T cells, compositions comprising T cells, and uses thereof

Abstract

The present invention relates to photo-porated T cells, where the homeostasis of T cells after photo-poration is not affected and is equivalent to that before photo-poration or is equivalent compared to non-photo-porated T cells. The present invention further relates to a population of T cells and a pharmaceutical composition comprising a therapeutically effective amount of T cells.

Description

Technical Field

[0001] The present invention relates to T cells optionally containing a (high) molecule, and a composition containing the T cells, which can be used in a therapeutic environment.

Background Art

[0002] Many biotechnology and biomedical applications rely on engineered cells, which require the delivery of macromolecules such as DNA, RNA, peptides or proteins into cells in vitro or ex vivo. Although several cell transfection methods and techniques for generating engineered cells are known in the art, there are some drawbacks and problems. Engineered cells transfected by chemical transfection reagents or viral vectors have safety concerns regarding therapeutic applications and are limited in flexibility in terms of the type and size of the (high) molecule. Furthermore, engineered cells transfected by physical transfection techniques are also known. However, known physical transfection techniques have the undesirable problems of high cytotoxicity, low cell viability, and changes in cell homeostasis after cell transfection.

[0003] U.S. Patent No. 10,131,876 describes an electroporation method for T cells for subsequent therapeutic use. Electroporation has been used to introduce foreign molecules into cells, but this technique has been hampered due to the fact that the viability of cells after electroporation is low and the homeostasis of the cells changes. This is a significant drawback when generating engineered cells for therapeutic applications.

[0004] In recent years, the FDA has approved several gene therapies using engineered T cells. Naturally, it is important to safely engineer T cells while minimizing the impact on their native capabilities. Historically, viral vectors have been preferentially used to transduce T cells, but there are safety concerns and limitations in flexibility in terms of the type and size of (macro)molecules. The need for such safely engineered T cells is increasing because the applications of approved gene therapies, especially adoptive T cell therapies such as cancer immunotherapies using T cells, are rapidly increasing.

[0005] In view of the above, there remains a need in the art for additional and / or improved engineered cells suitable for therapeutic use. The object of the present invention is to provide engineered T cells that can be safely used in a therapeutic setting.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The inventors were able to provide engineered cells that are safe to use in therapeutic therapies. For example, these experiments demonstrated the success of engineered T cells suitable for, for example, cell-based cancer immunotherapies.

[0007] For this purpose, the present invention relates in a first aspect to the T cells according to claim 1.

[0008] Examples show that T cells after photoporation maintain the same level of homeostasis and cell proliferation as T cells before photoporation.

[0009] Preferred embodiments of the T cells are shown in any of claims 2 to 13.

[0010] In a second aspect, the present invention relates to a population of T cells according to claim 14.

[0011] In a third aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of T cells as recited in claim 15.

[0012] In a fourth aspect, the present invention relates to T cells according to the first aspect of the present invention, a population of T cells according to the second aspect of the present invention, or a pharmaceutical composition according to the third aspect of the present invention, for therapeutic use as recited in claim 16.

[0013] The uses as described herein can have the advantageous effect that the T cells can contain a wide variety of molecules, resulting in a wide range of therapeutic applications. Examples show that the engineered T cells are safe and suitable for therapeutic use, solving problems known in the prior art regarding safety and regulatory concerns due to the presence of NPs in the cells.

[0014] Preferred embodiments of the fourth aspect of the present invention are shown in claims 17 to 24. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] A brief description of the drawings in FIG. 12 is as follows. JPEG2025521125000001.jpg55166

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The present invention relates to T cells, populations of T cells, and pharmaceutical compositions comprising a therapeutically effective amount of T cells.

[0018] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which the present invention pertains. Further guidance is provided by the definitions of terms included to better understand the teachings of the present invention.

[0019] As used herein, the following terms have the following meanings:

[0020] As used herein, the term "in vitro" refers to outside the body of an animal or human. The term "in vitro" as used herein should be understood to include "ex vivo".

[0021] The term "ex vivo" typically refers to tissue or cells that have been removed from the body of an animal or human and maintained or grown outside the body, such as in a culture vessel.

[0022] As used herein, the term "in vivo" means within or inside the body of an animal or human.

[0023] As used herein, "a", "an", and "the" refer to singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0024] As used herein, the term "about" when referring to a measurable value such as a parameter, amount, or time duration encompasses variations of ±20% or less, preferably ±10% or less, more preferably ±5% or less, still more preferably ±1% or less, and even more preferably ±0.1% or less of the specified value, as long as such variations are appropriate for practicing the invention as disclosed. It is to be understood, however, that the value itself to which the modifier "about" refers is also specifically disclosed.

[0025] As used herein, the terms "comprise", "comprising", "comprises", and "comprising of" are synonymous with "include", "including", "includes", or "contain", "containing", "contains", and are inclusive or open-ended terms that specify the presence of what follows a component and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps known in or disclosed in the art.

[0026] Further, in this specification and the claims, terms such as first, second, and third are used, unless otherwise specified, to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. Terms so used are interchangeable under appropriate circumstances, and it is to be understood that the embodiments of the invention described herein can operate in an order other than that described or illustrated herein.

[0027] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range as well as the recited endpoints.

[0028] The expressions "weight %", "percent by weight", "% wt", or "wt %" refer, unless otherwise defined, throughout this specification and the entire document, to the relative weight of each component with respect to the total weight of the formulation.

[0029] The terms "one or more" or "at least one", e.g., one or more or at least one of a group of members, are clear in themselves, while by way of further illustration, this term encompasses references to any one of the members, or any two or more of the members, e.g., any ≧3, ≧4, ≧5, ≧6 or ≧7, etc. of the members, and all of the members.

[0030] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the technical field to which the present invention pertains. As a further guideline, definitions of terms used herein are included to better understand the teachings of the present invention. The terms or definitions used herein are provided only to assist in understanding the present invention.

[0031] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, and although they do not necessarily all refer to the same embodiment, they may. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art from this disclosure. Additionally, some embodiments described herein include some features and do not include other features included in other embodiments, but combinations of features of different embodiments are also intended to be within the scope of the present invention and will be understood by those skilled in the art to form another embodiment. For example, in the following claims, any combination of the claimed embodiments can be used.

[0032] As used herein, the term "nucleic acid" typically refers to a polymer of any length (preferably a linear polymer) consisting essentially of nucleoside units. Nucleoside units generally include a heterocyclic base and a sugar group. As heterocyclic bases, in particular, purine bases and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), unnatural bases or derivatized bases are included. Exemplary modified nucleic acid bases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, but are not limited thereto. In particular, 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes and is considered a more preferred base substitution, for example, in antisense agents when combined with 2'-O-methoxyethyl sugar modification. As sugar groups, in particular, pentose (pentofuranose) groups, for example, preferably ribose and / or 2-deoxyribose common in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, as well as modified or substituted sugar groups (e.g., but not limited to, 2'-O-alkylated sugars such as 2'-O-methylated or 2'-O-ethylated sugars (e.g., ribose); 2'-O-alkyloxyalkylated sugars such as 2'-O-methoxyethylated sugars (e.g., ribose); or 2'-O,4'-C-alkylened sugars, 2'-O,4'-C-methylened or 2'-O,4'-C-ethylened sugars (e.g., ribose); 2'-fluoroarabinose, etc.) are included. A nucleic acid molecule containing at least one ribonucleoside unit can typically be called ribonucleic acid or RNA. Such ribonucleoside units contain a 2'-OH moiety, and -H may be substituted as known in the art for ribonucleosides (e.g., by methyl, ethyl, alkyl, or alkyloxyalkyl).Preferably, ribonucleic acid or RNA may mainly consist of ribonucleoside units. For example, ≧80%, ≧85%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or even 100% (in numerical units) of the nucleoside units constituting the nucleic acid molecule may be ribonucleoside units. A nucleic acid molecule containing at least one deoxyribonucleoside unit may typically be referred to as deoxyribonucleic acid or DNA. Such deoxyribonucleoside units contain 2'-H. Preferably, deoxyribonucleic acid or DNA may mainly consist of deoxyribonucleoside units. For example, ≧80%, ≧85%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or even 100% (in numerical units) of the nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units. Nucleoside units are linked to each other by any of a number of known internucleoside linkages, especially phosphodiester linkages common to naturally occurring nucleic acids, as well as modified phosphate or phosphonate-based linkages, such as phosphorothioate, alkylphosphorothioate, such as methylphosphorothioate, phosphorodithioate, alkylphosphonate, such as methylphosphonate, alkylphosphonothioate, phosphotriester, such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate, such as 3'-N-carbamate, morpholino, borano, thioether, 3'-thioacetal and sulfone internucleoside linkages. Preferably, the internucleoside linkage may be a phosphate-based linkage including a modified phosphate-based linkage, for example, more preferably, a phosphodiester, phosphorothioate or phosphorodithioate linkage or a combination thereof.The term "nucleic acid" also encompasses any other polymer containing nucleobases, such as nucleic acid mimics, including, but not limited to, peptide nucleic acid (PNA), peptide nucleic acid with a phosphate group (PHONA), locked nucleic acid (LNA), morpholino phosphorodiamidate backbone nucleic acid (PMO), cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having a backbone section with an alkyl linker or an amino linker (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). As used herein, "alkyl" specifically encompasses C1-C4 straight-chain or branched, saturated or unsaturated hydrocarbons such as lower hydrocarbon moieties, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl. Nucleic acids as contemplated herein can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. Modified nucleosides can include modified heterocyclic bases, modified sugar moieties, modified internucleoside linkages, or combinations thereof.

[0033] The term "nucleic acid" preferably includes DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA (gDNA), plasmid DNA (pDNA), amplification products, oligonucleotides and synthetic (e.g., chemically synthesized) DNA, RNA or DNA / RNA hybrids. RNA includes RNAi (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), tRNA (transfer RNA, whether filled or not with the corresponding acylated amino acid) and cRNA (complementary RNA). Nucleic acids may be naturally occurring, e.g., those present in nature or isolated from nature, may be recombinant, i.e., produced by recombinant DNA technology, and / or may be partially or wholly chemically or biochemically synthesized. "Nucleic acid" can be double-stranded, partially double-stranded, or single-stranded. In the case of single-stranded, the nucleic acid can be a sense strand or an antisense strand. Further, the nucleic acid may be circular or linear.

[0034] As used throughout this specification, the term "oligonucleotide" refers to a nucleic acid (including nucleic acid analogs and mimetics) oligomer or polymer as defined herein. Preferably, the oligonucleotide, more particularly an antisense oligonucleotide, is (substantially) single-stranded. The oligonucleotides contemplated herein can have a length of from about 10 to about 100 nucleoside units (i.e., nucleotides or nucleotide analogs), preferably from about 15 to about 50, more preferably from about 20 to about 40, and also preferably from about 20 to about 30 nucleoside units (i.e., nucleotides or nucleotide analogs). The oligonucleotides contemplated herein may contain one or more or all non-naturally occurring heterocyclic bases and / or one or more or all non-naturally occurring sugar groups and / or one or more or all non-naturally occurring internucleoside linkages, and by including these, properties such as, for example, increased stability in the presence of nucleases, increased hybridization affinity, and increased resistance to mismatches can be improved.

[0035] Nucleic acid binders, such as oligonucleotide binders, are typically at least partially antisense to the target nucleic acid of interest. The term "antisense" generally refers to an agent (e.g., an oligonucleotide) configured to specifically anneal (hybridize) to a given sequence in a target nucleic acid, such as in a target DNA, hnRNA, pre-mRNA, or mRNA, and typically comprises, consists essentially of, or consists of a nucleic acid sequence complementary or substantially complementary to the target nucleic acid sequence. Antisense agents suitable for use herein, such as hybridization probes or amplification or sequencing primers and primer pairs), can typically anneal (hybridize) to their respective target nucleic acid sequences under high stringency conditions and can hybridize specifically to the target under physiological conditions. The terms "complementary" or "complementarity" as used herein with respect to nucleic acids refer to the normal binding of single-stranded nucleic acids under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson-Crick base pairing. For example, complementary Watson-Crick base pairing occurs between the bases A and T, A and U, or G and C. For example, the sequence 5'-A-G-U-3' is complementary to the sequence 5'-A-C-U-3'. References to oligonucleotides can include, without particular limitation, hybridization probes and / or amplification primers and / or sequencing primers commonly used in nucleic acid detection techniques, etc.

[0036] As used herein, the term "ribozyme" or "ribonucleic acid enzyme" refers to an RNA molecule having the ability to catalyze specific biochemical reactions, such as RNA splicing in gene expression. The function of ribozymes is similar to that of protein enzymes. The most common activities of ribozymes are the cleavage or ligation of RNA and DNA, and the formation of peptide bonds. Within ribosomes, ribozymes function as part of the ribosomal RNA of the large subunit to ligate amino acids during protein synthesis. It is also involved in various RNA processing reactions, such as RNA splicing, viral replication, and the biosynthesis of transfer RNA. Examples of ribozymes include hammerhead ribozymes, VS ribozymes, leadzymes, and hairpin ribozymes.

[0037] As used herein, the term "protein" generally encompasses a macromolecule containing one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. This term can include proteins produced naturally, recombinantly, semi-synthetically, or synthetically. This term also includes, but is not limited to, proteins having one or more co-expression or post-expression modifications of the polypeptide chain, such as glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of proenzymes or prehormones to their active forms. This term further includes protein variants or mutants having amino acid sequence variations relative to the corresponding native protein, such as deletions, additions, and / or substitutions of amino acids. This term contemplates both full-length proteins and portions or fragments of proteins, such as portions of naturally occurring proteins resulting from the processing of such full-length proteins.

[0038] As used herein, the term "polypeptide" encompasses a polymeric chain of amino acid residues linked by peptide bonds. Thus, in particular, when a protein is composed of only a single polypeptide chain, in this specification, the terms "protein" and "polypeptide" may be used interchangeably to refer to such a protein. This term is not limited to any minimum length of the polypeptide chain. This term may encompass polypeptides produced naturally, recombinantly, semi-synthetically or synthetically. This term also encompasses polypeptides having one or more co-expression or post-expression modifications of the polypeptide chain, such as, but not limited to, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of a proenzyme or prehormone to its active form. This term further includes polypeptide variants or mutants having amino acid sequence variations relative to the corresponding native polypeptide, such as, for example, deletions, additions, and / or substitutions of amino acids. This term contemplates both full-length polypeptides and polypeptide portions or fragments, such as, for example, naturally occurring polypeptide portions resulting from the processing of such full-length polypeptides.

[0039] As used herein, the term "peptide" preferably refers to a polypeptide as used herein that consists essentially of 50 amino acids or less, such as 45 amino acids or less, preferably 40 amino acids or less, such as 35 amino acids or less, more preferably 30 amino acids or less, such as 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less amino acids.

