

[0444] As used herein, the term "T cell receptor" or "TCR" refers to a protein receptor on T cells, which is composed of heterodimers of alpha (α) and beta (β) chains, although in some cells the TCR is composed of γ and δ (γδ) chains. In some embodiments, the TCR can be derived from any cell containing the TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells. Each α, β, γ, and δ chain consists of two Ig-like domains: a variable domain (V) that confers antigen recognition via a complementarity-determining region (CDR), followed by a constant domain (C) that is anchored to the cell membrane via a linker peptide and a transmembrane (TM) region. The TM region is associated with a constant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with a complex formed by the binding of an antigen peptide to a protein encoded by the major histocompatibility complex (MHC).

[0445] Chimeric antigen receptor

[0446] Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anticancer therapy because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (modified) to express CARs that specifically target antigens on the patient's tumor cells, and then these cells can be reinfused into the patient.

[0447] In some implementations, the first antigen receptor is a CAR. In some implementations, both the first and second antigen receptors are CARs.

[0448] 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," referring to an artificial receptor comprising a single molecule or molecular complex that recognizes (i.e., binds) a target structure (e.g., antigen) on a target cell (e.g., binding to an antigen expressed on the surface of the target cell via an antigen-binding domain) and can confer specificity to immune effector cells (e.g., T cells expressing the CAR on their cell surface). Such cells do not necessarily require antigen processing and presentation to recognize the target cell, but may preferably specifically recognize any antigen present on the target cell. Preferably, CAR recognition of the target structure leads to activation of the immune effector cell expressing the CAR. The CAR may comprise one or more protein units containing one or more domains described herein. The term "CAR" does not include T-cell receptors.

[0449] A CAR contains a target-specific binding element, also known as an antigen-binding moiety or antigen-binding domain, which is typically part of the CAR's extracellular domain. The antigen-binding domain recognizes ligands that act as cell surface markers on target cells associated with a specific disease state. Specifically, the CAR disclosed herein can target antigens (such as tumor antigens) on diseased cells (such as tumor cells).

[0450] In one embodiment, the binding domain in the CAR specifically binds to an antigen. In one embodiment, the antigen bound to the binding domain in the CAR 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 native epitope of the antigen present on the surface of living cells.

[0451] In one embodiment of the invention, the antigen-binding domain comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) specific to the 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 by a peptide linker. Preferably, the antigen-binding portion in the CAR is scFv.

[0452] The CAR is designed as a transmembrane domain comprising an extracellular domain fused to 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 prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain can be derived from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful in this invention can be derived from (i.e., including at least the following transmembrane regions): 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, and CD154. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0453] In some cases, the CAR disclosed herein includes a hinged domain that forms a connection between the transmembrane domain and the extracellular domain.

[0454] The cytoplasmic domains, or intracellular signaling domains, of a CAR are responsible for activating at least one of the normal effector functions of the immune cells in which the CAR has been implanted. The term "effector function" refers to a specialized function of the cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector signals and directs the cell to perform its specialized function. While a complete intracellular signaling domain can often be used, in many cases, the entire chain is not necessary. If a truncated portion of an intracellular signaling domain is used, it can be used in place of the complete chain, provided that such a truncated portion can transduce effector signals. Therefore, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce effector signals.

[0455] It is known that the signals generated by the TCR alone are insufficient to fully activate T cells; secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0456] In one implementation, the CAR comprises a primary cytoplasmic signaling sequence derived from CD3-ζ. Furthermore, the cytoplasmic domain of the CAR may include a CD3-ζ signaling domain combined with a co-stimulatory signaling region.

[0457] The defining criteria for co-stimulatory domains are limited to their ability to enhance cell proliferation and survival when the CAR binds to its target region. Suitable co-stimulatory domains include CD28, CD137(4-1BB) (a member of the tumor necrosis factor receptor (TNFR) superfamily), CD134(OX40) (a member of the TNFR receptor superfamily), and CD278(ICOS) (a co-stimulatory molecule of the CD28 superfamily expressed on activated T cells). Those skilled in the art will understand that sequence variants of these indicated co-stimulatory domains can be used without adversely affecting the invention, wherein the variants have the same or similar activity as the domains they model. Such variants will have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments of the invention, the CAR construct comprises two co-stimulatory domains. While specific combinations include all possible variations of the four indicated domains, specific examples include CD28+CD137(4-1BB) and CD28+CD134(OX40).

[0458] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked together in a random or specified order. Optionally, short oligo- or polypeptide linkers (preferably between 2 and 10 amino acids in length) can form links. Glycine-serine duplexes provide particularly suitable linkers.

[0459] In one embodiment, the CAR includes a signal peptide that guides the nascent protein into the endoplasmic reticulum. In one embodiment, the signal peptide is located prior to the antigen-binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin, such as IgG.

[0460] CARs can contain the aforementioned domains, combined together as a fusion protein. Such fusion proteins typically contain an antigen-binding domain, one or more co-stimulatory domains, and a signal transduction sequence linked in an N-terminal to C-terminal orientation. However, the CARs of this invention are not limited to this arrangement; other arrangements are acceptable and include the binding domain, signal transduction domain, and one or more co-stimulatory domains. It should be understood that because the binding domain must be able to freely bind to the antigen, the placement of the binding domain in the fusion protein should generally allow that region to be displayed outside the cell. Similarly, because the co-stimulatory and signal transduction domains are used to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein typically displays these two domains inside the cell.

[0461] In one implementation, the CAR molecule comprises:

[0462] i) Target antigen (e.g., CLDN6 or CLDN18.2) binding domain;

[0463] ii) Transmembrane structural domains; and

[0464] iii) Intracellular domains, which include the 4-1BB co-stimulatory domain and the CD3-ζ signaling domain.

[0465] In one implementation, the antigen-binding domain includes scFv. In one embodiment, the transmembrane domain comprises a transmembrane domain selected from the following proteins or functional variants thereof: α, β, or ζ chains of T cell receptors, 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 , CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD1 8. LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD1 50, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C. In one embodiment, the transmembrane domain includes a CD8α transmembrane domain. In one embodiment, the antigen-binding domain is connected to the transmembrane domain via a hinge domain. In one embodiment, the hinge domain is a CD8α hinge domain.

[0466] In one embodiment, the CAR molecule of the present invention comprises:

[0467] i) Target antigen binding domain;

[0468] ii) CD8α hinge structural domain;

[0469] iii) CD8α transmembrane domain; and

[0470] iv) Intracellular domains, which include the 4-1BB co-stimulatory domain and the CD3-ζ signaling domain.

[0471] According to this disclosure, when a CAR is present on a T cell, it recognizes antigens (such as antigens on the surface of antigen-presenting cells or diseased cells (such as cancer cells), thereby stimulating the T cell and / or amplifying or performing the effector functions described above.

[0472] Immune effector cell activator molecules

[0473] The immune effector cells or particles of the present invention contain a second nucleic acid that encodes an immune effector activator molecule, also referred to as an "activator molecule" throughout this application.

[0474] In some implementations, the activator molecule is expressed transiently.

[0475] Different types of molecules can be activator molecules, as long as they enable immune effector cells (especially T cells) to be activated, expanded, differentiated and / or proliferated.

[0476] In some implementations, the activator molecule is a cytokine.

[0477] Examples of cytokines include interferons (such as interferon-α (IFN-α) or interferon-γ (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 factor. On the other hand, immunostimulants include adjuvant immunostimulants, such as APC Toll-like receptor agonists or co-stimulatory / cell adhesion membrane proteins. Examples of Toll-like receptor agonists include co-stimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0478] The term "cytokine" refers to proteins with a molecular weight of approximately 5-60 kDa that are involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, when released, cytokines exert an effect on cellular behavior around the site of release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to this disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that: (i) they generally exert their effects over a wider range of 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 prevent intracellular viral replication by binding to interferon receptors on the regulated cell surface, thereby altering and regulating intracellular gene transcription. Interferons can be classified into two types. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon α (IFNα), interferon β (IFNβ), interferon γ (IFFγ), 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), as well as their variants and derivatives.

[0479] According to this disclosure, cytokines can be naturally occurring cytokines or functional fragments or variants thereof. Cytokines can be human cytokines and can be derived from any vertebrate, especially any mammal.