[0040] As used herein, the term "antibody" is used in the broadest sense and generally refers to any immunological binding agent. This term specifically includes intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., bivalent, trivalent or more) and / or multispecific antibodies (e.g., bispecific or more specific antibodies) formed from at least two intact antibodies, as well as antibody fragments insofar as they exhibit the desired biological activity (in particular, the ability to specifically bind to the antigen of interest, i.e., antigen-binding fragments), and multivalent and / or multispecific complexes of such fragments. The term "antibody" does not include only antibodies made by methods involving immunization, but also any polypeptide made to include at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on the antigen of interest, e.g., a recombinantly expressed polypeptide. Thus, the term applies to such molecules whether generated in vitro or in vivo.

[0041] The antibody may be of any of the IgA, IgD, IgE, IgG, and IgM classes, preferably an antibody of the IgG class. The antibody may be a polyclonal antibody, e.g., an antiserum or an immunoglobulin purified therefrom (e.g., affinity purified). The antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target specific antigens or specific epitopes within an antigen with higher selectivity and reproducibility. By way of example and not limitation, monoclonal antibodies can be produced by the hybridoma method first described by Kohler et al. 1975 (Nature 256:495) or by recombinant DNA methods (e.g., as in U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597).

[0042] The antibody binding agent may be an antibody fragment. An "antibody fragment" includes a portion of an intact antibody that includes the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab´, F(ab´)2, Fv, and scFv fragments, single domain (sd) Fv, e.g., VH domain, VL domain, and VHH domain; diabodies; linear antibodies; single-chain antibody molecules, particularly heavy chain antibodies; and multivalent and / or multispecific antibodies formed from antibody fragments, e.g., dibodies, tribodies, and multibodies. The designations such as Fab, Fab´, F(ab´)2, Fv, scFv, etc. are intended to have the meanings established in the art.

[0043] The term "antibody" includes antibodies having origin in any animal species, preferably vertebrate species (including, for example, birds and mammals), or antibodies containing one or more portions derived therefrom. Without limitation, the antibody may be a chicken, guinea fowl, goose, duck, guinea pig, quail or pheasant. Also without limitation, the antibody may be a human, mouse (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., dromedary and Bactrian camel), llama (e.g., alpaca, llama or vicuña) or horse.

[0044] One of ordinary skill in the art will understand that an antibody may contain one or more amino acid deletions, additions, and / or substitutions (e.g., conservative substitutions) so long as binding to its respective antigen is maintained. An antibody may also contain one or more native or artificial modifications (e.g., glycosylation, etc.) of its constituent amino acid residues.

[0045] Methods for producing polyclonal and monoclonal antibodies, as well as fragments thereof, are well known in the art, similar to methods for producing recombinant antibodies or fragments thereof (see, for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; “Monoclonal Antibodies: A Manual of Techniques”, by Zola, ed., CRC Press 1987, ISBN 0849364760; “Monoclonal Antibodies: A Practical Approach”, by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: “Antibody Engineering: Methods and Protocols”, Lo, ed., Humana Press 2004, ISBN 1588290921).

[0046] As used herein, the term "lipid" refers to a macromolecule soluble in nonpolar solvents. Lipids can be divided into eight categories: fatty acids; glycerolipids; glycerophospholipids; sphingolipids; glycolipids; polyketides; sterol lipids or sterols; and prenol lipids or prenols.

[0047] As used herein, the terms "gene editing system" or "genome editing system" refer to tools that induce one or more nucleic acid modifications, such as DNA or RNA modifications, to specific DNA or RNA sequences within a cell. Targeted genome modification is a powerful tool for genetic manipulation of cells and organisms, including mammals. Genome modification or gene editing, including DNA insertion, deletion, or substitution in the genome, can be carried out using various known gene editing systems. Gene editing systems typically utilize agents that can induce nucleic acid modifications. In certain embodiments, the agent capable of inducing nucleic acid modifications can be an (endo)nuclease or a variant thereof with altered or modified activity. (Endo)nucleases typically include a programmable sequence-specific DNA or RNA binding module linked to a non-specific DNA or RNA cleavage domain. In DNA, these nucleases create site-specific double-strand breaks at desired positions in the genome. The induced double-strand breaks are repaired by non-homologous end joining or homologous recombination, resulting in a target mutation. In certain embodiments, the (endo)nuclease may be RNA-guided. In certain embodiments, the (endo)nuclease can be engineered using artificial nucleases such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated (Cas) (endo)nucleases such as Cas9, Cpf1, or C2c2, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, or modified versions thereof. Methods of using TALEN technology, zinc finger technology, and CRISPR / Cas technology are known to those of skill in the art.

[0048] The term "cell" refers to all types of biological cells, including eukaryotic and prokaryotic cells. As used herein, the terms "cell" and "biological cell" are used interchangeably.

[0049] The terms "blood cell", "hematopoietic cell", "hemocyte", or "hematocyte" generally refer to cells produced by hematopoiesis and mainly present in the blood. The main types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes).

[0050] The term "stem cell" generally refers to a non-specialized or relatively less specialized cell with the ability to proliferate, which can self-replicate, that is, it can proliferate without differentiating, and a stem cell or its progeny can give rise to at least one relatively specialized cell type. This term encompasses stem cells capable of substantially unlimited self-replication, that is, stem cells whose progeny or at least a part thereof substantially retain the non-specialized or relatively low-specialized phenotype, differentiation ability, and proliferation ability of the mother stem cell, as well as stem cells showing limited self-replication, that is, stem cells in which the further proliferation ability and / or differentiation ability of the progeny or a part thereof is clearly reduced compared to the mother cell. By way of example and not limitation, stem cells may give rise to progeny that differentiate along one or more lineages to produce increasingly relatively specialized cells, and such progeny and / or increasingly relatively specialized cells may themselves be stem cells as defined herein or may give rise to post-mitotic terminally differentiated cells, that is, fully specialized cells.

[0051] As used throughout this specification, the term "isolated" when used in connection with a particular component generally indicates that such a component exists in a state separated from one or more other components of its natural environment; for example, that such a component is separated from one or more other components of its natural environment, or is prepared in a separated state, and / or is maintained in a separated state. More particularly, the term "isolated" as used herein in connection with a cell or tissue indicates that such a cell or tissue does not form, or no longer forms, part of a plant, animal or human body.

[0052] The term "transfection" refers to the process of introducing nucleic acid into animal cells.

[0053] The terms "light-responsive", "photosensitive", and "photosensitizing" may be used interchangeably and refer to the ability to respond to electromagnetic radiation such as visible light.

[0054] As used herein, the term "delivery yield" refers to the ratio of the amount of viable cells containing one or more (macro)molecules after performing the methods taught herein (e.g., the amount of viable cells containing one or more (macro)molecules detected after the delivery method) to the amount of viable cells before performing the methods taught herein (e.g., the amount of viable cells detected before the delivery method).

[0055] The viability (%) of cells after performing the methods taught herein can be determined by dividing the amount (such as number) of viable cells obtained after performing the methods taught herein by the amount (such as number) of (total) viable cells before performing the methods taught herein, and then multiplying the resulting value by 100.

[0056] The efficiency (%) of the method taught herein can be determined by dividing the amount (such as number) of viable cells containing one or more (macro)molecules obtained after performing the method taught herein by the amount (such as number) of total viable cells obtained after performing the method taught herein, and then multiplying the resulting value by 100.

[0057] As used herein, the term "particle" refers to a particle having a dimension (more particularly, the maximum dimension of the particle) of about 1 nm to about 2000 nm (2 μm), or a group, aggregate or cluster of two or more particles.

[0058] As used herein, the term "microparticle" refers to a particle having a dimension (more particularly, the maximum dimension of the particle) greater than 1000 nm (>1 μm) and at most 2000 nm (≦2 μm), or a group, aggregate or cluster of two or more particles.

[0059] The term "nanoparticle" refers to a particle having a dimension (the maximum dimension of the particle) of at least 1 nm (≧1 nm) and at most 1000 nm (≦1 μm), or a group, aggregate or cluster of two or more particles.

[0060] The dimension of a particle, such as the width, height or diameter of the particle, can be measured using a transmission electron microscope (TEM), a scanning electron microscope (SEM) or an atomic force microscope (AFM).

[0061] The term "chimeric antigen receptor" or "CAR" (also known as chimeric immune receptor, chimeric T cell receptor or artificial T cell receptor) refers to a receptor protein engineered to give T cells the new ability to target specific proteins. The receptor is chimeric because it combines both an antigen-binding function and a T cell activation function in one receptor.

[0062] The terms "suspension" and "cell suspension" generally refer to a heterogeneous mixture containing cells dispersed in a liquid phase. Since the mixture is generally liquid, the cells can in principle precipitate or sediment from the mixture, but this is not necessary.

[0063] Cells such as animal cells containing human cells may be "adhesive", i.e., they may require a surface for growth, and usually grow as an adherent monolayer on the surface rather than as freely floating cells (suspension culture) in a culture medium. Adhesion of cells to a surface such as the surface of a tissue culture plastic container can be easily examined by visual inspection under an inverted microscope. Cells grown in adherent culture require periodic subculture, during which the cells are enzymatically (e.g., using trypsin) removed from the surface, suspended in a growth medium, and seeded again into a new culture container. Generally, the surface or substrate that enables cell adhesion can be any substantially hydrophilic substrate. As is known in the art, tissue culture containers, such as culture flasks, well plates, or dishes, etc., can usually be made of a variety of polymer materials that are appropriately surface-treated or coated after molding to provide a hydrophilic substrate surface.

[0064] The phrase "generation of vapor bubbles" includes the expansion of vapor bubbles, the collapse of vapor bubbles, or a combination of the expansion and collapse of vapor bubbles, as well as secondary effects that can result from the expansion and collapse of bubbles, such as pressure waves and flow of the surrounding medium. The terms "vapor bubble" or "bubble" as used herein refer to vapor nanobubbles and vapor microbubbles. Preferably, the vapor bubble can have a diameter in the range of 10 nm to 100 μm. The vapor bubble may include a water vapor bubble.

[0065] The terms "subject", "individual" or "patient" can be used interchangeably herein and typically and preferably refer to a human, but can also include references to non-human animals, preferably warm-blooded animals, more preferably mammals such as non-human primates, rodents, dogs, cats, horses, sheep and pigs. The term "non-human animal" includes all vertebrates, such as mammals including non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as amphibians, reptiles, and chickens. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a human subject. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses, are intended to be included regardless of whether they are male or female. Examples of subjects include humans, dogs, cats, cows, goats and mice. The term subject is further intended to include transgenic species.

[0066] Suitable subjects include, but are not limited to, subjects presented to a physician for screening of a disease or condition, subjects presented to a physician with symptoms and signs indicative of a disease or condition, subjects diagnosed with a disease state, and subjects who have received an alternative (unsuccessful) treatment for a disease or condition.

[0067] The term "therapeutically effective amount" refers to the amount of an active compound, such as a T cell as taught herein, that when administered results in a positive therapeutic response with respect to the treatment of a patient having the disease or condition being treated.

[0068] The terms "pharmaceutical composition", "pharmaceutical formulation" or "pharmaceutical product" may be used interchangeably herein and refer to a mixture containing an active ingredient. The terms "composition" or "formulation" may likewise be used interchangeably herein.

[0069] A pharmaceutical composition as contemplated herein can be formulated for essentially any route of administration, such as, but not limited to, oral administration (e.g., oral ingestion, etc.) and parenteral administration (e.g., subcutaneous, intravenous or intramuscular injection or infusion, etc.).

[0070] The term "homeostasis" as contemplated herein refers to the steady internal, physical and chemical states maintained by a cell. This is the optimal functional state for the cell and includes that many variables are kept within certain preset limits. The variables include, but are not limited to, the pH of the extracellular fluid and the concentrations of sodium, potassium and calcium ions. In the specific embodiments described herein, the term "homeostasis" refers to the unchanged state of the following markers, provided that the levels are not affected by the cargo brought into the cell: the levels of inflammatory cytokines in the time frame from 0 hour to 24 hours after photoporation, the cytokines being selected from tumor necrosis factor (TNF), interferon γ (IFNγ), IL-5, IL-6, IL-9, IL-10, IL-13 and IL-17A. Other markers include CD137, CD154 and PD1, provided that the levels are not affected by the cargo brought into the cell in this regard either.

[0071] In a first aspect, the invention relates to T cells, wherein the homeostasis of the T cells within at least 24 hours after photoporation is not affected and is equivalent to the homeostasis before photoporation or is equivalent compared to non-photoporated T cells. In further embodiments, the homeostasis is not affected for a period of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours; 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 hours or less.

[0072] Photoporation has been confirmed to be a technique suitable for manipulating T cells while minimizing the impact on cell properties.

[0073] As a result of photoporation, pores are formed in the cell membrane, through which metabolites and ions move. This movement depends on the existence of a concentration gradient between the intracellular environment and the extracellular environment. Components present at a higher concentration inside the cell (e.g., cell culture medium) move towards the extracellular environment. Alternatively, compounds present at a high concentration in the extracellular environment flow into the cell. As a result, photoporated cells are different from cells that normally exist in nature.

[0074] In certain embodiments, the photoporation is caused by photo-responsive organic or inorganic nanoparticles. The term "nanoparticle" refers to particles having a spherical equivalent diameter in the range of 1 nm to 1000 nm. The particles may have any shape. For example, they may have a spherical, ellipsoidal, rod-shaped, pyramid-shaped, branched, or irregular shape. The particles may include individual particles, or combinations or clusters of two or more particles arranged in proximity to each other.

[0075] The dimensions of the particles, such as the width, height, or diameter of the particles, can be measured using a transmission electron microscope (TEM), a scanning electron microscope (SEM), or an atomic force microscope (AFM).

[0076] The size of the particles is preferably defined by the spherical equivalent diameter d (also referred to as the equivalent volume spherical diameter).

[0077] In one embodiment, the particles are embedded in a structure. The material of the structure in which particles capable of absorbing electromagnetic radiation are embedded includes, for example, an inorganic material or an inorganic-based material, such as silica or a silica-based material, or a ceramic or a ceramic-based material. In another embodiment, the material is an organic material or an organic-based material, such as carbon or a carbon-based material, or a polymer or a polymer-based material. The material of the structure may also include a composite material including at least one of the above-described materials, for example, a composite material including an organic material and an inorganic material. Preferred materials for the structure include, or are based on, polystyrene, polycaprolactone, ethyl cellulose, cellulose acetate phthalate, polylactic acid, poly(lactic-co-glycolic acid), cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginic acid, hyaluronic acid, dextran, starch, polycarbonate or polyacrylate.

[0078] In one embodiment, the structure includes a surface-modified material, such as a surface-modified polymer material. The surface modification includes, for example, the application of a coating (such as collagen) to promote cell adhesion to the material of the structure.

[0079] In one embodiment, the photo-responsive nanoparticles are embedded in a solid structure, such as a fiber or a combination of fibers.

[0080] In one embodiment, the solid structure includes a non-porous structure, such as a polymer sheet or a polymer foil. Particularly preferred embodiments include a polymer sheet including, or based on, polystyrene, polycaprolactone, ethyl cellulose, cellulose acetate phthalate, polylactic acid, poly(lactic-co-glycolic acid), cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginic acid, hyaluronic acid, dextran, starch, polycarbonate or polyacrylate. Iron oxide particles and / or carbon particles are embedded in the polymer sheet, for example.

[0081] In another embodiment, the solid structure includes a porous polymer structure such as a structure containing fibers (e.g., polymer fibers), a structure containing fine particles (e.g., polymer fine particles), a structure containing a combination of fibers and fine particles (e.g., a combination of polymer fibers and / or polymer fine particles), and a structure containing a foam (e.g., a polymer foam).

[0082] In a particularly preferred embodiment, the solid structure includes photothermal electrospun nanofibers.

[0083] As used herein, the term "electrospun nanofiber" refers to nanofibers produced according to the electrospinning manufacturing method. Electrospinning is a fiber manufacturing method that uses an electric force to stretch a charged thread of a polymer solution or polymer melt to a fiber diameter of about several hundred nanometers.

[0084] The structure may include a porous structure or a non-porous structure. The porous structure has a high free surface area and thus may be preferred because it has the advantage of having a large surface on which cells introduced onto or in the vicinity of the structure can be exposed. Preferably, the porous structure has a pore size that allows partial or complete penetration of the cells introduced onto or in the vicinity of the structure into the pores. Preferably, the porous structure has a pore size that does not limit the access of molecules present in the cell culture medium to the cells. The porosity of the structure is defined as the ratio of the volume of the pores or voids of the structure to the total volume occupied by the structure, i.e., the sum of the volume V of the structure (the volume of the material and the particles embedded in the material) and the volume of the pores or voids of the structure. The porosity can range from 0% to 100%. When the structure includes a porous structure, the porosity of the structure is preferably at least 50%, at least 60%, at least 80%, at least 90%, at least 95% or at least 99%.

[0085] The photosensitive inorganic particles may include metal particles, metal oxide particles, carbon or carbon-based particles, particles containing one or more light-absorbing compounds, or particles supported or functionalized with one or more light-absorbing compounds.

[0086] Examples of metal particles include gold particles, silver particles, platinum particles, palladium particles, copper particles, and alloys thereof. Preferred metal particles include gold particles, silver particles, and alloys thereof.

[0087] Examples of metal oxide particles include iron oxide, titanium oxide, zirconium oxide, cerium oxide, zinc oxide, and magnesium oxide.

[0088] Examples of carbon or carbon-based particles include graphene quantum dots, (reduced) graphene oxide, and carbon nanotubes.

[0089] Examples of particles containing one or more light-absorbing compounds, or particles supported or functionalized with one or more light-absorbing compounds include particles containing synthetic organic or inorganic absorbers, particles supported or functionalized therewith, and particles containing naturally occurring absorbers or derivatives thereof, particles supported or functionalized therewith. Specific examples include liposomes, solid lipid nanoparticles, polymer-based particles containing, supporting, or functionalized with light-absorbing dye molecules such as indocyanine green, inorganic quantum dots (having a low fluorescence quantum yield), naturally occurring light-absorbing agents such as dyes (such as melanin, rhodopsin, photopsin, or iodopsin), and synthetic analogs such as polydopamine, or photosensitizers used in photodynamic therapy.

[0090] The photosensitive organic particles may be photosensitive polymer-based particles. In a further embodiment, the photosensitive organic particles may be photosensitive polymer-based particles selected from polydopamine (PD) particles, poly(N-phenylglycine) (PNPG) particles, poly-2-phenyl-benzobisthiazole (PPBBT) particles, porphyrin particles, phthalocyanine particles or polypyrrole particles. In a further embodiment, the photosensitive organic particles may comprise or consist of polydopamine, poly(N-phenylglycine), poly-2-phenyl-benzobisthiazole, porphyrin, phthalocyanine or polypyrrole. In a more preferred embodiment, the photosensitive organic particles can be prepared (manufactured or synthesized) from clinically approved monomers such as dopamine hydrochloride, thereby facilitating the clinical transition to the production of therapeutic cell products, such as CAR-T cells, engineered by the methods taught herein.

[0091] In another embodiment, the photo-responsive organic particles may be polymer-based particles, protein-based particles, lipid-based particles (e.g., liposomes or solid lipid particles) containing photo-absorbing molecules, or combinations thereof. In a further embodiment, the photo-responsive organic particles may be polymer-based particles containing photo-absorbing molecules. In an embodiment, the photo-responsive organic particles may be protein-based particles containing photo-absorbing molecules. In a further embodiment, the photo-responsive organic particles may be lipid-based particles containing photo-absorbing molecules. In a more preferred embodiment, the photo-responsive organic particles may be solid lipid particles containing photo-absorbing molecules. In an embodiment, the photo-responsive organic particles may be a combination of two or more of polymer-based particles, protein-based particles, and lipid-based particles containing photo-absorbing molecules. Such photo-responsive organic particles can be prepared using clinically approved molecules, thereby facilitating the clinical translation of the delivery methods taught herein to the manufacture of engineered therapeutic cell products such as CAR-T cells. In a further embodiment, the photo-responsive organic particles may be polymer-based particles, protein-based particles, or lipid particles carrying or functionalized with photo-absorbing molecules. In an embodiment, the photo-responsive organic particles may be polydopamine particles, preferably polydopamine particles coated with albumin.

[0092] In one embodiment, the photo-responsive organic particles may be present as individual particles in an aqueous solution, such as in cell culture medium. In another embodiment, the photo-responsive organic particles may form a group, aggregate, or cluster of two or more particles in an aqueous solution, such as in cell culture medium. The particles, group of particles, aggregate, or cluster can have any shape. For example, the particles, group of particles, aggregate, or cluster may have a spherical, ellipsoidal, rod-shaped, pyramidal, branched, or irregular shape.

[0093] The term "based on" as used in the context of the materials of the organic particles defined above is understood primarily as particles that contain or are made from said materials. In other words, the protein-based particles should be understood primarily as particles that contain or consist entirely of one or more proteins or peptides. Lipid-based or "lipid particles", which may be used interchangeably herein, refer to particles that contain, consist essentially of, or consist of one or more lipids.

[0094] In a preferred embodiment, the photoporation is photothermal electrospun nanofiber (PEN) photoporation.

[0095] In one embodiment, the photoporation introduces one or more (macro)molecules. In a further embodiment, the (macro)molecule(s) would not otherwise be present in the native T cells prior to photoporation. In another embodiment, the amount of one or more (macro)molecules present in the T cells after photoporation is greater than before photoporation.

[0096] The one or more (macro)molecules are preferably selected from the group consisting of nucleic acids, proteins, peptides, chemicals, polysaccharides, or any combination thereof. In a more preferred embodiment, the combination of the one or more (macro)molecules may be a gene editing system, such as a CRISPR / Cas system. In one embodiment, the macromolecule may be a nucleic acid such as DNA or (m)RNA encoding a CAR. In one embodiment, the one or more (macro)molecules may be a negatively charged protein at physiological pH (e.g., a pH of about 6 to about 8). (IEP). In a more preferred embodiment, the one or more (macro)molecules may be neutral proteins at physiological pH (e.g., a pH of about 6 to about 8).

[0097] In certain embodiments, the molecular weight of the one or more polymers is at least 100 Da, such as from 0.1 to 5000 kDa. In certain embodiments, the molecular weight of the one or more (high) polymers is at most 1000 kDa, more preferably at most 500 kDa. In certain embodiments, the one or more (high) polymers can be nucleic acids such as (m)RNA or (plasmid)DNA having a size of at least 0.5 kilobases (kb). For example, the one or more (high) polymers can be nucleic acids such as (m)RNA or (plasmid)DNA having a size of at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, or more. For example, the one or more (high) polymers can be nucleic acids such as (m)RNA or (plasmid)DNA having a size of at least 3.0 kb, at least 4.0 kb, at least 5.0 kb, at least 6.0 kb, at least 7.0 kb, at least 8.0 kb, at least 9.0 kb, at least 10.0 kb, or more.

[0098] In certain embodiments, the one or more (high) polymers can be proteins, polysaccharides, or combinations thereof.

[0099] As detailed above, the cells used for photoporation can preferably be cultured or propagated in vitro. The cells may be isolated cells or tissues. The term "culture" or "cell culture" is common in the art and broadly means the maintenance of cells and potential cell expansion (proliferation, propagation) in vitro. Typically, animal cells, such as mammalian cells like human cells, are cultured by exposing (i.e., contacting) them to an appropriate cell culture medium under conditions known in the art that assist in in vitro cell culture in a suitable vessel or container (e.g., 96-, 24- or 6-well plates, T-25, T-75, T-150 or T-225 flasks, or a cell factory).

[0100] As described above, the homeostasis of T cells after photoporation changed little. This was further reflected by minimal or no changes in cell size, calcium level, proliferation, and marker profile. In certain embodiments, the cell size of the T cells within at least 24 hours or within 24 hours after photoporation differs by at most 3%, preferably at most 2%, and most preferably at most 1% compared to the cell size of the T cells before photoporation or compared to non-photoporated T cells. The cell size can be measured by conventional means in the art, such as microscopy. In certain embodiments, a confocal microscope is used, the cells are labeled (e.g., by calcein AM), and the cell size is then measured by confocal imaging using a 10x objective lens. Image processing can be used as the area of the cells in the image.

[0101] In certain embodiments, the calcium level in the T cells at intervals of 0 to 24 hours, or within at least 24 hours after photoporation, is at most 2%, preferably at most 1.5%, more preferably at most 1.25%, and most preferably at most 1% different compared to the calcium level of the T cells before photoporation or compared to non-photoporated T cells. The intracellular calcium level was measured using the Fluo-4Direct™ Calcium Assay Kit (#F10471, Invitrogen) according to the manufacturer's instructions.

[0102] In certain embodiments, a significant increase in inflammatory cytokines is not detected within at least 24 hours after photoporation, or in the time frame of 0 hours to 48 hours, more preferably 24 hours to 48 hours. In certain embodiments, the inflammatory cytokine is selected from the group of TNF, IFNγ, IL-5, IL-6, IL-9, IL-10, IL-13 or IL-17A.

[0103] In certain embodiments, the growth N / N0 of photoporated T cells in the time frame of 0 to 72 hours is similar to that of non-photoporated T cells. In another embodiment, the growth N / N0 of photoporated T cells at time intervals up to 72 hours after photoporation increases from at least 1 to at least 2, preferably from at least 1 to at least 3, more preferably from at least 1 to at least 4, and most preferably from at least 1 to at least 5. Cell growth includes an increase in the number of cells as a result of cell growth and cell division. As a result, cell growth N / N0 is a measure of high cell viability and unchanged cell homeostasis. The growth N / N0 of healthy cells includes exponential growth similar to the cell growth of non-photoporated T cells. Thereafter, the photoporated T cells according to the above-described embodiments can grow exponentially and exhibit unchanged cell homeostasis and high cell viability even after photoporation.

[0104] The marker profile of the photoporated cells hardly changes. In an embodiment, photoporation did not result in upregulation of CD137, PD1 and / or CD154 within at least 24 hours or 48 hours after photoporation as compared to the levels before photoporation. The markers CD137, PD1 and CD154 are activation markers. Upregulation of said markers after photoporation suggests an undesirable phenotypic change of T cells. Subsequently, the unchanged marker profile reconfirms that the homeostasis of the cells after photoporation has not changed.

[0105] In one embodiment, the T cells are CAR T cells. In a further embodiment of the invention, the CAR T cells after photoporation maintain a tumor cell lysis ability similar to that of their non-photoporated counterparts. In a further embodiment, the tumor cell lysis ability is similar when the ratio of effector to target is at least 5 / 1, preferably at least 4 / 1, more preferably at least 3 / 1, even more preferably at least 2 / 1, and most preferably at least 1 / 1.

[0106] As used herein, the term "similar tumor cell lysis ability" refers to a tumor cell lysis ability that is at least 75%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% similar when the effector / target ratio is at least 5 / 1, where the effector is a T cell and the target is a tumor cell.

[0107] In certain embodiments, the CAR T cells are engineered to target at least one selected from the following molecules: CD70, TNFRSF17, ILR3A, SDC1, EGFRvIII, MUC1, FAP, CD44, CD19, MS4A1, CD22, EPCAM, PDCD1, CA9, CD174, TNFRSF8, CD33, CD38, EPA2, CD274, FOLR1, SLAMF7, CD5, NCAM1, ERBB2, KDR, L1CAM, GD2, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, EGFR, MSLN, FOLH1, CD23, CD276, CSPG4, CD133, TEM1, GPNMB, PSCA.

[0108] Also disclosed herein are populations of T cells or pharmaceutical compositions comprising T cells as described herein. Preferably, the compositions or populations comprising the T cells disclosed herein comprise at least 10 3 cells, 10 6 cells, 10 9 cells or more (e.g., for each dose or administration, 5 million to 500 million, or 5 million to 250 million, or 50 million to 500 million, or 50 million to 250 million, or 100 million to 500 million, or 100 million to 250 million cells).

[0109] Such compositions or populations can also include other agents of biological origin (e.g., antibodies or growth factors) or chemical origin (e.g., drugs, cell preservation or labeling compounds) that can provide additional therapeutic, diagnostic, or other useful effects. This document further shows some examples of any additives, excipients, vehicles, and / or carriers suitable for cell-based pharmaceutical compositions that may further include specific buffers, growth factors, or adjuvants, and the amounts of each component of the composition are defined (in micrograms / milligrams, volume, or percentage), and the means of combining with hepatic progenitor cells are also defined.