[0480] In some implementations, the activator molecule is a second cell surface antigen receptor as described herein, preferably a CAR or TCR.

[0481] In some embodiments, the activator molecule is an antigen, particularly an antigen that specifically binds to an antigen receptor expressed on the first cell surface. In some embodiments, the antigen is an antigen as described herein.

[0482] The term "antigen" can also be referred to as "antigen receptor-targeted antigen", "homologous antigen molecule", or simply "antigen molecule".

[0483] In one embodiment, the homologous antigen molecule comprises an antigen or fragment thereof expressed by a target cell and targeted by an antigen receptor, or a variant of the antigen or fragment thereof.

[0484] In one implementation, homologous antigen molecules are expressed on the cell surface.

[0485] The binding of an antigen to an antigen receptor preferably leads to the stimulation, priming, and / or amplification of immune effector cells. The stimulated, primed, and / or amplified immune effector cells are preferably targeted at a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., disease-related antigens. Therefore, an antigen may comprise a disease-related antigen or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to a disease-related antigen. In the context of this disclosure, the terms "fragment of antigen" or "variant of antigen" refer to a substance that causes the stimulation, priming, and / or amplification of immune effector cells targeting an antigen, i.e., a disease-related antigen, particularly when presented by diseased cells, tissues, and / or organs. Therefore, an antigen may correspond to or may comprise a disease-related antigen, may correspond to or may comprise a fragment of a disease-related antigen, or may correspond to or may comprise an antigen homologous to a disease-related antigen or a fragment thereof. If an antigen comprises a fragment of a disease-related antigen or an amino acid sequence homologous to a fragment of a disease-related antigen, then said fragment or amino acid sequence may comprise an epitope of the disease-related antigen targeted by a first antigen receptor and / or a second antigen receptor of an immune effector cell, or a sequence homologous to an epitope of the disease-related antigen. Therefore, according to this disclosure, an antigen may comprise an immunogenic fragment of a disease-related antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-related antigen. The “immunogenic fragment of an antigen” of this disclosure preferably relates to an antigen fragment capable of stimulating, priming, and / or amplifying immune effector cells carrying antigen receptors of cells that bind or express the antigen. Preferably, the antigen (similar to a disease-related antigen) provides a relevant epitope for binding to an antigen-binding domain present on the immune effector cell. In one embodiment, the antigen (similar to a disease-related antigen) is expressed on the surface of an immune effector cell, thereby providing a relevant epitope for binding by other immune effector cells. In one embodiment, the antigen (similar to a disease-related antigen) is expressed by immune effector cells and presented on the surface of immune effector cells in the context of MHC, thereby providing a relevant epitope for binding by other immune effector cells. The antigen may be a recombinant antigen.

[0486] In one embodiment of all aspects of the invention, a second nucleic acid encoding an antigen is expressed in immune effector cells to provide the antigen or a processed product thereof for binding to an antigen receptor expressed by other immune effector cells, said binding leading to stimulation, sensitization and / or amplification of the other immune effector cells.

[0487] The term "immunologically equivalent" means that immunologically equivalent molecules (such as immunologically equivalent amino acid sequences) exhibit the same or substantially the same immunological properties and / or produce the same or substantially the same immunological effects (e.g., in terms of the type of immunological effect). In the context of this disclosure, the term "immunologically equivalent" is preferably used in terms of the immunological effects used for immunization or the properties of an antigen or antigen variant. For example, when an amino acid sequence is exposed to the immune system of a subject (e.g., T cells that bind to a reference amino acid sequence or cells expressing that reference amino acid sequence), if the amino acid sequence induces an immune response with specificity for the reference amino acid sequence, then the amino acid sequence is immunologically equivalent to that reference amino acid sequence. Therefore, molecules immunologically equivalent to antigens exhibit the same or substantially the same properties as antigens targeted by T cells, and / or produce the same or substantially the same effects as antigens targeted by T cells in terms of T cell stimulation, sensitization, and / or amplification.

[0488] As used herein, “activation” or “stimulation” refers to a state in which immune effector cells (such as T cells) have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signaling pathways, the production of induced cytokines, and detectable effector function. The term “activated immune effector cells” specifically refers to immune effector cells undergoing cell division.

[0489] The term "priming" refers to the process in which immune effector cells (such as T cells) come into contact with their specific antigens for the first time, leading to their differentiation into effector cells (such as effector T cells).

[0490] The term "clonal expansion" or "expansion" refers to the process in which a specific entity proliferates. In the context of this disclosure, the term is preferably used in the context of an immune response, in which lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes that recognize said antigen are expanded. Preferably, clonal expansion leads to lymphocyte differentiation.

[0491] The term "antigen" refers to a substance containing an epitope that can generate an immune response. The term "antigen" particularly includes proteins and peptides. In one embodiment, the antigen is presented by or present on the surface of immune system cells (e.g., antigen-presenting cells such as dendritic cells or macrophages). In one embodiment, the antigen or a processed product thereof (e.g., a T-cell epitope) is bound to an antigen receptor. Therefore, the antigen or its processed product can specifically react with immune effector cells (e.g., T lymphocytes (T cells)). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, and the epitope is derived from such antigen.

[0492] The term "disease-associated antigen" is used in its 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 produce an antigen-specific cellular immune response and / or humoral antibody response against the disease. Disease-associated antigens or their epitopes can be used for therapeutic purposes. Disease-associated antigens may be associated with microbial (usually microbial antigen) infection or with cancer (usually tumors).

[0493] The term "tumor antigen" or "tumor-associated antigen" refers to components of cancer cells that can originate from the cytoplasm, cell surface, and nucleus. Specifically, it refers to those antigens produced within tumor cells or acting as surface antigens of tumor cells. Tumor antigens are typically preferentially expressed by cancer cells (e.g., their expression levels are higher in cancer cells than in non-cancer cells), and in some cases, they are expressed only by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, and Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the tight junction protein 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, LDLR / F UT, 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 M AGE-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, p190 minor BCR-abL, Pm1 / 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 from the tight junction protein family, such as CLAUDIN-6 or CLAUDIN-18.2.

[0494] The term "viral antigen" refers to any viral component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0495] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Bacterial antigens can originate from the bacterial cell wall or cytoplasmic membrane.

[0496] 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 cell's plasma membrane, wherein 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 extracellular antibody. In this context, the portion is preferably at least 4 amino acids, preferably at least 8 amino acids, preferably at least 12 amino acids, and more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure found on the extracellular leaflet of the plasma membrane. For example, a molecule associated with the cell surface can be a transmembrane protein having an extracellular portion, or it can be a protein that is associated with the cell surface through interaction with another protein, which is also a transmembrane protein.

[0497] The term "cell surface" or "the surface of the cell" is used in its usual sense in the art and therefore includes the extracellular space that can be approached and bound by proteins and other molecules. If an antigen is located on the cell surface and can be approached and bound by, for example, an antigen-specific antibody added to the cell, the antigen is expressed on the cell surface. In one embodiment, the antigen expressed on the cell surface is an integrated membrane protein having an extracellular portion that can be recognized by a CAR.

[0498] In the context of this invention, the term "extracellular portion" or "extracellular domain" refers to a portion of a molecule (such as a protein) that faces the extracellular space of the cell and is preferably accessible from outside the cell, for example, by a binding molecule (such as an antibody) located outside the cell. Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

[0499] 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 may include a continuous or discontinuous portion of the antigen and may be about 5 to about 100 amino acids in length, such as about 5 to about 50 amino acids, more preferably about 8 to about 30 amino acids, and most preferably about 10 to about 25 amino acids. For example, the length of an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T-cell epitopes.

[0500] 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 MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" encompasses MHC class I and MHC class II molecules and refers to complexes of genes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present T cells with both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). In the case of class I MHC / peptide complexes, the length of the bound peptide is typically about 8 to about 10 amino acids, although longer or shorter peptides can be effective. In the case of class II MHC / peptide complexes, the length of the bound peptide is typically about 10 to about 25 amino acids, particularly about 13 to about 18 amino acids, while longer and shorter peptides can be effective.