[0110] In certain embodiments, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Thus, the pharmaceutically acceptable carriers, excipients, and / or diluents are selected such that the cells as described herein maintain a viable state and retain their properties. The carrier can be, for example, a pharmaceutically acceptable solvent or dispersion medium including water, saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0111] In the practice of the therapeutic methods or uses provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease, disorder, or condition to be treated. In some embodiments, the mammal is a human. In other embodiments, the mammal is non-human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent being used, and other factors. The therapeutic agents described herein, and optionally the compositions, can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0112] A problem related to the therapeutic use of cells as described herein is the amount of cells necessary to obtain an optimal effect. The dosage can be variable and may include an initial dose and subsequent doses, and can be determined by one of ordinary skill in the art given the present disclosure. Typically, the dosage administered provides a therapeutically effective amount of cells, i.e., one that achieves the desired local or systemic effect and performance. In addition, one of ordinary skill in the art can readily determine any additives, vehicles, and / or carriers in the pharmaceutical compositions of the invention to be administered to a subject.

[0113] In some embodiments, the pharmaceutical compositions described herein comprise at least one additional active agent described herein. In some embodiments, the at least one additional active agent is a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitor, antihormonal agent, antiangiogenic agent or checkpoint inhibitor. In some embodiments, the at least one additional active agent is an adjuvant for enhancing the effectiveness of vaccination.

[0114] The pharmaceutical composition or population must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for this purpose and known to those skilled in the art.

[0115] The pharmaceutical compositions or populations described herein can be administered to a subject by a suitable route of administration including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes. The compositions described herein can include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, immediate release formulations, controlled release formulations, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate.

[0116] In further embodiments, the compositions or populations described herein can be used either fresh or in a formulation suitable for long-term storage (e.g., cryopreserved cells) in a method of treatment for in vivo administration (in a human or animal model) or in vitro application. These pharmaceutical compositions can be provided in a form suitable for the desired method of treatment, the selected route of administration, and / or storage, and in a preferred means for providing such pharmaceutical compositions (e.g., within a kit). Other agents of biological origin (e.g., antibodies or growth factors) or chemical origin (e.g., drugs, preservatives or labeling compounds) that can provide any other useful effect can also be combined in such compositions.

[0117] In a final aspect, methods for therapy and therapeutic use are described herein based on the T cells, populations, and compositions as described above.

[0118] Cells such as T cells can be obtained (e.g., isolated, derived) from a biological sample, preferably a biological sample of a mammalian subject.

[0119] As used herein, the term "biological sample" or "sample" refers to a sample obtained from a biological source, such as an organism, animal or human subject, cell culture, tissue sample, etc. A biological sample of an animal or human subject refers to a sample removed from an animal or human subject and containing its cells. A biological sample of an animal or human subject may contain one or more tissue types or cells of one or more tissue types. Methods for obtaining a biological sample of an animal or human subject are well known in the art, such as tissue biopsy or blood collection.

[0120] In certain embodiments, the T cells, populations, or compositions can be used for the treatment of a wide range of diseases and conditions. In essence, any disease associated with the specific expression or overexpression of a particular antigen can be treated by targeting T cells to the antigen. For example, autoimmune diseases, infectious diseases, and cancers can be treated using the T cells, populations, and / or compositions of the present invention. These include cancers such as primary, metastatic, recurrent, treatment-sensitive, treatment-resistant cancers (e.g., chemotherapy-resistant cancers). Cancers can be cancers of the blood, lung, brain, colon, prostate, breast, liver, kidney, stomach, cervix, ovary, testis, pituitary, esophagus, spleen, skin, and bone (e.g., B cell lymphoma or melanoma). In the case of cancer treatment, T cells typically target cancer cell antigens, also known as tumor-associated antigens.

[0121] In certain embodiments, the T cells, populations or compositions are used to treat a subject having minimal residual disease, such as a cancer patient in a state of apparent remission. Using new sensitive diagnostic techniques, cancer-related antigens (or cancer cells) can be detected even in patients who do not exhibit overt cancer symptoms. Such patients can be treated by an on-demand method using antigen-targeted T cells to eliminate the residual disease. In preferred embodiments, CAR T cells are used. In further embodiments, the treatment further comprises the expression of a membrane-bound growth cytokine, since these cells retain the ability to proliferate in vivo despite low levels of the target antigen.

[0122] In certain embodiments, the T cells, populations or compositions can be used to treat a cell proliferative disease, fungal, viral, bacterial or parasitic infection. Targetable pathogens include, but are not limited to, protozoa, trypanosoma, aspergillus, candida, HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogen. Further examples of antigens that can be targeted by the T cells of the embodiments include, but are not limited to, CD19, CD20, cancer fetal antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-related antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, myosin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2.

[0123] In certain embodiments, the therapeutic use of T cells described herein involves stimulating a universal chimeric antigen receptor-mediated immune response in a mammal. Preferably, the present invention provides for the use of T cells as a therapeutic treatment, more preferably, the use of T cells as a treatment for cancer or autoimmune diseases. Autoimmune diseases result from an abnormal immune response of the body against substances and tissues that are normally present in the body (autoimmunity).

[0124] In certain embodiments, the therapeutic use involves T cells expanded in vitro to provide a sufficient population of T cell-derived effector cells attenuated for further in vivo expansion in a subject undergoing adoptive T cell therapy.

[0125] In a preferred embodiment of the present invention, the T cells of the T cell population or composition are allogeneic to the patient.

[0126] In a preferred embodiment of the present invention, the T cells of the T cell population or composition are autologous to the patient.

[0127] In a preferred embodiment of the present invention, the patient has a cell proliferative disorder.

[0128] In a more preferred embodiment of the present invention, the cell proliferative disorder is an autoimmune disease and the T cells target autoimmune cells.

[0129] In another preferred embodiment of the present invention, the cell proliferative disorder is cancer and the T cells target cancer cell antigens.

[0130] In a preferred embodiment of the present invention, the patient is human.

[0131] The present invention is further illustrated by the following non-limiting examples which are not intended to limit the scope of the invention and should not be construed as limiting.

Examples

[0132] For the purpose of better explaining the characteristics of the present invention, the following are examples and do not limit any other potential uses of the present invention in any way.

[0133] Example 1: Use of Photothermal Electrospun Nanofibers (PEN) for Safe and Efficient Delivery of (Macro)Molecules into Cells Method Preparation of Photothermal Nanofibers Polycaprolactone (PCL, molecular weight = ~70,000 g / mol), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and iron oxide (Fe3O4) nanopowder (#MKBW3262, Sigma-Aldrich, Belgium) were purchased from Sigma-Aldrich (Belgium). The iron oxide nanopowder was redispersed in a 2 mL 1:1 DMF / THF solution, and 480 mg of dry PCL was added thereto. This mixture was used for electrospinning the fibers collected on a microscope slide (#1000912, Marienfeld, Germany) attached to a grounded rotating collector (Figure 1). During electrospinning, unless otherwise specified, the applied voltage, flow rate, and electrospinning distance were fixed at 10 kV, 0.3 mL / h, and 20 cm, respectively. The rotation speed of the grounded rotating collector was set at 500 rpm. After 30 minutes (or after the time specified otherwise), the electrospinning process was stopped, the slide glass with the nanofiber web was detached from the rotating collector, and sterilized by UV irradiation for 45 minutes in a laminar flow cabinet.

[0134] Preparation of a Self-Made PEN Cell Culture Substrate An 8-well Secure-Seal™ double-sided adhesive spacer (#S24737, Invitrogen) was sterilized by UV irradiation for 45 minutes in a laminar flow cabinet. After peeling off the protective seal from one side of the adhesive spacer, it was gently applied onto the nanofiber web. Next, these samples were immersed in DI water for 3 minutes and the web (with the adhesive spacer on top) was easily removed from the slide glass. The web was manually cut into small pieces with one or four adhesive wells per piece (in which cells can be grown) and stored in PBS buffer.

[0135] Next, these PEN cell culture substrates were further modified with collagen for optimal cells. Poly(allylamine hydrochloride) (PAH, Mw = 17,560 g / mol, #MKBZ2824V, Sigma-Aldrich, Bornem, Belgium) and concentrated sulfuric acid solution (96%) were purchased from Sigma-Aldrich. Collagen I rat protein was purchased from ThermoFisher Scientific (#A1048301, Gibco™, Belgium). The 4-well PEN cell culture substrates were immersed in 32% sulfuric acid solution (3 mL per well of a 6-well plate) for 3 minutes. After washing with distilled water, they were immersed in an aqueous solution of the polyelectrolyte PAH (2 mg / mL, 0.5 M NaCl) for 15 minutes and rinsed three times with distilled water. The physical adsorption of PAH onto the nanofiber surface charged the fibers positively. Next, the PAH-coated fibers were immersed in an aqueous solution of 0.5 mg / mL collagen I rat tail protein for 15 minutes and rinsed with PBS solution. Hydrated fibers were formed by surface hydrolysis, and the PCL fibers were immersed in 0.1 M NaOH at 4 °C for over 1 hour and rinsed with PBS solution. Finally, the modified PEN substrates were stored in PBS until further use.

[0136] HeLa cells and Jurkat cells were cultured or harvested on PEN cell substrates for photoporation treatment HeLa cells (#CCL-2) and Jurkat clone E6.1 (#TIB-152) were obtained from ATCC and used as models for transfection of adherent and suspension cells by PEN photoporation. Human lung epithelial cells (H1299) stably expressing enhanced green fluorescent protein (GFP) were used for verification of siRNA knockdown experiments. The HeLa cell culture medium was prepared by adding 2 mM glutamine, 100 U / mL penicillin / streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) to DMEM / F-12. The H1299 and Jurkat cell culture media consisted of RPMI 1640 supplemented with 2 mM glutamine, 100 U / mL penicillin / streptomycin, and 10% FBS.

[0137] To grow adherent cells, PEN cell culture substrates were placed in 6-well titration plates (#10062-892, VWR), and HeLa or H1299 was added thereto (~1×10 6 cells, in 2 mL cell culture medium). The cells were allowed to adhere and grow for 24 hours in a cell incubator at 37 °C under a humidified atmosphere of 5% CO2. Immediately before the photoporation treatment, the target molecule to be delivered into the cells was added to the cell culture medium.

[0138] Jurkat cells were cultured at a cell density of 1×10 2 to 1×10 2 cells / mL in 75 cm 5 or 182.5 cm 6 flasks (#734-2313, #734-2315, VWR). For photoporation, the target molecule was added to the cell culture medium, and the cells were transferred to PEN cell substrates at ~2×10 5 cells / well. After allowing the cells to settle on the fiber web for 5 minutes, photoporation laser scanning was initiated.

[0139] Laser irradiation of cells on PEN substrates In photoporation, it is necessary to irradiate cells with laser light. Here, a previously reported custom optical setup was used with some modifications (Xiong, R. H. et al., ACS Nano, 8, 6288-6296 (2014); Xiong, R. H. et al., Nano Lett., 16, 5975-5986 (2016)). A pulsed laser with a pulse duration of 7 ns was tuned to a wavelength of 647 nm (Opolette™ HE355LD, OPOTEK Inc, California), applied, and the PEN substrate was irradiated. The collimated pulsed laser beam was irradiated through a 1° Light Shaping Diffuser (Physical Optics Corporation, Torrance, California), and the combination of an achromatic lens and a 20x objective lens (PlanFluor, Nikon) in front of the microscope entrance resulted in a laser beam diameter of ~250 μm at the sample. The energy of the laser pulse was monitored with an energy meter (J-25MB-HE&LE, Coherent) synchronized with the pulsed laser. To scan all cells on the PEN substrate (diameter ~9 mm), a motorized microscope stage was used to scan the sample line by line with a fixed laser beam. Since the laser repetition rate was 20 Hz, the scanning speed was set at 3 mm / s, and the distance between subsequent lines was 0.15 mm. In this way, all cells received at least one laser pulse, up to a maximum of four, in the overlapping region between adjacent irradiation zones. In some experiments using Jurkat cells or human T cells, the cells were scanned multiple times as shown in the text. In this case, the cells were resuspended in the PEN well and allowed to sediment again between each scan, randomly adhering the cells to the nanofibers at new positions.

[0140] IONP-sensitized photoporation of traditional cells. Polyethyleneimine (PEI)-functionalized iron oxide nanoparticles (IONP) were prepared by dispersing 100 mg of iron oxide powder (iron oxide Fe3O4 nanopowder, #MKBW3262, Sigma-Aldrich, Belgium) in 10 mL of a 10 wt% solution of branched PEI (bPEI, 25 kDa, Sigma-Aldrich), followed immediately by sonication for 1 minute with a tip sonicator (10%A, Branson Digital Sonifier, Danbury, United States of America). The mixture was further sonicated for an additional 1 hour with a bath sonicator (Branson2510 Branson Ultrasonics, Danbury, Connecticut, United States of America) and then stirred vigorously overnight to adsorb PEI molecules onto the IONP surface. Next, unbound bPEI was removed by performing several washing steps by centrifugation (4000Xg, 10 minutes) with HyClone water (VWR). Finally, PEI-coated IONP of the appropriate size was selected by fractionation centrifugation. Physicochemical characterization (i.e., hydrodynamic diameter, zeta potential, and particle concentration) was performed using dynamic light scattering (DLS, Zetasizer Nano-ZS, Malvern instruments Co., Ltd) (= hydrodynamic diameter and zeta potential) and / or nanoparticle tracking analysis (NTA, NanoSight LM10, Malvern Panalytical, United Kingdom) (= hydrodynamic diameter and particle concentration), respectively.

[0141] In IONP-sensitized photoporation, HeLa cells were cultured in a 96-well plate (#10062-900, VWR®, United States of America) at a density of 1×10 4 cells per well. Next, the cells were incubated with PEI-coated IONP at the indicated various concentrations for 30 minutes at 37 °C. Subsequently, the cells were photoporated at the indicated laser fluence in the presence of 2 mg / mL of FD10 dissolved in cell culture medium.

[0142] Detection of vapor nanobubbles The generation of vapor nanobubbles was detected by dark-field microscopy because they scatter light efficiently. VNBs generally have a very short lifespan (<1 μs), depending on their size. Therefore, an electron pulse generator (BNC575, Berkeley Nucleonics Corporation, California, USA) was used to synchronize a camera (EMCCD camera, Cascade II:512, Photometrics, Tucson, USA) with a pulsed laser. The pulsed laser sends a Q-switch signal to the pulse generator, which triggers the camera with a set delay.

[0143] Detection of Reactive Oxygen Species (ROS) The formation of ROS was evaluated using the probe 2´,7´-dichlorofluorescein (DCFH) as a fluorescence indicator. Briefly, DCFH was prepared by mixing 0.5 mL of 1 mM DCFH-DA (2´,7´-dichlorofluorescein diacetate, purchased from Sigma (#D6883)) in methanol and 2.0 mL of 0.01 N NaOH at room temperature for 30 minutes. The mixture was neutralized to pH 7.2 with 10 mL of 25 mM NaH2PO4. All reactions were carried out in a total volume of 1 mL of 40 mM Tris-HCl containing 25 μl of the DCFH solution and 10 μM Fe 2+ (from FeSO4).