[0501] In one embodiment, the target antigen of the first antigen receptor is a tumor antigen, and the antigen, as an activator molecule or a fragment thereof (e.g., an epitope), is derived from the tumor antigen. The tumor antigen can be a “standard” antigen, which is generally known to be expressed in various cancers. The tumor antigen can also be a “neoantigen,” which is specific to an individual’s tumor and has not previously been recognized by the immune system. A neoantigen or neoepitope can be caused by one or more cancer-specific mutations in the genome of a cancer cell, resulting in amino acid changes. If 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.

[0502] Cancer mutations vary from person to person. Therefore, cancer mutations encoding novel epitopes (neo-epitopes) represent attractive targets for developing vaccine compositions and immunotherapies. The efficacy of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes capable of inducing an effective immune response within the host.

[0503] The length of peptides and protein antigens can be 2-100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, peptides can be longer than 50 amino acids. In some embodiments, peptides can be longer than 100 amino acids.

[0504] In one embodiment of all aspects of the invention, the antigen is expressed in diseased cells (such as cancer cells). In one embodiment, the antigen is expressed on the surface of diseased cells (such as cancer cells). In one embodiment, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope in the extracellular domain. In one embodiment, the CAR binds to a natural epitope of the antigen present on the surface of a living cell. In one embodiment, when a T cell expresses a CAR and / or a CAR is present on the T cell, the binding of the CAR to the antigen present on the diseased cell (such as cancer cell) leads to cell lysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzyme.

[0505] Particles

[0506] The particles disclosed herein comprise at least a first nucleic acid molecule and a second nucleic acid molecule. In some embodiments, the particles may comprise other nucleic acids. In some embodiments, the particles comprise a third nucleic acid, preferably more than one third nucleic acid. In some embodiments, the particles comprise a third nucleic acid and a fourth nucleic acid, preferably more than one third nucleic acid and / or a fourth nucleic acid. In some embodiments, the particles comprise a third nucleic acid and a fifth nucleic acid, preferably more than one third nucleic acid and / or a fifth nucleic acid. In some embodiments, the particles comprise a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid, preferably more than one third nucleic acid, a fourth nucleic acid, and / or a fifth nucleic acid.

[0507] To overcome safety and efficacy barriers in nucleic acid delivery, nucleic acids can be administered co-administered with one or more delivery vehicles that protect the nucleic acids from degradation, maximize delivery to target cells, and minimize exposure to non-target cells. Such nucleic acid delivery vehicles can compound or encapsulate nucleic acids and include a range of substances, including polymers and lipids. In some embodiments, such nucleic acid delivery vehicles can form particles with nucleic acids (preferably DNA and / or RNA).

[0508] The DNA or RNA, particularly mRNA, described herein can be present in particles comprising (i) DNA and / or RNA, and (ii) at least one cationic or ionizable cationic compound, such as a polymer or lipid complexing DNA and / or RNA. Electrostatic interactions between positively charged molecules (such as polymers and lipids) and negatively charged DNA and / or RNA are involved in particle formation. This leads to the complexation and spontaneous formation of nucleic acid (particularly DNA and / or RNA) particles.

[0509] Different types of nucleic acid-containing particles have been previously described for use in the delivery of DNA and / or RNA in particulate form (see, for example, Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). For non-viral DNA and / or RNA delivery vectors, the encapsulation of nucleic acid nanoparticles physically protects the nucleic acid from degradation and, depending on specific chemical properties, can facilitate cellular uptake and endosome escape.

[0510] In the context of this disclosure, the term "particle" refers to a structured entity formed of molecules or molecular complexes (particulate-forming compounds). In some embodiments, the particle contains a coating (e.g., one or more layers or thin layers) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance is both hydrophilic and lipophilic. The coating may also contain additional substances that are not necessarily amphiphilic (e.g., additional lipids). Thus, the particle can be a monolayer or multilayer structure, wherein the substance constituting one or more layers or thin layers comprises one or more types of amphiphilic substances (particularly selected from amphiphilic lipids), optionally combined with additional substances that are not necessarily amphiphilic (e.g., additional lipids). In some embodiments, the term "particle" refers to a micrometer or nanometer-scale structure, or a micrometer or nanometer-scale dense structure. According to this disclosure, the term "particle" includes nanoparticles.

[0511] "DNA particles," "RNA particles," or "DNA and RNA particles" can be used to deliver DNA and / or RNA to target sites of interest (e.g., cells, tissues, organs, etc.). DNA and / or RNA particles can be formed from lipids comprising at least one cationic or ionizable cationic lipid. Without being bound by any theory, it is believed that cationic or ionizable cationic lipids combine with nucleic acids to form aggregates, and that such aggregation results in colloidally stable particles.

[0512] The DNA and / or RNA particles described herein include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPXs).

[0513] The lipid complex (LPX) described herein can be obtained by mixing two aqueous phases: a phase containing RNA and a phase containing lipid dispersions. In some embodiments, the lipid phase contains liposomes.

[0514] In some embodiments, liposomes are self-enclosed monolayer or multilayer vesicle particles, wherein the layers comprise lipid bilayers and the encapsulated cavities contain an aqueous phase. The prerequisite for using liposomes to form nanoparticles is that the lipids in the desired mixture are capable of forming a layered (bilayer) phase in the aqueous environment in which the application is performed.

[0515] In some embodiments, liposomes comprise a monolayer or multilayer phospholipid bilayer enclosing an aqueous core (also referred to herein as an aqueous cavity). They can be prepared from materials having polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, the cationic lipids used to formulate liposomes intended for delivery of DNA and / or RNA are inherently amphiphilic and consist of a positively charged (cationic) amino head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

[0516] In some embodiments, the lipid complex is a multilayered liposome-based formulation formed by the electrostatic interaction of cationic liposomes with nucleic acids. In some embodiments, the formed lipid complex has a unique internal molecular arrangement resulting from the transformation from a liposome structure to a dense DNA and / or RNA-liposome.

[0517] In some embodiments, LPX particles comprise amphiphilic lipids, particularly cationic or ionizable cationic amphiphilic lipids, and DNA and / or RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or ionizable cationic amphiphilic lipids) and negatively charged RNA (especially mRNA) lead to the spontaneous formation of complex and RNA-lipid complex particles. Positively charged liposomes can typically be synthesized using cationic or ionizable cationic amphiphilic lipids (such as DOTMA and / or DODMA) and optionally additional lipids (such as DOPE or DSPC).

[0518] Generally, lipid nanoparticles (LNPs) can typically be 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 can be used for particle formation, which does not form a layered (bilayer) phase in water.

[0519] In some embodiments, the LNP comprises or is composed of cationic / ionizable cationic lipids and accessory 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, DNA and / or RNA (particularly mRNA) are bound to ionizable cationic lipids occupying a central core of the LNP. In some embodiments, polymer-conjugated lipids, together with phospholipids, form the surface of the LNP. In some embodiments, charged and uncharged forms of cholesterol and ionizable cationic lipids may be distributed throughout the LNP.

[0520] In some embodiments, the DNA and / or RNA (e.g., mRNA) described herein may be non-covalently associated with the particles described herein. In embodiments, the DNA and / or RNA (especially mRNA) may be attached to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or may be contained within the particle (encapsulated DNA and / or RNA (especially encapsulated mRNA)).

[0521] In some embodiments, the size (e.g., diameter) of the particles described herein (e.g., LNP and LPX) is in the range of about 10 to about 2000 nm, such as 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 at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, to (Approximately 950nm, up to approximately 900nm, up to approximately 850nm, up to approximately 800nm, up to approximately 750nm, up to approximately 700nm, up to approximately 650nm, up to approximately 600nm, up to approximately 550nm, or up to approximately 500nm), such as in the range of approximately 20-approximately 1500nm, such as approximately 30-approximately 1200nm, approximately 40-approximately 1100nm, approximately 50-approximately 1000nm, approximately 60-approximately 9 .... The sizes of the particles described herein (e.g., LNP and LPX) are in the range of approximately 70-800 nm, approximately 80-700 nm, approximately 90-600 nm, or approximately 50-500 nm or approximately 100-500 nm, such as in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, 50-250 nm, 60-200 nm, 70-150 nm or 80-150 nm. In some embodiments, the sizes (e.g., diameters) of the particles described herein (e.g., LNP and LPX) are in the range of approximately 40 nm to approximately 200 nm, such as approximately 50 nm to approximately 180 nm, approximately 60 nm to approximately 160 nm, approximately 80 nm to approximately 150 nm or approximately 80 nm to approximately 120 nm.

[0522] In some embodiments, the average diameter of the particles described herein (e.g., LNP and LPX) is in the following ranges: 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. m, approximately 50nm-approx. 250nm, approximately 50nm-approx. 200nm, approximately 100nm-approx. 1000nm, approximately 100nm-approx. 800nm, approximately 100nm-approx. 700nm, approximately 100nm-approx. 600nm, approximately 100nm-approx. 500nm, approximately 100nm-approx. 450nm, approximately 100nm-approx. 400nm, approximately 100nm-approx. 350nm, approximately 100nm-approx. 300nm, approximately 100nm-approx. 2 50nm, approximately 100nm-approx. 200nm, approximately 150nm-approx. 1000nm, approximately 150nm-approx. 800nm, approximately 150nm-approx. 700nm, approximately 150nm-approx. 600nm, approximately 150nm-approx. 500nm, approximately 150nm-approx. 450nm, approximately 150nm-approx. 400nm, approximately 150nm-approx. 350nm, approximately 150nm-approx. 300nm, approximately 150nm-approx. 250nm, approximately 15 The average diameter of the particles described herein (e.g., LNP and LPX) is within the range of approximately 200 nm, 200 nm to approximately 1000 nm, approximately 200 nm to approximately 800 nm, approximately 200 nm to approximately 700 nm, approximately 200 nm to approximately 600 nm, approximately 200 nm to approximately 500 nm, approximately 200 nm to approximately 450 nm, approximately 200 nm to approximately 400 nm, approximately 200 nm to approximately 350 nm, approximately 200 nm to approximately 300 nm, approximately 200 nm to approximately 250 nm, or approximately 80 nm to approximately 150 nm. In some embodiments, the average diameter of the particles described herein (e.g., LNP and LPX) is within the range of approximately 40 nm to approximately 200 nm, approximately 50 nm to approximately 180 nm, approximately 60 nm to approximately 160 nm, approximately 80 nm to approximately 150 nm, or approximately 80 nm to approximately 120 nm.

[0523] The DNA and / or RNA particles (especially mRNA particles) described herein can 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. For example, DNA and / or RNA particles can exhibit a polydispersity index in the range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0524] The N / P ratio indicates the ratio of nitrogen groups in lipids to phosphate groups in nucleic acids. It relates to the charge ratio, as nitrogen atoms (depending on pH) are generally positively charged, while phosphate groups are negatively charged. When charge balance is present, the N / P ratio depends on pH. Lipid formulations are typically formed with N / P ratios greater than 4, ranging from 12, because positively charged nanoparticles are considered beneficial for transfection. In this case, complete binding of DNA and / or RNA to the nanoparticles is assumed.

[0525] This disclosure describes compositions comprising DNA and / or RNA (especially mRNA) and at least one cationic or ionizable cationic lipid associated with the DNA and / or RNA to form DNA and / or RNA particles, as well as formulations comprising such particles. The DNA and / or RNA particles may comprise DNA and / or RNA complexed with the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they cannot virally infect cells.

[0526] Suitable cationic or ionizable cationic lipids are lipids that 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 be part of a DNA and / or RNA particle.

[0527] In some implementations, DNA and / or RNA particles (especially mRNA particles) contain more than one type of DNA and / or RNA molecules, wherein the molecular parameters of the DNA and / or RNA molecules may be similar to or different from each other, for example, in terms of molar mass or basic structural elements, such as molecular structure, capping, coding regions or other features.

[0528] In granule formulations, each DNA and / or RNA substance can be formulated separately as an independent granule formulation. In this case, each independent granule formulation contains one DNA and / or RNA substance. The independent granule formulations can exist as separate entities, for example, in separate containers. Such formulations can be obtained by providing each DNA and / or RNA substance (typically in the form of a DNA and / or RNA-containing solution) separately with a granule-forming agent, thereby enabling granules to form. The corresponding granules will contain only the specific DNA and / or RNA substance provided during granule formation (the independent granule formulation). In some embodiments, the composition (such as a pharmaceutical composition) comprises more than one independent granule formulation. The corresponding pharmaceutical composition is referred to as a mixed granule formulation. The mixed granule formulations of this disclosure can be obtained by the steps of separately forming independent granule formulations and then mixing the independent granule formulations. Through the mixing step, a formulation comprising a mixed population of DNA- and / or RNA-containing granules is obtained. The independent granule population can coexist in a single container containing the mixed population of independent granule formulations. Alternatively, all DNA and / or RNA substances of the pharmaceutical composition can be formulated together as a combined granule formulation. Such formulations are obtained by providing a combination of all DNA and / or RNA substances (typically a combined solution) along with a particle-forming agent, thereby enabling particle formation. Unlike mixed particle formulations, combined particle formulations typically comprise particles containing more than one type of DNA and / or RNA substance. In combined particle compositions, different DNA and / or RNA substances are usually co-present within a single particle.

[0529] Polymer

[0530] Given their high chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically aggregate negatively charged DNA and / or RNA into particles, particularly nanoparticles. These positively charged groups are often composed of amines, which alter their protonation state in a pH range of 5.5–7.5, presumably causing ionic imbalance and leading to endosome disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers like chitosan, have all been used for nucleic acid delivery and are suitable as cationic polymers for this paper. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) have gained widespread use in nucleic acid delivery due to their ease of synthesis and biodegradability. These synthetic polymers are also suitable as cationic polymers for this paper.

[0531] As used herein, “polymer” has its usual meaning, namely, a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may be all identical, or in some cases, more than one type of repeating unit may be present within a polymer. In some cases, polymers are biologically derived, i.e., biopolymers, such as proteins. In some cases, additional portions may be present in the polymer, such as targeting moieties.

[0532] A polymer is called a "polymer" if it contains more than one type of repeating unit. It should be understood that the polymer 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., containing 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), and so on. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0533] In some embodiments, the polymer is biocompatible. A biocompatible polymer is one that typically does not cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is capable of chemical and / or biological degradation within a physiological environment (e.g., in vivo).

[0534] In some embodiments, the polymer may be protamine or polyalkyleneimine.

[0535] The term "protamine" refers to any variety of relatively low molecular weight, strongly basic proteins rich in arginine that are found in the sperm cells of various animals (such as fish) and associate with DNA in place of somatic cell histones. Specifically, the term "protamine" refers to proteins found in fish sperm that are strongly basic, soluble in water, do not coagulate upon heating, and primarily produce arginine upon hydrolysis. In purified form, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0536] According to this disclosure, as used herein, the term "protamine" means any protamine amino acid sequence obtained from or derived from a natural or biological source, including fragments thereof, as well as multimeric forms of said amino acid sequence or fragments thereof, and (synthetic) polypeptides that are artificial, specifically designed for a particular purpose, and cannot be isolated from a natural or biological source.

[0537] In one embodiment, the polyalkylene imide comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkylene imide is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 × 10⁻⁶. 2 -10 7 Da, preferably 1000-10 5 Da, more preferably 10000-40000Da, more preferably 15000-30000Da, and even more preferably 20000-25000Da.

[0538] According to this disclosure, linear polyalkylene imides, such as linear polyethyleneimine (PEI), are preferred.

[0539] The cationic polymers (including polycationic polymers) considered for use herein include any cationic polymer capable of electrostatically binding nucleic acids. In some embodiments, the cationic polymers considered for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or forming vesicles therein encapsulating or encapsulating the nucleic acid.

[0540] The particles described herein may also contain polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

[0541] Lipid

[0542] In this document, the terms "lipid" and "lipid-like substance" are broadly defined as molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules comprising both hydrophobic and hydrophilic moieties are also frequently referred to as amphiphilic molecules. Lipids are generally insoluble or sparingly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into ordered structures and different phases. One such phase consists of lipid bilayers, which exist in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by introducing nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can comprise polar and / or charged groups and include sugars, phosphates, carboxyls, sulfates, aminos, thiols, nitros, hydroxyl groups, and other similar groups.

[0543] As used herein, the term "hydrophobic" refers to any molecule, part, or group that is substantially immiscible or insoluble in aqueous solutions. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. The monovalent group of a hydrocarbon is referred to herein as a hydrocarbon group. Hydrophobic groups may have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, provided that the group satisfies the condition of being substantially immiscible or insoluble in aqueous solutions.