[0144] To measure the amount of ROS generated by laser irradiation on the PEN substrate, 150 μL of DI water was added to the PEN well before starting the laser scanning procedure. After treatment, the DI water was recovered from the PEN well again and added to the DCFH solution. Positive control samples, including a negative control without laser treatment, were prepared from 150 μL of H2O2 added to the DCFH solution.

[0145] After further incubation at 37 °C for 2 hours, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a Victor3 microplate reader (#1420-040, PerkinElmer, Turku, Finland). The relative fluorescence intensity (FI) was calculated by Equation (1):

Equation

[0146] Electron microscope and confocal microscope. For TEM imaging, electrospinning was directly performed onto a Cu grid (200 mesh) with carbon coating for nanofibers. After laser irradiation of the nanofibers, visualization was carried out by operating a JEM1400plus transmission electron microscope (JEOL, Tokyo, Japan) at 20 - 60 kV. For SEM imaging, first, the samples were coated with 5 nm of platinum using a Quorum Q150T ES sputter coater. The images of the scanning electron microscope were taken with a Zeiss Crossbeam540 electron microscope using a SE2 detector at 20 kV.

[0147] For visualization by confocal microscopy, fluorescent PCL nanofibers were fabricated by electrospinning a PCL solution mixed with the fluorescent dye 3-(2-benzothiazolyl)-7-(diethylamino)coumarin (coumarin-6, #12779, Sigma-Aldrich). The fluorescent PCL nanofibers were imaged using a confocal laser scanning microscope (C1si, Nikon, Japan) and a 60x water lens (Plan Apo VC, Nikon). HeLa cells and H1299 cells grown on a PEN substrate were imaged with a C1si confocal equipped with a 10x lens (CFI Plan Apochromat, Nikon). For confocal imaging of Jurkat cells, the cell membrane was stained with 10 μg / mL of deep red fluorescent CellMask (#C10046, ThermoFisher Scientific). A series of z-stack confocal images were acquired in two channels (the green channel recording the nanofibers and the deep red channel recording the cells) using a 60x water lens.

[0148] Quantification of intracellular delivery by flow cytometry The photoporation efficiency was quantified by flow cytometry. For HeLa, as model compounds, FITC-dextrans of 10, 40, 70, 150, 500 kDa or Alexa Fluor® 647-labeled dextran of 10 kDa were used and added to the cells at final concentrations of 2 mg / mL or 0.5 mg / mL, respectively. Prior to 24-hour laser treatment, 1 million HeLa cells in 2 mL of cell culture medium were added to a 6-well plate containing four PEN substrate well dishes. After photoporation on the PEN substrate, HeLa or H1299 cells were detached by treatment with 0.25% trypsin-EDTA (Invitrogen, Belgium) and recovered by centrifugation at 300×g for 5 minutes. To recover Jurkat or human CD3+ T cells, the PEN substrate was simply washed once or twice with PBS.

[0149] Next, the recovered cells were resuspended in flow buffer (PBS supplemented with 5% FBS) and measured by flow cytometry (CytoFLEX Cytometer, Beckman Coulter, Belgium) until at least 10,000 events per sample were detected. Cells loaded with FITC-dextran or Alexa Fluor® 488-labeled siRNA were excited with a 488-nm laser, and fluorescence was recorded in the 525 / 40 channel. On the other hand, when cells were loaded with Alexa Fluor® 647-labeled dextran or labeled with a PD1 APC antibody (see below), the cells were excited with a 638-nm laser, and fluorescence was detected in the 660 / 10 channel.

[0150] The following antibodies were used for flow cytometry analysis of human CD3+ T cells: CD3 BV421 (Pacific Blue), CD4 BB700 (PERCP-Cy5.5), CD8 APC-Cy7 and PD1 APC (Invitrogen, Belgium). Briefly, T cells were washed with PBS (PBS, Gibco-invitrogen) and resuspended in FACS buffer supplemented with 5% bovine serum albumin, BSA (Sigma-Aldrich, Belgium, Bornem). After incubation with the indicated antibodies for 30 minutes at 4°C, the cells were washed and analyzed by flow cytometry. Pacific Blue and PERCP-Cy5.5 were excited with 405 nm and 488 nm lasers using 450 / 50 and 690 / 50 filters, respectively. APC-Cy7 and APC were excited with a 638 nm laser using 660 / 20 and 780 / 60 filters, respectively. Control samples were used to define the threshold of positive cell loading, which was defined as the 95% level of control cells.

[0151] Evaluation of cell viability Two methods were employed to evaluate cell viability. Calcein AM (#C3100MP, Invitrogen™) was used as a viability stain to visualize dead cells with a confocal microscope or to exclude them from flow cytometry analysis. Live cells become positive for calcein fluorescence, while dead cells do not. Prior to analysis, cells were incubated with calcein AM for 30 minutes at room temperature. To more accurately quantify cell viability, the CellTiter-Glo® Luminescent cell viability assay (#G7571, Promega, Belgium), which is based on the quantification of ATP, was used. After photoporation treatment, the cell culture medium was removed, and 100 μL of CellTiter-Glo reagent solution was added to each sample together with 100 μL of fresh cell culture medium. Samples were shaken on a shaker at 100 rpm for 10 minutes at room temperature. Finally, 100 μL of the solution was removed again from each sample and transferred to a 96-well plate (#655075, GreinerBio-one, Germany) and analyzed with a microplate reader (GloMax®, Progmega, Belgium).

[0152] Quantification of cell loading and viability by the imaging process After laser treatment, three to five confocal images were acquired with a confocal laser scanning microscope (C1si, Nikon, Japan) using a 10× lens (CFI Plan Apochromat, Nikon, The Netherlands, Badhoevedorp). Each image consists of green fluorescence (viability) and red fluorescence (loading efficiency) channels. A Matlab (The MathWorks, Inc., Natick, Massachusetts, USA) program was created for the automatic quantification of cell loading and cell viability. Untreated cells were used to define the threshold for positive cell loading, and the threshold was defined as the 95% level of untreated cells. Similarly, if the green fluorescence intensity is higher than the 95% level of dead cells, the cell is considered to be alive.

[0153] Quantitative iron assay by ICP-MS / MS The measurement of iron by inductively coupled plasma mass spectrometry (ICP-MS) is hampered by the occurrence of spectral interferences. Therefore, tandem ICP mass spectrometry (ICP-MS / MS) was used instead, and by relying on chemical separation using a reactive gas mixture of NH3 / He (1:9), interference-free conditions were obtained. Through method optimization, it was revealed that the mass shift approach of monitoring iron in the form of the reaction product ion Fe(NH3)2 + provides the best conditions. Using this method, the potential release of IONPs from the fibers was evaluated regardless of the presence of cells. In the case of no cells, pure water was added to the PEN substrate and recovered after laser treatment. Samples with cells were prepared as described above. After laser irradiation, cells were recovered by washing with PBS for suspended cells and by trypsin treatment for adherent cells, respectively. Finally, 100 μL of aqua regia (3:1 HCl / HNO3) was added to the samples to decompose the cells and other potentially present organic substances. The sample solution was diluted 100-fold with 2% HNO(3) to a final volume of 10 mL in a metal-free tube, and Y was added as an internal standard at a final concentration of 1 μg / L (1,000 mg / L Y standard stock solution, Inorganic Ventures, Christiansburg, Virginia, USA) to correct for instrument instability and / or signal drift. External calibration standards (0, 0.5, 1, 2.5, 5, 10 μg / L Fe + 1 μg / L Y) were prepared by appropriately diluting a 1,000 mg / L Fe standard stock solution (Inorganic Ventures, Christiansburg, Virginia, USA) with 2% HNO3 to mimic the matrix of the sample solution. In all steps of sample preparation, the solutions were thoroughly mixed using a vortex mixer. The tandem ICP-MS instrument (Agilent 8800 triple quadrupole ICP-MS, Agilent Technologies, Japan) was adjusted daily to achieve optimal conditions for highly sensitive measurements over the entire mass range, with low formation of oxide ions and interference-free measurement of Fe. The measurement of iron was 56 Fe(NH3)2 + performed by monitoring the signal intensity of 89 Y(NH3)6 +External calibration was performed based on internal normalization normalized using signal intensity. The methodological detection limit of 80 μg / L was determined by multiplying the device background equivalent concentration (BEC) by the dilution factor (100-fold).

[0154] Simulation of PEN photothermal reaction In order to better understand the photothermal response of PEN fibers to nanosecond pulsed laser irradiation, numerical simulations were performed. First, the laser-induced heating of IONP was calculated using the generalized multi-particle Mie theory (GMM). This accurately describes the interaction between spherical particles (aggregates) whose composition is determined by the real and imaginary parts of the dielectric constant and electromagnetic waves. In the GMM method, the scattered fields from individual spheres are solved in a reference system centered on each sphere. To solve multi-sphere scattering by the Mie-type multipole superposition approach, the incident plane wave is expanded in vector spherical wave functions in each sphere center coordinate system, and the total electromagnetic field incident on each sphere within the particle cluster is sought. Here, it consists of two parts: (1) the initial incident plane wave and (2) the scattered waves from all other spheres within the aggregate. In the next step, a single-field representation of the total scattered field from the entire aggregate is generated by expanding in vector spherical wave functions. Finally, based on the available total scattered field and the analytical expression of the amplitude scattering matrix of the sphere aggregate, it is possible to derive exact expressions for other basic scattering characteristics such as extinction, absorption, and scattering cross-sections. In all calculations presented in this study, the dielectric function of iron oxide (magnetite) represented by Querry was adopted. The calculations were performed for 160 nm particles in water (n = 1.33) or PCL (n = 1.46). Since the GMM code is limited to applications in homogeneous media, the effective medium approximation was used for calculations at the polymer-water interface. Here, considering that half of the IONP is exposed to the aqueous medium, the particles have an effective refractive index n effIt was considered to be immersed in a dielectric environment with a dielectric constant of 1.40. The calculation of the linear arrangement of IONPs was carried out with a particle - to - particle distance of 1 nm. The heat transfer from IONPs to the nanofiber PCL matrix and the surrounding medium was simulated using a commercially available CFD (Computation Fluid Dynamics) software package (ANSYS FLUENT) that can numerically solve the heat transfer equation. The simulation procedure is as follows. A three - dimensional shape model with a simulation domain of 6 μm×6 μm×36 μm was constructed, which included a cylindrical region representing the nanofiber (diameter = 0.32 μm, length = 30 μm) and a spherical region representing a single IONP (diameter = 0.16 μm). The simulation domain was discretized into a grid of a total of 2.85 million elements (the minimum mesh size was 30 nm). The boundary condition was an infinite boundary condition. The initial temperature of the IONP was set according to the Mie - theory calculation described above. The temperature of the IONP was maintained for 7 nanoseconds, which is the duration of the laser pulse used in this study. The specific heat and thermal conductivity of the PCL polymer were 1250 J / kg - K and 0.175 W / m - K, respectively. For the water surrounding the fiber, a specific heat of 4182 J / kg - K and a thermal conductivity of 0.6 W / m - K were used.

[0155] Calculation of the temperature rise of bulk water The total absorbed energy by the IONPs embedded in the fiber was simply calculated as follows:

Equation

Equation

[0156] siRNA Transfection for GFP Downregulation in H1299 For siRNA transfection of H1299 cells, 21-nucleotide siRNA duplexes targeting enhanced green fluorescent protein (siGFP) and a negative control duplex (siCTRL) were ordered from Eurogentec (Seraing, Belgium).

[0157] siGFP: Sense strand = 5´-CAAGCUGACCCUGAAGUUCtt-3´; Antisense strand = 5´-GAACUUCAGGGUCAGCUUGtt-3´.

[0158] siCTRL: Sense strand = 5´-UGCGCUACGAUCGACGAUGtt-3´; Antisense strand = 5´-CAUCGUCGAUCGUAGCGCAtt-3´.

[0159] To quantify intracellular delivery after PEN photoporation, the siCTRL duplex was labeled with Alexa Fluor® 488 (Eurogentec). Prior to 24 h of laser treatment, 1 million H1299 cells in 2 mL of cell culture medium were added to a 6-well plate containing four PEN substrate well dishes. The amount of siRNA was added to the cells at a final concentration of 1 μM unless otherwise indicated.

[0160] To calculate the siRNA gene silencing efficiency, the GFP knockdown efficiency was calculated according to Equation (4):

Equation

[0161] Intracellular Delivery of RNPCas9 for GFP Knockout in H1299 Cells The crRNA:tracrRNA duplex was prepared by mixing individual crRNAs with tracrRNA at a 1:1 molar ratio, heating at 95 °C for 5 minutes, and annealing at room temperature for 5 - 10 minutes. Next, the Cas9RNP complex was obtained by mixing any of the crRNA:tracrRNA duplexes with Cas9 endonuclease at a 2.5:1 molar ratio and assembling the complex at room temperature for at least 10 minutes before delivery. H1299 cells were seeded on a PEN cell culture substrate as described above before PEN photoporation. On the day of photoporation, Cas9RNP was prepared as described above. The RNP complex was diluted with Opti-MEM at the final concentration shown in the text, added to the cells, and then photoporation was performed by laser scanning. After laser treatment, the cells were washed once with DPBS-, supplied with fresh culture medium, and further incubated at 37 °C and 5% CO2, and then GFP knockout was analyzed by confocal microscopy or flow cytometry. The knockout efficiency of the RNP gene was calculated by Equation (5):

Number

[0162] PEN Photoporation and Electroporation of Human Embryonic Stem Cells The H9 human embryonic stem cell (hESC) line (WA09, WiCell, feeder-free cultures obtained from Prof. Verfaillie, KULeuven, Belgium) was used for all PEN and EP experiments. Cultures were performed feeder-free on Geltrex coating (#A1413302, Invitrogen) in Essential 8 medium (#A1517001, Invitrogen) supplemented with 1:100 penicillin / streptomycin (#15140-122, Invitrogen). Passage of hESCs was performed using TrypLE Select (#12563011, Invitrogen).

[0163] Prior to cell seeding, PEN cell culture substrates were coated overnight with 1:100 Geltrex on an orbital shaker. Next, 5×10 4 hESCs were seeded onto PEN cell culture substrates. After incubation at 37 °C for 1 h in a humidified atmosphere of 5% CO₂ and 5% O₂, 1 mL of E8 Essential medium supplemented with 1:100 RevitaCell (A2644501, Invitrogen) was added to the 12 wells. After 24 h, the medium was replaced with Essential 8 medium and refreshed daily until the cell density reached the required density in 3 - 4 days.