[0544] The term "hydrocarbon" includes acyclic, e.g., linear (straight-chain) or branched hydrocarbon groups, such as alkyl, alkenyl, or alkynyl groups as defined herein. It should be understood that one or more hydrogen atoms in an alkyl, alkenyl, or alkynyl group may be substituted with other atoms (e.g., halogen, oxygen, or sulfur). Unless otherwise stated, hydrocarbon groups may also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0545] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety that may have 1 to 30, typically 1 to 20, and usually 6 to 18 carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc.

[0546] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond, wherein the total number of carbon atoms can be 6-30, typically 6-20, and often 6-18. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded down to the nearest integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1-6 (e.g., 1-4), that is, 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0547] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total number of carbon atoms can be 6-30, typically 6-20, and often 6-18. The alkynyl group may optionally have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is odd, the result of the division is rounded down to the nearest 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 1-6 (e.g., 1-4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.

[0548] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety that may have 1 to 30, typically 2 to 20, and usually 4 to 12 carbon atoms. Exemplary nonpolar alkylenes include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecylene, tetradecamethylene, hexadecylene, octadecylene, etc.

[0549] The term "alkenyl" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond, wherein the total number of carbon atoms can be 2-30, typically 2-20, and often 4-12. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded down to the nearest integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1-6 (e.g., 1-4), that is, 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0550] The term "cycloalkyl" represents a cyclic non-aromatic form of "alkyl" and "alkenyl," preferably having 3-14 carbon atoms, such as 3-12 or 3-10 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 3-7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclododecyl, cyclododecenyl, and adamantyl. A cycloalkyl group can consist of one ring (monocyclic), two rings (bicyclic), or more rings (polycyclic).

[0551] The term "aryl" refers to a monovalent group in an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3-14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, 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, anthraceneyl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl groups do not cover fullerenes.

[0552] In the context of hydrocarbons, the term "aromatic" means that the entire molecule must be aromatic. For example, if a monocyclic aryl group is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of this disclosure. Similarly, if a bicyclic or polycyclic aryl group (such as naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of this disclosure (even if only one ring (such as in 1,2-dihydronaphthyl) is aromatic).

[0553] As used herein, the term "amphiphilic" refers to a molecule having both a polar and a nonpolar moiety. Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have either a positive or a negative charge. Alternatively, the polar moiety may have both a positive and a negative charge and be an amphoteric ion or an inner salt. For purposes of disclosure, the amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0554] The terms "lipid-like substances," "lipid-like compounds," or "lipid-like molecules" refer to substances that are structurally and / or functionally related to lipids but may not be strictly considered lipids, particularly amphiphilic substances. For example, the term includes compounds capable of forming amphiphilic layers (such as those present in vesicles, multilayer / monolayer liposomes, or membranes) in an aqueous environment, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. Generally, the term includes molecules containing hydrophilic and hydrophobic portions with different structural organization, which may be similar to or dissimilar to the structural organization of lipids. Examples of lipid-like compounds capable of spontaneously integrating into cell membranes include functional lipid constructs, such as synthetic functional-spacer-lipid constructs (FSL), synthetic functional-spacer-sterol constructs (FSS), and artificial amphiphilic molecules. Lipids comprising 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. These lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Conventional surfactant monomers, consisting of only a linear alkyl chain and a hydrophilic head group, are generally conical. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into water-soluble spherical or elliptoid micelles. While lipids also have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—lipids differ from surfactants in the shape of the monomer, the type of aggregates formed in solution, and the concentration range required for aggregation. As used herein, the term "lipid" is interpreted to encompass both lipids and lipid-like substances, unless otherwise stated herein or clearly contradicted by the context.

[0555] Lipids are generally classified into eight classes: fatty acids, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subclass of lipid called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholesterolanol, cholesterol ketone, cholesterol, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0556] Fatty acids, or fatty acid residues, are a group of distinct molecules formed by hydrocarbon chains ending with carboxylic acid groups; this arrangement imparts polar, hydrophilic ends and water-insoluble, hydrophobic ends to the molecule. The carbon chains, typically 4–24 carbons in length, can be saturated or unsaturated and can be linked to functional groups containing oxygen, halogens, nitrogen, and sulfur. If fatty acids contain double bonds, they may exhibit cis or trans geometric isomers, which significantly affect the molecular configuration. Cis double bonds cause the fatty acid chain to bend, an effect exacerbated by the presence of more cis double bonds in the chain. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0557] Glycerides are composed of mono-, di-, and tri-substituted glycerols, the most well-known being fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically esterified by different fatty acids. Other subclasses of glycerides are represented by glycosylglycerols, characterized by the presence of one or more sugar residues linked to glycerol via glycosidic bonds.

[0558] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) with a glycerol core linked to two fatty acid-derived "tails" via ester bonds and to a "head" group via phosphate ester bonds. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids) include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0559] Sphingolipids are a complex family of compounds sharing a common structural feature: the sphingoid base skeleton. The main sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are the major subclass of sphingoid base derivatives, consisting of fatty acids linked by an amide. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The main phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi possess phytoceramide phosphoinositol and a mannose-containing head group. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples include simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0560] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, together with glycerophospholipids and sphingomyelins, are important components of membrane lipids.

[0561] Glycolipids describe compounds in which fatty acids are directly linked to a glycosyl backbone, forming structures compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerides and glycerophospholipids. The most familiar glycolipids are acylated glucosamine precursors of the lipid A component of lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, derived from up to seven fatty acyl chains. The smallest lipopolysaccharide required for growth in *E. coli* is Kdo2-lipid A, a hexaacylated glucosamine disaccharide glycosylated from two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0562] Polyketides are synthesized through the polymerization of acetyl and propionyl subunits via classical enzymes and iterative and modular enzymes sharing mechanistic features with fatty acid synthases. They comprise a wide range of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and exhibit high structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified through glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0563] According to this disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist as uncharged or neutral zwitterions at a selected pH.

[0564] Cationic / ionicizable cationic lipid

[0565] In some embodiments, the DNA and / or RNA compositions, formulations, and nucleic acid particles described herein comprise at least one cationic or ionizable cationic lipid as a particle-forming agent. Cationic or ionizable cationic lipids considered for use herein include any cationic or ionizable cationic lipid (including lipid-like substances) capable of electrostatically binding nucleic acids. In some embodiments, the cationic or ionizable cationic lipids considered for use herein can associate with nucleic acids, for example, by forming a complex with nucleic acids or forming vesicles therein encapsulating or encapsulating nucleic acids.

[0566] As used herein, “cationic lipid” refers to lipids or lipid-like substances that have a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Typically, cationic lipids have a lipophilic moiety, such as a sterol, acyl chain, diacyl chain, or chain of more than one acyl group, and the head group of the lipid is usually positively charged.

[0567] In some embodiments, the cationic lipids have a net positive charge only at certain pH levels, particularly at acidic pH levels, while at different, preferably higher pH levels such as physiological pH levels, they preferably have no net positive charge, or more preferably no charge, i.e., they are neutral. Compared to particles that remain cationic at physiological pH levels, this ionizable property is believed to enhance potency by facilitating endosome escape and reducing toxicity.

[0568] As used herein, "ionizable cationic lipid" refers to a lipid or lipid-like substance having a net positive charge or being neutral, i.e., it is not a permanent cation. Therefore, depending on the pH of the composition in which the ionizable cationic lipid is dissolved, the ionizable cationic lipid is either positively charged or neutral. For the purposes of this disclosure, unless contradicted by the context, the term "cationic lipid" includes ionizable cationic lipids.

[0569] In some embodiments, the cationic or ionizable cationic lipid comprises a head group, said head group including at least one positively charged or protonable nitrogen atom (N).

[0570] Examples of cationic or ionizable cationic lipids include, but are not limited to, N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and 1,2-di-O-octadecenyl-3-trimethylammonium propane. Propagated oxalis (DOTMA), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride 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 propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), and dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-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 (DLi) n-K-XTC2-DMA), 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 (N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide) bromide, DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide, GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide, GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide,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 (N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide) 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 (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 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-glycerol-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide) 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(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (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)-{ ... -Ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-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 (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).

[0571] In some embodiments, the cationic or ionizable cationic lipid is DOTMA. In some embodiments, the cationic or ionizable cationic lipid is DODMA.

[0572] DOTMA is a cationic lipid with a quaternary ammonium head group. The structure of DOTMA is shown below:

[0573]

[0574] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA is shown below:

[0575]

[0576] In some embodiments, cationic or ionizable cationic lipids may comprise about 10 mol% to about 95 mol%, about 20 mol% to about 95 mol%, about 20 mol% to about 90 mol%, about 30 mol% to about 90 mol%, about 40 mol% to about 90 mol%, or about 40 mol% to about 80 mol% of the total lipids present in the particles.

[0577] Additional lipid

[0578] The DNA and / or RNA compositions, formulations, and DNA and / or RNA particles described herein may also contain lipids other than cationic or ionizable cationic lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-ionizable cationic lipids or lipid-like substances). Anionic lipids and neutral lipids or lipid-like substances are collectively referred to herein as non-cationic lipids. Optimizing the formulation of DNA and / or RNA particles by adding other hydrophobic portions (such as cholesterol and lipids) besides cationic or ionizable cationic lipids can enhance particle stability and nucleic acid delivery efficiency.

[0579] One or more additional lipids may or may not affect the total charge of DNA and / or RNA particles. In some embodiments, the one or more additional lipids are noncationic lipids or lipid-like substances. Noncationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, “anionic lipid” means any lipid that carries a negative charge at a selected pH. As used herein, “neutral lipid” means any of a variety of lipid species that are present at a selected pH in an uncharged or neutral zwitterionic form.

[0580] In some embodiments, the DNA and / or RNA compositions, formulations, and DNA and / or RNA particles described herein comprise cationic or ionizable cationic lipids and one or more additional lipids.

[0581] Without being bound by theory, the amount of cationic or ionizable cationic lipids, compared to the amount of one or more additional lipids, may affect important characteristics of DNA and / or RNA particles, such as charge, particle size, stability, tissue selectivity, and the biological activity of DNA and / or RNA. Therefore, in some embodiments, the molar ratio of cationic or ionizable cationic lipids 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.

[0582] In some embodiments, the DNA and / or RNA compositions, formulations, and one or more additional lipids contained in the DNA and / or RNA particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.

[0583] In some embodiments, one or more additional lipids comprise neutral lipids, which are phospholipids. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. These phospholipids specifically include diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, and dipalmitoylphosphatidylcholine. atidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), diilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso) PC) and phosphatidylethanolamine, especially diacylphosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), and dimyristoyyl-phosphatidylethanolamine (dimyristoy). l-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphate-(1′-rac-glycerol)(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 other phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0584] In some embodiments, the additional lipid comprises one of the following: (1) phospholipids, (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholesterol ketones, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0585] Therefore, in some embodiments, the DNA and / or RNA compositions, formulations and DNA and / or RNA particles described herein comprise (1) a cationic or ionizable cationic lipid and a phospholipid (such as DSPC or DOPE) or (2) a cationic or ionizable cationic lipid and a phospholipid (such as DSPC or DOPE) and cholesterol.

[0586] In some embodiments, the DNA and / or RNA particles described herein (especially those containing mRNA) contain (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0587] DSPC is a neutral phospholipid. The structure of DSPC can be shown below:

[0588]

[0589] DOPE is a neutral phospholipid. The structure of DOPE can be shown below:

[0590]

[0591] The structure of cholesterol can be seen as follows:

[0592]

[0593] In some embodiments, the DNA and / or RNA compositions, formulations, and DNA and / or RNA particles described herein do not include polymer-conjugated lipids, such as PEGylated lipids. The term "PEGylated lipid" refers to a molecule that simultaneously comprises a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art.

[0594] In some embodiments, additional lipids (e.g., one or more phospholipids and / or cholesterol) may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 2 mol% to about 80 mol%, about 5 mol% to about 80 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 50 mol%, about 7.5 mol% to about 50 mol%, or about 10 mol% to about 40 mol%. In some embodiments, additional lipids (e.g., one or more phospholipids and / or cholesterol) comprise about 10 mol%, about 15 mol%, or about 20 mol% of the total lipids present in the particles.

[0595] In some embodiments, the additional lipid comprises a mixture of: (i) phospholipids, such as DOPE; and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of phospholipids (such as DOPE) to cholesterol or a derivative thereof is about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0596] Polymer-conjugated lipid

[0597] In some embodiments, the DNA and / or RNA compositions, formulations, and DNA and / or RNA particles described herein may comprise at least one polymer-conjugated lipid. The polymer-conjugated lipid is typically a molecule comprising a lipid moiety and a polymer moiety conjugated thereto. In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as a polyethylene glycol-based lipid or PEG-lipid. The term "polyethylene glycol-based lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-based lipids are known in the art. In some embodiments, the polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as a sarcosine-based lipid or pSar-lipid. The term "sarcosine-based lipid" refers to a molecule comprising both a lipid moiety and a polysarcosine moiety.

[0598] In some embodiments, polymer-conjugated lipids are designed to spatially stabilize lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, the polymer-conjugated lipids can reduce their association with serum proteins and / or the resulting uptake by the reticuloendothelial system. Polyethylene glycol (PEG)-conjugated lipid

[0599] In some embodiments, the DNA and / or RNA compositions / formulations and DNA and / or RNA particles described herein contain PEG-conjugated lipids.

[0600] In some embodiments, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is a lipid having the following general formula:

[0601]

[0602] Or its pharmaceutically acceptable salts, tautomers, or stereoisomers, wherein:

[0603] R 12 and R 13 Each of them is independently a straight or branched alkyl or alkenyl chain containing 10-30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and the average value of w is 30-60.

[0604] In some implementations of this formula, R 12 and R 13 Each of them is independently a straight-chain alkyl chain containing 10-18 carbon atoms, preferably 12-16 carbon atoms.

[0605] In some implementations of this formula, R 12 and R 13 The same. In some implementations, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 12 carbon atoms. In some embodiments, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 14 carbon atoms. In some embodiments, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 16 carbon atoms.

[0606] In some implementations of this formula, R 12 and R 13 Different. In some implementations, R 12 and R 13 One of them is a straight-chain alkyl chain containing 12 carbon atoms, and R 12 and R13 Another one is a straight-chain alkyl chain containing 14 carbon atoms.

[0607] In some implementations of this formula, the average value of w is 40-50, such as an average value of 45.

[0608] In some embodiments of this formula, w is within a certain range, such that the average molecular weight of the PEG portion of the polyethylene glycol-modified lipid is about 400 to about 6000 g / mol, such as about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. In some embodiments, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 14 carbon atoms, and the average value of w is 45.

[0609] Various PEG-conjugated lipids are known in the art, including but not limited to PEG-DAG such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), PEG-DAG such as PEG-PE, PEG-Succinate, etc. diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), polyethylene glycol-modified ceramide (PEG-cer), or PEG-dialkoxypropylcarbamate (PEG-S-DAG) Diethylcarbamate, such as w-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(wmethoxy(polyethoxy)ethyl)carbamate, etc.

[0610] In some embodiments, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is or contains 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the polyethylene glycol-modified lipid has the following structure:

[0611]

[0612] In some embodiments, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is DMG-PEG 2000, for example, having the following structure:

[0613]

[0614] In some embodiments, the PEG-conjugated lipids (polyethylene glycol-modified lipids) have the following structure:

[0615]

[0616] The average value of n is 30-60, such as about 50. In one embodiment, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is PEG2000-C-DMA, which preferably refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

[0617] In some embodiments, the DNA and / or RNA compositions / formulations described herein may comprise one or more PEG-conjugated lipids or polyethylene glycol-modified lipids as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0618] In some embodiments, the polyethylene glycol-modified lipids comprise about 1 mol% to about 10 mol% of the total lipids present in the DNA and / or RNA compositions / formulations described herein and in the DNA and / or RNA particles, preferably about 1 mol% to about 5 mol%, more preferably about 1 mol% to about 2.5 mol%.

[0619] Embodiments of lipid complex particles

[0620] In some embodiments of this disclosure, the DNA and / or RNA described herein may be present in DNA and / or RNA lipid complex particles.