[0164] Prior to PEN photoporation, 0.5 mg / mL of dextran labeled with 10 kDa Alexa Fluor® 647 in cell medium was added to the cells. After laser scanning at the indicated laser fluence, the cells were cultured for an additional 2 h before recording confocal microscope images. Cell viability was measured by Cell Titer-Glo at the indicated times after treatment. Cell proliferation was quantified from confocal microscope images as described below.

[0165] Electroporation using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza, Cologne, Germany) equipped with Nucleofector™ 4D (Lonza, Cologne, Germany) was used to deliver dextran labeled with Alexa Fluor® 647 of 10 kDa according to the manufacturer's protocol. Briefly, 2×10 5 single hESCs were resuspended in Nucleofector™ solution supplemented with dextran labeled with Alexa Fluor® 647 at a final concentration of 0.5 mg / mL. This solution containing the cells was transferred to 20 μL Nucleofector™ strips and electroporated using the indicated program. For electroporation, hESCs were detached with TrypLE, transferred to an electroporation cuvette, and processed with the selected program (Costa, M. et al., Nat. Protoc., 2, 792-796 (2007);. Helledie, T., Nurcombe, V. & Cool, S. M., Stem Cells Dev., 17, 837-848 (2008)).

[0166] After electroporation, the cells were washed with cell culture medium, transferred to a 48-well plate and further incubated at 37 °C. Finally, the delivery efficiency was quantified from confocal microscopy images and the cell viability was measured at the indicated times after treatment with Cell Titer-Glo. Inductive differentiation into cardiomyocytes was performed using a PSC cardiomyocyte differentiation kit (#A2921201, Invitrogen) according to the manufacturer's protocol. The staining protocols for hESCs and cardiomyocytes were performed as follows. hESCs and cardiomyocytes were fixed with 4% paraformaldehyde for 20 minutes at room temperature. hESCs were permeabilized with 0.1% Triton X-100 diluted in phosphate-buffered saline (PBS) for 30 minutes. Subsequently, they were incubated with a blocking solution consisting of 5% goat serum (#16210-064, Invitrogen) in PBS for 30 minutes. The cells were incubated overnight at 4 °C with a primary antibody diluted in PBS containing 0.05% Tween 20 and 1% bovine serum albumin (BSA). The next day, the cells were incubated for 30 minutes at room temperature with a secondary antibody diluted in PBS containing 0.05% Tween 20 and 1% BSA, followed by incubation with a 0.1% Hoechst solution (#H3570, Invitrogen) for 10 minutes. Immunostaining of CMs was performed except that the primary antibody was incubated overnight at 4 °C.

[0167] The single-guide RNA targeting the IL-2R gamma gene (sequence: 5´-GGTAATGATGGCTTCAACA-3´) was purchased from Synthego. The Cas9 RNP complex was simply prepared by mixing any of the sgRNAs with Cas9 endonuclease at a molar ratio of 2.5:1 and assembling the complex at room temperature for at least 10 minutes before delivery. Genomic DNA extraction was performed using the innuPREP DNA Mini Kit (AnalytikJena, Jena, Germany) according to the manufacturer's protocol. Genomic DNA of H9 stem cells was extracted using the innuPREP DNA mini kit (AnalytikJena, Jena, Germany) according to the manufacturer's instructions. Next, using 100 ng of genomic template DNA and KAPA HiFi HotStart ReadyMix (Roche Diagnostics Belgium, Diegem, Belgium), the target DNA region of the IL-2R gamma gene was amplified using the forward primer 5´-ACCACCTTACAGCAGCACC-3´ and the reverse primer 5´-ATGATGGTCAGAAGGAGGAGG-3´. The PCR cycling conditions consisted of an initial denaturation at 98 °C for 2 minutes, followed by 35 cycles of denaturation at 98 °C (10 seconds), annealing at 65 °C (30 seconds), extension at 72 °C (21 seconds) and final extension at 72 °C (10 minutes). The amplified PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, California, USA) according to the manufacturer's protocol. The sequence of the PCR amplicon was finally determined by Sanger sequencing using the sequencing primer 5´-AGGACTTAGCCCGTGTC-3´ by the GATC Lightrun service (Eurofins Genomics, Ebersberg, Germany). The knockout level was determined by Inference of CRISPR Edits (ICE) analysis (Synthego), using the untreated sample as the unedited control and ensuring a model fit of R 2 >0.9 was sought.

[0168] PEN Photoporation and Electroporation of Human CD3+ T Cells Human T cells were obtained from the University Hospital of Ghent. Buffy coats were obtained from healthy donors after informed consent and approval. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Lymphoprep (Alere Technologies, Oslo, Norway). Next, PBMCs were incubated in IMDM (Gibco, Invitrogen, Belgium) supplemented with 10% fetal calf serum (FCS, Bovogen), 100 U / mL penicillin (Gibco, Invitrogen), 100 μg / mL streptomycin (Gibco, Invitrogen), 2 mM glutamine, 5 ng / mL IL-2 (Roche, Vilvoorde, Belgium) and stimulated with CD23 / CD28 Activator (Stemcell Technologies, Vancouver, Canada) at a bead-to-cell ratio of 1:1. After 7 days, the cells were harvested and re-incubated in complete IMDM supplemented with 1 μg / mL phytohemagglutinin (Remel Europe, Kent, UK) with irradiated (40 Gy) (SARRP) PBMCs (ratio 1:2) and irradiated (50 Gy) JY (ratio 5:1) feeder cells. After a further 14 days, CD3+ cells were harvested and used in the experiments shown further on. Feeder cells were irradiated using the Small Animal Radiation Research Platform (Xstrahl, Surrey, UK). For the photoporation procedure, T cells were transferred to the culture substrate at a density of ~8×10 5 cells / well. Before starting the laser treatment, the cells were allowed to sediment on the fiber web for 5 minutes.

[0169] CD70-specific CAR T cells were produced. Briefly, PBMCs were isolated with Lymphoprep and T cells were stimulated with Imunocult Human CD3 / CD28 / CD2 Activator in complete IMDM supplemented with 10 ng / mL of IL-12 (PeproTech, Hamburg, Germany). Cells were harvested 72 hours after stimulation and resuspended in the retroviral supernatant. Next, the cells were centrifuged at 1000×g (32 °C) for 90 minutes on a retronectin-coated plate (TaKaRa, Saint-Germain-en-Laye, France). Irradiated PBMCs (40 Gy) and irradiated JY cells (50 Gy) were used as allogeneic feeder cells for expanding the transduced cells in complete IMDM supplemented with 1 μg / mL of phytohemagglutinin (PHA, Sigma-Aldrich). IL-2 at 5 ng / mL was added on days 5 and 10, and the cells were restimulated every 7 - 14 days. For photoporation, CD3+ T cells or CAR T cells were transferred to the culture substrate in the presence of the transfection molecule at a density of ~1.0×10 6 cells / well. The cells were allowed to sediment on the fiber web for 5 minutes before starting the laser treatment.

[0170] FD10 kDa and siRNA were delivered to human T cells by electroporation using the P3 Primary Cell 4D-Nucleofector™ X kit (Lonza, Cologne, Germany) with a Nucleofector™ 4D (Lonza, Cologne, Germany) according to the manufacturer's protocol. Briefly, 1×10 6Individual CD3+ T cells or CAR T cells were resuspended in Nucleofector™ solution supplemented with FD10 at a final concentration of 2 mg / mL or siRNA at 1 μM. The cell-containing solution was transferred to 20 μL Nucleofector™ strips and electroporated using the EO-100, EO-115 or FI-115 program. After electroporation, the cells were washed with cell culture medium and transferred to a 96-well plate at 200K cells per well and further incubated at 37°C. For siRNA transfection, viable human T cells were stimulated 4 hours after treatment with Immunocult CD3 / CD28 activator and 5 ng / mL of IL-2. After incubation for 24 hours, 48 hours or 72 hours, the cells were washed with PBS and analyzed using flow cytometry or confocal microscopy.

[0171] siRNA Transfection and Analysis of PD1 Expression in Transfected T Cells For siRNA transfection of human T cells, siRNA duplexes targeting programmed cell death protein 1 (PD-1) and negative control duplexes (siCTRL) were obtained from various manufacturers (Figure 8). Human T cells were photoporated or electroporated as described above in the presence of the indicated concentrations of siRNA. After treatment, the cells were washed twice with PBS and transferred to a 96-well plate (#10062-900, VWR®, United States of America) at 2 × 10 5Resuspended in individual complete IMDM. After 4 hours, human T cells were stimulated with Immunocult Human CD3 / CD28 Activator (Stemcell Technologies, Vancouver, Canada) and 5 ng / mL of IL-2 to upregulate the expression of PD1 (e.g., unstimulated conditions) unless otherwise specified. The expression of PD1 was evaluated using flow cytometry at the indicated time points. Briefly, human T cells were washed with PBS and resuspended in FACS buffer. Next, the T cells were incubated with PD1PE (Milteny Biotec, Germany) at 4°C for 30 minutes, after which the cells were washed and incubated with TO-PRO™-3-iodide for 10 minutes. The data obtained by flow cytometry were post-processed with the FlowJo software package (Treestar Inc, Ashland, USA). TO-PRO™-3-iodide (APC channel) was used to exclude dead cells from further flow cytometry analysis. The knockdown efficiency of PD1 expression was calculated according to Equation (6): [Number] Here, MFI サンプル is the mean fluorescence intensity of cells treated with PD1 siRNA, and MFIunstimulated is the mean fluorescence intensity of unstimulated T cells under the same experimental conditions; MFI siCTRL is the mean fluorescence intensity of cells treated with negative control siRNA.

[0172] Characterization of T cell phenotype and intracellular Ca 2+ analysis The following antibodies were used for flow cytometry analysis of human CD3+ T cells: CD3BV421 (Pacificblue, Invitrogen, Belgium), CD4 BB700 (PERCP-Cy5.5, Invitrogen, Belgium), CD8 APC-Cy7 (Invitrogen, Belgium), CD137 PE (Biolegend, United States of America), CD154 FITC (Biolegend, United States of America), PD1 APC (Invitrogen, Belgium) and PD1 PE (MiltenyBiotec, Germany). Briefly, T cells were washed with PBS (PBS, Gibco-in vitrogen) and resuspended in FACS buffer supplemented with 5% bovine serum albumin (BSA, Sigma-Aldrich, Belgium, Bornem). After incubation with the indicated antibodies for 30 minutes at 4°C, the cells were washed and analyzed by flow cytometry. Unstimulated cells were used to set a 90% threshold for considering cells as positive%. Intracellular Ca 2+ was measured using the Fluo-4 Direct™ Calcium Assay Kit (#F10471, Invitrogen) according to the manufacturer's instructions.

[0173] Analysis of T cell proliferation using a confocal microscope After performing PEN photoporation or electroporation using the optimized delivery protocol (see text), T cells were washed twice and seeded at 2 × 10 per well in a 96-well plate 5Cells were seeded. After 4 hours, T cells were stimulated with 5 ng / mL of IL-2 and Immunocult human CD3 / CD28 activator in complete IMDM. At the indicated time points, T cells were washed and stained with Calcein AM and TO-PRO-3 iodide in cell medium for 30 minutes. Live cells were detected using an A1R confocal microscope (Nikon, Badhoevedorp, the Netherlands) equipped with a Perfect Focus System and a X20 objective lens (CFIPlanApochromat, Nikon, Badhoevedorp, the Netherlands), and quantified based on the fluorescence levels of green (Calcein AM positive, live cells) and red (TO-PRO-3 negative, dead cells). For image processing, the software package ImageJ with the Analyze Particles plugin was used.

[0174] Cytokine expression analysis of human T cells To analyze the cytokine secretion profiles of electroporated T cells or PEN-photoporated T cells, human T cells were seeded at 1 × 10 6 cells per well in a 96-well plate and seeded until 48 hours after treatment. At the indicated time points, supernatants were collected for cytokine secretion analysis. The secretion of 10 cytokines including IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IFN-γ, and TNF-α was quantified using a multiplex bead assay (LEGENDplex, Biolegend) according to the manufacturer's instructions.

[0175] 51 Chromium release cytotoxicity assay The cytotoxic killing of CAR-transduced T cells (not containing one or more (high) molecules not present in native T cells) that had received electroporation or PEN-photoporation was measured as described above 51 using a chromium release assay 50As target cancer cell lines, both SKOV3 cells and H1650 cells were used. CD70-specific CAR T cells were either photo-porated or electroporated as described above, or left untreated, and then cultured for 48 hours in complete IMDM supplemented with 5 ng / mL of IL-2. Target cells were 51 labeled with chromium (PerkinElmer, Belgium, Zaventem) at 37 °C for 90 minutes. After several washes, 10 3 target cells per well were added to a 96-well V-bottom plate (NUNC, Thermo Fisher Scientific, Belgium, Merelbeke). Various amounts of CAR T cells were added at the indicated effector-to-target cell ratios. Next, the supernatant was collected after 4 hours and measured using a 1450 LSC & Luminescence Counter (PerkinElmer, Belgium, Zaventem). Specific lysis was calculated using the following formula: (experimental release - negative control release) / (positive control release - negative control release) × 100%. Here, the negative control release is the release induced only by target cells in normal cell culture medium; the positive control release is the release obtained by completely lysing target cells by adding 2% Triton X-100 to the cell culture medium; and the experimental release is the release of the sample under the experimental conditions.

[0176] CAR-T Therapy Against SKOV3 Mouse Tumor Model Buffy coats from healthy donors were obtained from the Belgian Red Cross and used following informed consent according to the Helsinki Declaration and the guidelines of the Medical Ethics Committee of Ghent University Hospital. PBMCs were isolated by Lymphrop (StemCell Technologies) gradient centrifugation. The percentage of CD3+ cells was measured by flow cytometry, and T cells were stimulated with Immunocult Human CD3 / CD28 / CD2 T Cell Activator (StemCell Technologies) according to the manufacturer's instructions. Cells were harvested 48 h after stimulation, resuspended in retroviral supernatant, and centrifuged on RetroNectin (TaKaRa)-coated plates. Cells were harvested 2 days after transduction and cultured for 8 days in the presence of 10 ng ml−1 IL7 and IL15 (Miltenyi). CAR T cells were harvested on day 11 after stimulation, washed with sterile PBS, diluted in PBS, and injected intravenously into mice. The expressed CAR was composed of an anti-hCD70 VHH, a CD8α-based hinge, a co-stimulatory domain of 4-1BB (CD137), and a signaling domain CD3ζ derived from the T cell receptor.

[0177] NSG mice were subcutaneously injected with 2 × 10 6 SKOV3 cells. When the tumors reached a size of 4–7 mm in diameter, mice were intravenously injected with PBS or 5 × 10 6 non-transfected or transfected CAR T cells (either transfected or untreated). The next day, mice were intraperitoneally injected with PBS or 100 μg of nivolumab (Opdivo, Bristol Myers Squibb). Tumor size was measured with calipers.