[0621] Lipid complexes (LPX) are electrostatic complexes that are typically formed by mixing pre-formed cationic lipid liposomes with anionic nucleic acids. The resulting lipid complexes have a unique internal molecular arrangement resulting from the transformation from a liposome structure to a tight DNA and / or RNA lipid complex.

[0622] In some embodiments, the RNA-lipid complex particles comprise cationic lipids and additional lipids. In an exemplary embodiment, the cationic lipid is DOTMA, and the additional lipid is DOPE.

[0623] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0624] In some embodiments, the average diameter of the DNA and / or RNA lipid complex particles described herein is about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, the average diameter of the DNA and / or RNA lipid complex particles is about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the average diameter of the DNA and / or RNA lipid complex particles is about 250 nm to about 700 nm. In some embodiments, the average diameter of the DNA and / or RNA lipid complex particles is about 300 nm to about 500 nm. In one exemplary embodiment, the average diameter of the DNA and / or RNA lipid complex particles is about 400 nm.

[0625] The DNA and / or RNA lipid complex particles described herein, as well as compositions containing DNA and / or RNA lipid complex particles, can be used to deliver DNA and / or RNA to target tissues after parenteral administration (especially after intravenous administration).

[0626] RNA-lipid complex particles targeting the spleen are described in WO 2013 / 143683, which are incorporated herein by reference. RNA-lipid complex particles with a net negative charge have been found to be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Therefore, DNA and / or RNA accumulation and / or DNA and / or RNA expression occur in the spleen following administration of the DNA and / or RNA lipid complex particles. Thus, the DNA and / or RNA lipid complex particles of this disclosure can be used to express DNA and / or RNA in the spleen. In one embodiment, DNA and / or RNA accumulation and / or DNA and / or RNA expression do not occur or substantially do not occur in the lungs and / or liver following administration of the DNA and / or RNA lipid complex particles. In some embodiments, DNA and / or RNA accumulation and / or DNA and / or RNA expression occur in antigen-presenting cells (such as professional antigen-presenting cells) in the spleen following administration of the DNA and / or RNA lipid complex particles. Therefore, the DNA and / or RNA lipid complex particles of this disclosure can be used to target DNA and / or RNA (e.g., DNA and / or RNA encoding an antigen or at least one epitope) to the lymphatic system, particularly secondary lymphatic organs, more specifically the spleen. Targeting the lymphatic system (particularly secondary lymphatic organs, more specifically the spleen) is particularly preferred if the administered DNA and / or RNA is DNA and / or RNA encoding a vaccine antigen. In some embodiments, the target cells are spleen cells. In some embodiments, the target cells are antigen-presenting cells (e.g., professional antigen-presenting cells) in the spleen. In one embodiment, the target cells are dendritic cells in the spleen.

[0627] The charge of the DNA and / or RNA lipid complex particles disclosed herein is the sum of the charges present in at least one cationic lipid and the charges present in DNA and / or RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in DNA and / or RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in DNA and / or RNA is calculated using the following equation: Charge ratio = [(Cationic lipid concentration (moles)) * (Total positive charge in cationic lipids)] / [(DNA and / or RNA concentration (moles)) * (Total negative charge in DNA and / or RNA)]. The concentration of DNA and / or RNA and the amount of at least one cationic lipid can be determined by those skilled in the art using conventional methods.

[0628] In some embodiments, the charge ratio of positive to negative charge in the DNA and / or RNA lipid complex particles is about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2, at physiological pH. In specific embodiments, the charge ratio of positive to negative charge in the DNA and / or RNA lipid complex particles is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0, at physiological pH.

[0629] Embodiments of lipid nanoparticles (LNP)

[0630] In some embodiments, the DNA and / or RNA described herein are present in the form of lipid nanoparticles (LNPs). LNPs typically comprise four components: ionizable cationic lipids, neutral lipids (such as phospholipids), steroids (such as cholesterol), and polymer-conjugated lipids (such as PEG-lipids). LNPs can be prepared by mixing lipids dissolved in ethanol with DNA and / or RNA in an aqueous buffer.

[0631] In some embodiments, in the DNA and / or RNA LNPs described herein, DNA and / or RNA are bound to ionizable cationic lipids occupying the central core of the LNP. The polymer-conjugated lipids, together with phospholipids, form the surface of the LNP. In some embodiments, cholesterol and ionizable cationic lipids may be distributed throughout the LNP.

[0632] In some embodiments, the LNP comprises one or more ionizable cationic lipids and one or more stabilized...

Claims

1. An immune effector cell comprising a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, the 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 2, wherein the first nucleic acid molecule is DNA or RNA.

4. The immune effector cell of any one of claims 1-3, wherein the first nucleotide sequence is integrated into a genomic nucleic acid molecule of the immune effector cell.

5. The immune effector cell of any one of claims 1-4, wherein the genomic nucleic acid molecule is a chromosome, an episome, or a non-viral episome.

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

7. The immune effector cell of any one of claims 1-6, wherein the first nucleotide sequence is comprised within a transposable element.

8. The immune effector cell of any one of claims 1-7, 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 9, wherein the third nucleic acid molecule is mRNA.

11. The immune effector cell of any one of claims 8-10, 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 8-12, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.

13. The immune effector cell of any one of claims 8-12, 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. The immune effector cell of any one of claims 1-13, wherein the first cell surface-expressed antigen receptor is stably expressed.

15. The immune effector cell of any one of claims 1-14, wherein the activator molecule enables the immune effector cell to activate, expand, differentiate, and / or proliferate.

16. The immune effector cell of any one of claims 1-15, wherein the activator molecule is a non-coding RNA or a protein.

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

18. The immune effector cell of any one of claims 1-16, wherein the activator molecule is a cytokine.

19. The immune effector cell of any one of claims 1-16, wherein the activator molecule is a second cell surface-expressed antigen receptor, wherein the extracellular portions of the first cell surface-expressed antigen receptor and the second cell surface-expressed antigen receptor do not bind to the same binding target.

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

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

22. The immune effector cell of claim 20 or 21, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises 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.

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

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

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

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

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

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

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

30. The immune effector cell of any one of claims 19-30, wherein the second cell surface-expressed antigen receptor binding target is a cell surface-expressed protein or a 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. The immune effector cell of claim 30 or 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. The immune effector cell of any one of claims 19-34, wherein the second cell surface-expressed antigen receptor binding target is a cell surface protein expressed on a blood cell, preferably another immune effector cell.

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

36. The immune effector cell of claim 34 or 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. The immune effector cell of any one of claims 1-37, wherein the second nucleic acid molecule is DNA or RNA.

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

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

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

42. 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. The immune effector cell of claim 43 or 44, wherein the 5' cap structure is one of: capO, capl, cap2, cap3, cap4, ARCA (anti-reverse cap analog), modified ARCA, inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

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

47. The immune effector cell of claim 45, wherein the 5' cap structure is capl, which is m7 G(5')ppp(5')(N1 2'-OMe ).

48. The immune effector cell of claim 47, wherein the Nl is selected from A, C, G, or U.

49. The immune effector cell of claim 47 or 48, wherein the capl further comprises a second nucleotide N2, which is a cap-proximal A, G, C, or U at position +2, and the capl is represented as m7 G(5')ppp(5')(N1 2'-OMe )pN2.

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

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

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

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

54. 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 a tumor- associated antigen; (ii) an mRNA molecule encoding a second T cell receptor or chimeric antigen receptor that binds a different target than 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 a tumor-associated antigen, which is integrated into the genome of the immune effector cell or is comprised in 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 a different target than the tumor-associated antigen, which is not 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 a tumor- associated antigen, which is integrated into the genome of the immune effector cell; and (ii) an mRNA molecule encoding a second chimeric antigen receptor that binds a different target than the tumor-associated antigen.

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

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

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

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

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

62. The immune effector cell of any one of claims 1-61, wherein the immune effector cell has reduced cell surface expression of an endogenous HLA complex to a level that prevents host versus graft activity in a subject to which the immune effector cell has been 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. The immune effector cell of any one of claims 1-62, wherein the immune effector cell does not express its endogenous HLA complex on its cell surface.