[0178] Statistical analysis Differences between two datasets were evaluated using one-way analysis of variance, and multiple comparisons were adjusted by Bonferroni correction. Statistical significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0179] Results Synthesis and Characterization of Photothermal Electrospun Nanofibers (PEN) A mixture of polycaprolactone (PCL) and iron oxide nanoparticles (IONP) was dissolved in N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) solution at various weight ratios to prepare nanofibers. As shown in Figs. 1a and b, the fibers were collected on microscope slides. It was revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) that the average fiber diameter was ~300 nm regardless of the IONP concentration (Figs. 1b-d). Confocal microscopy showed that the thickness of the PEN web gradually increased to 4 μm after 1 hour of electrospinning (Figs. 1e, f). Since the web did not change much even after 30 minutes, this electrospinning time was selected for all fiber webs to be prepared hereafter. Increasing the amount of IONP added to the nanofibers did not significantly change the thickness of the PEN web (Fig. 1g).

[0180] JPEG2025521125000008.jpg130166

[0181] PEN Photoporation Enabling Safe and Efficient Intracellular Delivery to Adherent Cells Intracellular delivery by PEN photoporation was first tested in HeLa cells. As shown in Fig. 15, cell culture wells were prepared from PEN webs. The fibers were coated with collagen to promote cell adhesion (Fig. 16a), and the cell density and cell area increased significantly compared to cells grown on bare PCL fibers (Fig. 16b, c). Depending on the IONP content, the average number of IONP clusters per cell ranged from 0.26 (0.02%) to 159 (5.0%) (Fig. 16d). To investigate the success of intracellular delivery by PEN photoporation, red fluorescently labeled 10 kDa dextran (RD10) was added to cells cultured in PEN wells. After scanning once with a 7 ns pulsed laser beam, the cells were washed and Calcein AM viability stain was added to the cells. An example of a confocal image is shown in Fig. 16e, and it can be seen that the intracellular delivery of RD10 increases with increasing laser fluence. Quantification of the confocal images revealed that increasing the laser fluence or IONP content generally increased intracellular delivery, but also gradually increased cytotoxicity (Fig. 2a). Since optimal delivery efficiency can be obtained with the minimum laser energy, we chose to continue working with 1% IONP PEN webs and a laser fluence of 0.08 J / cm 2 2.

[0182] Conventional NP-sensitized photoporation uses gold NPs, which tend to fragment already after the first laser pulse, and as a result, the photothermal function is lost, so it can only be activated once. The same is true for photoporation using free IONPs. In this case, the optimal delivery efficiency is at a laser pulse fluence of 1.26 J / cm 2Obtained in (Fig. 17a). The free IONPs clearly lost their activity after the first laser pulse (Fig. 17b). At such a high laser fluence, vapor nanobubbles are formed (Fig. 17d), resulting in fragmentation of the particles (Fig. 17c). Considering that a much lower laser fluence is sufficient to obtain optimal delivery in the PEN web with IONPs embedded in PCL nanofibers, it is interesting to check whether the PEN web can be activated multiple times by repeating the scanning of the laser beam. We hypothesized that this might be possible because the sensitizing particles are stabilized by the surrounding polymer material and are irradiated with a laser fluence less than 1 / 10 of that commonly used in conventional photoporation. We first began by irradiating the cells on the PEN substrate twice in succession. For the first PEN photoporation, RD10 was irradiated as usual. Then, the cells were washed and the second irradiation was performed on the same PEN substrate, but this time in the presence of the 10 kDa green fluorescent FITC-dextran polymer (FD10). Subsequently, when the cells were observed with a confocal microscope, it was revealed that most cells emitted both red and green fluorescence (Fig. 18a). It can be seen that cells showing strong green fluorescence do not necessarily show strong red fluorescence, and vice versa. This is presumably because there was a time interval of about 10 minutes between the two experiments. This is the time required for the first photoporation treatment, subsequent cell washing, addition of the new dextran, and initiation of the second photoporation procedure. During that time, the cells may have moved slightly or changed the shape of their cell surface again, potentially coming into contact with the fibers at different locations, which could lead to some variation in the delivery efficiency between the two photoporation treatments. Nevertheless, quantitative analysis by flow cytometry confirmed that 90% of the cells were positive for both RD10 and FD10 (Fig. 18b). To further prove that photoporation was repeated on the same PEN web, HeLa cells were continuously photoporated up to 4 times with FD10.The FD10 concentration was doubled (from 0.2 mg / mL to 1.6 mg / mL) between each round of photoporation to more easily confirm the increase in intracellular delivery (which remains diffusion-driven). The percentage of positive cells increased from ~70% to ~90% (Figure 18c), but the increase in delivery was most evident from the relative mean fluorescence per cell (rMFI), which increased almost linearly with each round of photoporation (Figure 18d). Since the 5PEN web was found to be repeatedly activatable, we wanted to more accurately investigate how much the photoporation ability is lost each time the PEN web is scanned repeatedly. For this purpose, PEN photoporation was repeated again, but this time only FD10 was added to the cells immediately before the last scan. For example, in the case of N = 4 scans, the first 3 laser scans were performed with the cells in normal culture medium (without FD10), and FD10 was added to the cell medium before the 4th and last scan. Looking at the percentage of transfected cells, it was observed that the functionality of the PEN substrate decreased slightly when photoporation was performed more than 2 times. However, the data clearly showed that the PEN web was activated at least 6 times and still 60 - 70% of the cells were transfected (Figure 18e). Interestingly, repeated photoporation of the cells had little effect on cytotoxicity, and even after 6 rounds of PEN photoporation, 75% of the cells survived (Figure 18f). The slight decrease in photoporation efficiency with repeated activation is thought to be due to morphological changes occurring in the IONP (clusters) or the release of IONP (a part of it) from the fibers. Since the non-release of IONP is a basic premise of this study, this will be investigated separately and described later. At this point, it was already confirmed that IONP release did not occur, so here we focus on the potential morphological changes of the embedded IONP after (repeated) laser irradiation, investigated by SEM and TEM. As shown by the SEM and TEM images in Figure 19a, at the lowest fluence of 0.04 J / cm2, the IONP did not change even after 1 laser irradiation. At higher fluences, the IONP probably melted by reaching a high temperature and tended to form larger spherical structures.For example, the laser pulse fluence is 0.12 J / cm². 2 In the case of 2 , the temperature of the particles reaches ~1800 °C (see subsequent simulations), which already exceeds the melting temperature of iron oxide (1565 °C). A similar phenomenon was also observed when the PEN web was exposed to multiple laser scans (0.08 J / cm² 2 ), and it was found that gradually more IONP clusters were reformed into large spherical particles (Figure 19b). As a result, the photoporation efficiency decreases slightly when laser irradiation is repeated because the photothermal effective area decreases to some extent (Figure 19c). So far, the intracellular delivery of a 10 kDa model marker, which is of a similar size to, for example, antisense oligonucleotides or siRNA, has been evaluated. Furthermore, it is also interesting to evaluate to what extent larger macromolecules, close to the molecular weight of proteins or mRNA, can be delivered. For this purpose, FITC-dextrans (FD40, FD70, FD150, and FD500) with molecular weights of 40 kDa, 70 kDa, 150 kDa, and 500 kDa were used as model molecules and delivered to HeLa cells by 1×, 2×, 4× PEN photoporation. As shown in Figures 20a and b, the delivery efficiency gradually decreased as the molecular weight increased. This is due to the combination of the molecule becoming larger than the pore size and the slow diffusion of the molecule. When the photoporation procedure was repeated, generally the number of positive cells increased slightly, but the amount of delivery per cell was not improved. Nevertheless, it was concluded that PEN6 photoporation successfully transfected compounds up to at least 500 kDa into cells, and very high results were obtained with 65 - 90% of the cells being transfected, depending on the molecular size.

[0183] As detailed above, contrary to free IONPs (Figure 17), it was found that the PEN substrate can be repeatedly laser-activated and the delivery efficiency gradually improves (Figures 18, 19). This was found to be most useful for the delivery of large macromolecules (Figure 21).

[0184] Efficient Intracellular Delivery into Suspension Cells by PEN Photoporation Next, we tested whether compounds could be delivered into suspension cells by PEN photoporation. For this purpose, Jurkat cells, an immortalized line of human T lymphocytes that is widely used as a model for primary human T cells that are difficult to transfect, were used. Cells were added to PEN culture wells in the presence of 10 kDa FITC-dextran (FD10) and sedimented onto the fiber for 5 minutes (Figure 21a). Depending on the IONP content, the number of IONPs per cell was 7.7 - 28.4 IONP / cell (Figure 21b). In the first delivery experiment, it was shown that positively charged nanofibers gave better results than those coated with collagen (Figure 21c). From image analysis, the delivery efficiency increased with increasing laser fluence or IONP content, but the cell viability measured by calcein orange AM viability staining was sacrificed (Figure 2b). When setting a threshold of minimum viability of 80%, PEN substrates with 2% IONP (~12 IONP / cell, Figure 21) and a laser fluence of 0.16 J / cm 2 gave the best transfection efficiency (~75% positive cells). Finally, we repeated the experiment of repeating PEN photoporation (lower left panel of Figure 1b) and found that the proportion of positive cells could be increased by repeating the procedure with little effect on cell viability. In this experiment, note that a PEN substrate containing 2% IONP was used at a sub-optimal laser fluence of 0.08 J / cm 2 to better show the progressive improvement.

[0185] ICP-MS / MS was used to confirm that no IONP leaked from the PEN substrate upon laser irradiation An important premise of this study is to prevent the sensitizing NPs from coming into direct contact with cells during photoporation. To verify whether this condition is met, the iron concentration in cells was measured using ICP-MS / MS (tandem ICP mass spectrometry) after PEN photoporation. HeLa cells and Jurkat cells were photoporated using PEN substrates containing 1% or 2% IONP, respectively. Next, as schematically shown in Fig. 2c, the cells were detached from the nanofibers and decomposed with aqua regia (a 3:1 mixture of hydrochloric acid and nitric acid) prior to ICP-MS / MS analysis. As a positive control, cells incubated with 500 μg / mL of 30 nm IONP coated with polyethylene glycol at 37 °C for 4 h were included. As shown in Fig. 2d, the positive control actually had a significantly higher iron concentration in both cell types compared to the negative control (untreated cells). Importantly, however, the iron content in PEN photoporated cells showed no significant difference from that in untreated cells, regardless of the laser fluence or the number of laser scans. This demonstrates that there is no measurable increase in the intracellular iron content, although it can also be said that since the endogenous iron content in cells is already quite high, a slight increase may not be easily detected. Therefore, we decided to measure the potential release of iron from the PEN substrate when immersed in pure DI water (in the absence of cells, Fig. 2e). The iron content in DI after laser activation of the PEN substrate showed no significant increase, regardless of the IONP content, the number of scans, or the laser fluence, remaining below the detection limit of 0.08 mg / L (Fig. 2f). Instead, when the PEN fibers were intentionally decomposed with aqua regia to release the IONP, very high iron concentrations (1, 2, or 5% IONP) proportional to the embedded IONP content were actually measured. From the above, it can be concluded that IONP is not released from the PEN substrate by laser activation, and cells are not directly exposed to potentially toxic sensitizing NPs or their components. Nevertheless, detailed numerical simulations showed that due to the proximity of the IONP to the fiber surface, efficient heat transfer to specific locations on the cell membrane where pores are formed is possible, enabling efficient cell permeabilization (Figs. 22 - 26).