64. A cell composition comprising the immune effector cell of any one of claims 1-63.

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

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

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

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

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

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

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

72. The immune effector cell, cell composition or pharmaceutical composition for use according to any one of claims 67-71, wherein the immune effector cell is autologous or allogeneic 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, 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, wherein the second nucleotide sequence is not comprised within a transposable element.

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

75. A particle comprising (i) a DNA episome comprising a first nucleotide sequence encoding a first cell surface expressed antigen receptor, preferably a non-viral episome; 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 provides for transient expression of the activator molecule when present in a cell.

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

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

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

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

80. The particle according to claim 78, wherein the third nucleic acid molecule is mRNA.

81. The particle according to any one of claims 74 or 76-80, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol 1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl or a functional equivalent variant thereof having transposase / transposition activity.

82. The particle according to any one of claims 74 or 76-81, wherein the molecule having transposase activity is Sleeping Beauty transposase SB 100X.

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

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

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

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

87. The particle of any one of claims 73-86, wherein the activating agent molecule enables the immune effector cell to activate, expand, differentiate, and / or proliferate.

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

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

90. The particle of any one of claims 73-88, wherein the activating agent molecule is a cytokine.

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

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 comprised within a transposable element.

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

94. The particle of claim 92 or 93, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprises 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.

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

96. The particle of any one of claims 92-95, wherein the fourth nucleic acid molecule and / or the fifth nucleic acid molecule is an mRNA.

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

98. A particle comprising the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule as set forth in any one of claims 1-72.

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

100. A particle comprising a first nucleic acid molecule, a second nucleic acid molecule, a third nucleic acid molecule, and a fourth nucleic acid molecule as set forth in any one of claims 19-72.

101. The particle of claim 100, further comprising a fifth nucleic acid molecule as set forth in any one of claims 19-72.

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

103. The particle of any one of claims 73-102, wherein the particle comprises a lipid, preferably a lipid having a cationic head group.

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

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

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

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

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

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

110. The particle of any one of claims 73-110, wherein the particle is a nanoparticle, wherein: (i) the number of positive charges in the nanoparticle is no more than the number of negative charges in the nanoparticle, and / or (ii) the nanoparticle has a neutral or net negative charge, and / or (iii) the zeta potential of the nanoparticle is 0 or less.

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

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 nucleic acid molecule, the second nucleic acid molecule, and the optional third nucleic acid molecule (N:P ratio) is 2.0-15.0, preferably 6.0-12.0; 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 nucleic acid molecule, the second nucleic acid molecule, and the optional third nucleic acid molecule (N:P ratio) is at least about 48, optionally about 48-300, about 60-200, or about 80-150.

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

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

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

116. The particle of claim 115, wherein the hydrophobic moiety having a binding moiety covalently attached thereto and the particle are non-covalently associated with one another.

117. The particle of claim 115 or 116, wherein the hydrophobic moiety having a binding moiety covalently attached thereto is a constituent part of the particle.

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

119. The particle of any one of claims 115-118, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula I L-X1-P-X2-B (I) wherein P comprises a polymer; L comprises a hydrophobic moiety attached to a first terminus of the polymer; B comprises a binding moiety attached to a second terminus of the polymer; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety.

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

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

122. The particle of any one of claims 115-121, wherein the hydrophobic moiety is or is comprised in a lipid.

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

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

125. The particle of any one of claims 115-124, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula II wherein B comprises the binding moiety.

126. The particle of claim 125, wherein B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.

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

128. The particle of any one of claims 73-127, wherein the particle is complexed with and / or the particle encapsulates the nucleic acid molecule. ​ ​ ​ ​ ​ ​ ​ ​ 129. A pharmaceutical composition comprising the particle of any one of claims 73-128, and a pharmaceutically acceptable carrier.

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

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

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

133. The particle or pharmaceutical composition for use of claim 130, wherein the disease, disorder, or condition is an infection.

134. The particle for use of claim 133, wherein the infection is a viral infection.

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

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

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

138. The complex of any one of claims 135-137, wherein (i) the moiety that binds the binding moiety covalently attached to the hydrophobic moiety and (ii) the moiety that targets a cell surface antigen are linked to one another.

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

140. The complex of any one of claims 135-139, wherein the moiety that targets a cell surface antigen comprises an antibody or antibody derivative.

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

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

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

144. The complex of any one of claims 135-143 for use in a method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of a target of the first cell surface-expressed antigen receptor.

145. A method of generating an immune effector cell expressing a first antigen receptor at the cell surface, the method comprising contacting an immune effector cell with (i) a first nucleic acid molecule 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 nucleotide sequence is not comprised within a transposable element, and wherein the first cell surface-expressed antigen receptor is stably expressed in the cell, the activator molecule is transiently expressed in the cell.

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

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

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

149. The method of any one of claims 145-148, 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 comprised within a transposable element.

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

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

152. The method of any one of claims 149-151, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Tol 1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functional equivalent variant thereof having transposase / transposition activity.

153. The method of any one of claims 149-151, wherein the molecule having transposase activity is Sleeping Beauty transposase SB 100X.

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

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

156. The method of any one of claims 145-155, wherein the activator molecule enables the immune effector cell to activate, expand, differentiate, and / or proliferate.

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

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

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

160. The method of any one of claims 145-159, 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. 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. The method of claim 160 or 161, wherein the method further comprises contacting the immune effector cell with a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a binding target of the second cell surface-expressed antigen receptor.

163. The method of claim 161 or 162, wherein the fourth or fifth nucleic acid molecule comprises 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.

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

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

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

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

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

169. The method of any one of claims 145-168, 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. The method of any one of claims 145-169, wherein the second nucleic acid molecule is DNA or RNA.

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

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

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

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

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

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

177. The method of claim 175 or 176, wherein the 5’ cap structure is one of: capO, capl, cap2, cap3, cap4, ARCA (anti-reverse cap analog), 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. The method of any one of claims 145-177, wherein the immune effector cell is a T cell, a B cell, a dendritic cell, or an NK cell.

179. The method of any one of claims 145-178, wherein the immune effector cell is a CD8+ and / or CD4+ T cell.

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

181. The method of any one of claims 145-180, wherein the second nucleic acid is not inherited in the same manner as a chromosome to progeny cells of the immune effector cell, and / or, wherein the second nucleic acid is diluted in each generation of progeny cells of the immune effector cell, and / or, wherein the amount of the second nucleic acid molecule in each progeny cell is less than the amount in the parent cell after one round of cell division.

182. A method of generating an immune effector cell expressing a first antigen receptor at the cell surface, the method comprising contacting an immune effector cell with a particle of any one of claims 73-134 or a complex of any one of claims 135-144.

183. The method of any one of claims 145 or 182, wherein the contacting is performed in vitro.

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

185. A method of generating an immune effector cell expressing two antigen receptors at the cell surface, the method comprising contacting an immune effector cell in vitro or ex vivo with: (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface expressed antigen receptor, the first nucleotide sequence being comprised within a transposon element; (ii) an RNA molecule comprising a second nucleotide sequence encoding a second cell surface expressed antigen receptor, the second nucleotide sequence not being comprised within a transposon element; and (iii) an RNA molecule comprising a third nucleotide sequence encoding a transposase, the third nucleotide sequence not being comprised within a transposon element, wherein the extracellular domain of the first and second cell surface expressed antigen receptor bind to different targets, preferably wherein 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 at the surface of a blood cell.

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

187. A method of generating an immune effector cell expressing two antigen receptors at the cell surface, the method comprising contacting an immune effector cell with a particle comprising (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface expressed antigen receptor, the first nucleotide sequence being comprised within a transposon 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 domain of the first and second cell surface expressed antigen receptor bind to different targets, preferably wherein 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 at the surface of a blood cell.

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

189. A method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, the method 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 the antigen associated with the disease, disorder, or condition, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of a cell of the subject or is not contained within an episome present in a cell of the subject, and / or (ii) the activator molecule is transiently expressed in the subject.

190. The method of claim 189, wherein the nucleic acid molecules are in a particle comprising a lipid.

191. A method of treating a subject having a disease, disorder, or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject a particle comprising (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell surface-expressed antigen receptor, the first nucleotide sequence being contained within a transposable element, and wherein the binding target of the first cell surface-expressed antigen receptor is the 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 ectodomains of the first and second cell surface-expressed antigen receptors bind to different targets.

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

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

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

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

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

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

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

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

Images