[0186] Since such good results were obtained with adherent and suspension cells, and it was confirmed that the delivery process occurs in a method that does not involve NPs, we continued to study in more detail the mechanism behind cell membrane permeabilization by PEN photoporation. In conventional NP-sensitized photoporation, membrane permeability can be induced by the photothermal effect (heat or mechanical energy) or a photochemical process 1. The photochemical process involves the generation of reactive oxygen species (ROS), which mainly occurs when femtosecond or picosecond ultra-fast pulsed lasers 2, 3, 4, 5 irradiate the sensitizing NPs. In this study, since a larger pulse width (7 ns) was used, it was considered that permeabilization was less likely to be caused by the photochemical process. Indeed, when 2´,7´-dichlorofluorescein (DCFH) was used as a fluorescent ROS indicator, no significant ROS was observed after laser irradiation of the PEN web up to an IONP content of 5% and a laser fluence of 0.16 J / cm 2 2. After laser irradiation of the PEN web up to a laser fluence of 0.16 J / cm 2 , no significant ROS was observed (Figure 22a). This means that the photothermal mechanism must be the cause of cell membrane permeabilization, and either mechanical damage due to the formation of vapor nanobubbles or thermal damage due to direct heating of the cell membrane is considered. We investigated the potential formation of VNBs using a dark-field microscope that can easily visualize VNBs, as we and other researchers have reported previously 6, 7. In fact, VNBs are visualized as short-lived local bursts of light in dark-field images. At a relatively low laser fluence of 0.14 J / cm 2 similar to the optimal conditions for Hela (0.08 J / cm 2 ) and Jurkat (0.16 J / cm 2) Only at [a certain condition], VNB began to appear as observed with free IONP (Figure 4d). By counting the number of VNBs in the laser irradiation area while increasing the laser fluence, the VNB threshold, defined as the laser pulse fluence at 90% of the plateau for generating VNB, can be determined. The VNB generation threshold was almost the same in PEN webs containing 0.02% and 2% IONP, and the value was 1.4 - 1.5 J / cm2 (Figure 22c). Since this is approximately 10 times higher than the highest laser fluence used in PEN photoporation, VNB formation can be safely excluded as the dominant permeabilization mechanism. From this, the pure heating mechanism is the most plausible mechanism remaining as the mechanism of membrane permeabilization. It should be noted that when a single laser pulse with a fluence of 0.16 J / cm2 is irradiated on a fiber containing 5% IONP, the bulk temperature only rises by 0.005 K, indicating that it is not simple bulk heating (see "Methods" for details of the calculation). Therefore, the most likely scenario is that the temperature rises rapidly and locally at the IONP (cluster) sites within the fiber, locally permeabilizing the cell membrane in contact with the hot spots of the fiber. Due to the extremely short time scale and small spatial scale, it is practically impossible to experimentally investigate this. Therefore, as schematically shown in Figure 23a, theoretical calculations and numerical simulations of heat transfer from IONP to the surroundings following laser pulse absorption were used to gain insights into this. The optical properties of IONP were calculated from generalized multi-particle Mie theory simulations, the absorption cross-section was calculated, and this was further used to calculate the initial IONP temperature. The 3D heat transfer differential equation was numerically solved to simulate heat transport from IONP to the fiber and the surrounding cell medium. Although potential phase changes during heat diffusion were ignored, this seems to be a reasonable assumption in the absence of VNB generation. The UV-VIS extinction spectrum calculated from Mie theory was in good agreement with the experimental spectrum of IONP dispersed in DI water (Figure 23b). Since it has already been shown that IONP tends to exist in clusters within the fiber, the absorption cross-section spectra of pairs of adjacent IONP were calculated.As can be seen in Fig. 24a, this does not significantly affect the value of the absorption cross-section at 647 nm, probably because the electromagnetic coupling between individual IONPs is weak. Also, the absorption cross-section spectra of IONPs in different media (PCL polymer, water, or the average of both) were calculated, and it was found that there was also no significant change in the value of the absorption cross-section at 647 nm (Fig. 24b). Therefore, using the theoretical absorption cross-section of a single IONP at 647 nm, the initial temperature (T0) when a single 7 ns laser pulse (λ = 647 nm) is absorbed was calculated for various laser fluences. As shown in Fig. 23c, the initial temperature easily exceeds the melting temperature of iron oxide, 1565 °C. For example, already at a laser fluence of 0.12 J / cm², a temperature of ~1600 °C is reached. Next, a 3D model was constructed to simulate the heat transfer of a single IONP of 160 nm surrounded by water in a surrounding environment composed of PCL fibers with a diameter of 320 nm (Fig. 24c). Simulations were performed for the absorption of a laser pulse of 0.08 J / cm² by the IONP located approximately h = 40 nm away from the fiber surface (see Fig. 1m). Since the duration of a single pulse is 7 ns, the simulation was started by setting the temperature of the IONP to 1069 °C during this duration. The subsequent heat transfer is shown in Fig. 23d, where temperatures >60 °C are colored red, which is reported to be the temperature at which the cell membrane becomes completely permeable8,9,10. As can be seen, a significant portion of the fiber surface reaches >60 °C, although only for a very short time interval of several tens of nanoseconds. The area of this surface was analyzed as a function of time for the upper side of the fiber. In Figs. 23e, f, the time evolution of the area A representing the size of the region >60 °C is plotted together with the average temperature T of these regions. The average temperature maintains >60 °C over a time of 137 ns, reaches a maximum of 110.1 °C after 27 ns, and the time-averaged value 2 of the laser fluence of 0.12 J / cm² reaches a temperature of ~1600 °C. Next, a 3D model was constructed to simulate the heat transfer of a single IONP of 160 nm surrounded by water in a surrounding environment composed of PCL fibers with a diameter of 320 nm (Fig. 24c). Simulations were performed for the absorption of a laser pulse of 0.08 J / cm² by the IONP located approximately h = 40 nm away from the fiber surface (see Fig. 1m). Since the duration of a single pulse is 7 ns, the simulation was started by setting the temperature of the IONP to 1069 °C during this duration. The subsequent heat transfer is shown in Fig. 23d, where temperatures >60 °C are colored red, which is reported to be the temperature at which the cell membrane becomes completely permeable8,9,10. As can be seen, a significant portion of the fiber surface reaches >60 °C, although only for a very short time interval of several tens of nanoseconds. The area of this surface was analyzed as a function of time for the upper side of the fiber. In Figs. 23e, f, the time evolution of the area A representing the size of the region >60 °C is plotted together with the average temperature T of these regions. The average temperature maintains >60 °C over a time of 137 ns, reaches a maximum of 110.1 °C after 27 ns, and the time-averaged value 2 is T = 85.4 °C. The area >60 °C is on average A = 0.087 μm², and the maximum size reaches 0.145 μm after 57 ns is T = 85.4 °C. The area >60 °C is on average A = 0.087 μm², and the maximum size reaches 0.145 μm after 57 ns 2 is T = 85.4 °C. The area >60 °C is on average A = 0.087 μm², and the maximum size reaches 0.145 μm after 57 ns 2We then investigated how T depends on the laser pulse fluence (I = 0.04-0.32 J / cm2), the number of locally clustered IONPs (N = 1, 2, 4, 8) and the distance h from the fiber surface (5, 40, 80 nm). Note that a maximum value of h = 80 nm was chosen because more than 90% of the IONPs reached h277°C, and additional calculations of IONPs at different depths below the fiber surface (h = 5, 10, 20, and 40 nm) were performed. The simulations were performed with a laser pulse of 0.08 J / cm2, which is the optimal setting for adherent and suspended cells, respectively. 2 or 0.16 J / cm 2 These calculations were further repeated for clusters of IONPs consisting of 1 to 8 individual nanoparticles (N = 1, 2, 4, 8). From these simulations, the ratios of fiber surface areas reaching temperatures above 277 °C (As) and above 60 °C (Ae) were calculated. The ratio As / Ae indicates the relative importance of membrane pore formation by steam nanobubbles (T > 277 °C) or direct heating (T > 60 °C). Figure 26 shows the relative importance of membrane pore formation by steam nanobubbles (T > 277 °C) or direct heating (T > 60 °C). 2 It is shown that for a laser fluence of 0.16 J / cm, the As / Ae is less than 5% in all cases, and even 0% for h ≥ 20 nm. 2 At laser fluences of 1000 nm, As / Ae is less than 10% in all cases. Taken together, these theoretical considerations indicate that, even if bubble formation cannot be completely excluded, it is highly unlikely to occur under the conditions used in our study, or at least does not contribute substantially to the almost purely thermally mediated cell permeabilization process. These calculations were further supported by dark-field microscopy experiments, where no vapor nanobubble generation was observed under similar conditions (Figure 22b,c).

[0187] Efficient gene silencing or knockout in adherent cells by PEN photoporation After successfully delivering the model polymer, to test the delivery of siRNA as a functional polymer, we began by delivering anti-GFP siRNA to adherent H1299 cells that stably express green fluorescent protein (GFP). As shown in Fig. 3a, after growing the cells on a PEN web (1% IONP) coated with collagen at 37 °C for 24 h, PEN photoporation (0.08 J / cm 2 ) was performed with the control and anti-GFP siRNA. By confocal microscopy, when 5 μM siRNA was used, it was confirmed qualitatively that siRNA knockdown was successful after 24 h (Fig. 3b), and quantitatively confirmed by flow cytometry (Fig. 3c). The knockdown efficiency increased with the siRNA concentration (0.5, 1, 2, 5 μM), but did not affect cell viability (measured here by the cell Titer-Glo luminescence assay) (Fig. 3d-f). When laser scanning was repeated while keeping the siRNA concentration fixed (0.5 μM), the knockdown efficiency also improved and reached a maximum of 70% after 4 laser scans. This is the same as one scan with 5 μM siRNA.

[0188] Next, we investigated the delivery of CRISP-Cas9 ribonucleoprotein (RNP). After PEN photoporation of H1299 cells with 0.5 - 4 μM RNP, the cells were grown for an additional 48 h before analysis. Examples of confocal images and flow cytometry histograms are shown in Figs. 3g, h respectively, confirming that GFP knockout was successful. The GFP knockout efficiency increased with the RNP concentration (Figs. 3i, j), and at the highest RNP concentration, the knockout efficiency reached as high as 80%. When PEN photoporation was repeated while keeping the RNP concentration fixed (0.5 μM) (N = 2, 3, 4), the knockout efficiency improved. Summing this up, it was found that PEN photoporation can deliver not only relatively small biological molecules such as siRNA, but also very large polymer complexes such as RNP.

[0189] Gene knockout via CIRSPR / Cas9 achieved in human embryonic stem cells without affecting cell function by PEN photoporation Next, human pluripotent stem cells related to stem cell therapy were focused on. Human embryonic stem cells (hESC) were cultured on PEN nanofibers (1% IONP) modified with Geltrex coating to promote adhesion and proliferation. After 3 - 4 days, hESC were subjected to PEN photoporation with RD10 (0.5 mg / mL) to examine the delivery efficiency. By quantifying confocal images, it was revealed that the delivery efficiency gradually increased, and along with it, the cell viability determined by live - dead staining here decreased. Calculating the delivery yield, which is the ratio of live cells and transfected cells compared to the initial cell number, a maximum delivery yield of 61% was obtained at I = 0.08 J / cm 2 and it increased to 71% when laser scanning was performed twice (N = 2) (Figure 4a). As a comparison, RD10 was delivered to hESC by electroporation, a non - viral transfection method commonly used for stem cells. In electroporation, only a delivery yield of 53% was obtained with the most functional electroporation program (CE - 118) (Figure 4b). Figure 4c shows an example of confocal images of control hESC and hESC subjected to PEN photoporation and electroporation under the most optimal conditions. The cell yield 24 hours after treatment was 63% for PEN photoporation and 25% for electroporation, and the difference was even more significant (Figure 4d). This decrease in the delivery yield in electroporation was due to the fact that the viability decreased from 72% 2 hours after treatment to only 34% 24 hours after treatment, indicating a long - term adverse effect on electroporated hESC. To further investigate this, the proliferation of electroporated hESC and PEN - photoporated hESC was compared. PEN - photoporated cells recovered immediately and proliferated exponentially like untreated cells. Instead, electroporated cells took 4 days to recover and resume exponential growth (Figure 4e).

[0190] Since it was confirmed that PEN photoporation does not seem to have a significant impact on the survival rate and proliferation ability of hESCs, next, we examined the pluripotency transcription factors Oct4 (Pou5f1), Sox2, and Nanog, which are important for maintaining the identity of pluripotent cells. Since we were interested in examining the effect of the permeabilization method itself, these experiments were conducted according to the optimized conditions, but in a state lacking one or more (high) molecular substances that do not exist in native T cells. Based on immunostaining and confocal images, no significant difference was observed in PEN photoporation cells compared to untreated hESCs (Figure 4f, g). Furthermore, based on the immunostaining of TNNT2 and NKX2.5, which are cardiomyocyte-specific markers, the differentiation ability of PEN photoporation hESCs into cardiomyocytes did not change compared to control cells (Figure 4h, i). This is expected to be beneficial for downstream applications such as differentiation into hESC-derived cardiomyocytes and subsequent transplantation.

[0191] Finally, PEN photoporation was applied to the intracellular delivery of CRISPR / Cas9 RNP in hESCs to knockout the IL-2Rγ (IL-2R) gene on the X chromosome involved in X-linked severe combined immunodeficiency. Sanger sequencing of PEN-photoporated hESCs with 2 μM RNP revealed that the knockout efficiency was >60%, indicating the success of CRISPR / Cas9-mediated gene knockout in human embryonic stem cells that are difficult to transfect (Figure 4j, k).

[0192] PEN photoporation that achieved efficient gene knockdown by siRNA delivery in human primary T cells PEN photoporation was applied to human donor-derived T cells (Figure 13). First, the PEN photoporation conditions were optimized by FD10 delivery. When using neutral PEN fibers, an IONP content of 5% was 0.16 J / cm 2It was demonstrated to be optimal at the laser fluence of (Figure 14). Using these optimized settings, a direct comparison was made between neutral nanofibers and hydrated nanofibers treated with sodium hydroxide to enhance hydrophilicity and cell adhesion. The hydrated nanofibers showed a viable transfected cell yield of 40.7% with three laser scans, resulting in the best outcome (Figure 5a). A comparison was made with electroporation, the most commonly used non-viral transfection tool for nucleic acid delivery to T cells. Based on the manufacturer's recommendations, several protocols were tried (EO-100, EO-115, FI-115). The electroporation protocol EO-100 yielded the highest FD10 delivery rate (19.3%) with a viability of 26.2% and a delivery efficiency of 76.0% (Figure 5b). Considering the manufacturer's claim that a T cell viability of >70% can be expected with the EO-100 program, such a low cell viability after electroporation may seem unexpected, but this is based on the cell viability measured by live-dead staining and quantification by flow cytometry, and it should be noted that this may lead to an overestimation of cell viability (Figure 7).

[0193] However, in flow cytometry analysis, there is a tendency to overestimate cell viability because fragmented cells that are "lost" against the background of debris are not considered. In fact, when measuring the viability of cells electroporated by flow cytometry after calcein AM staining, a seemingly high cell viability of up to 80% was observed (Figure 7a). However, when the same cells were measured by the Cell Titre Glo assay, which measures the remaining metabolic activity of the cells compared to the initial population, it was found that the viability was much lower, at most 20 - 30%. To confirm that this obvious discrepancy is due to the complete fragmentation of the cells, absolute cell numbers were measured by cell counting before and after electroporation of T cells labeled with calcium AM (green) and propidium iodide (red) (Figure 7b). Quantification of microscopic images showed that more than 30% of the cells were actually lost due to fragmentation after EP at both 1 hour and 24 hours after treatment (Figure 7c). The fact that the difference in viability between flow cytometry and the Cell Titre Glo assay is even greater indicates that many of the remaining "intact" cells are not actually very healthy and have reduced metabolic activity even after 24 hours of recovery.

[0194] Next, to suppress the expression of the PD1 receptor, optimized PEN photoporation and electroporation protocols were applied to siRNA delivery to human T cells. The expression of PD1 normally increases in stimulated T cells and is considered an important mediator of T cell immunosuppression in the tumor microenvironment. Human T cells were cultured for 7 days, transfected by PEN photoporation or electroporation according to pre-optimized conditions, and stimulated with CD3 / CD28 tetramer antibody complex and IL-2 to increase PD1 expression. When several siRNA constructs were tested (Figure 8), it was found that the D2siRNA construct functioned best (Figure 9) and was selected for further optimization of PD1 gene silencing. The expression of PD1 could be silenced by both photoporation and electroporation in human T cells. Silencing became more effective as the concentration of siPD1 increased, and reached ~80% knockdown with 4 μM siPD1 by both PEN photoporation and electroporation (Figure 5c, d). This also shows that PEN photoporation not only fails to obtain more viable cells and transfected cells, but also results in a similar level of down-regulation per cell as electroporation.

[0195] In contrast to electroporation, PEN photoporation does not change T cell homeostasis and functionality in vitro The optimal intracellular delivery technology must minimally inhibit the normal functions and homeostasis of cells, especially when applied to therapeutic cells. Therefore, the downstream effects of PEN photoporation and electroporation on the morphology, phenotype, and activation state of T cells were compared (Figure 5e-h). To examine the effects induced by the delivery technology itself, human donor-derived T cells were subjected to PEN photoporation and electroporation in the absence of one or more (macro) molecules that are not present in native T cells. First, it was noted that electroporated cells decreased in size 1 hour after treatment, while photoporated cells did not (Figure 5e). This morphological change after electroporation was accompanied by a strong and persistent increase in Ca 2+ levels up to 6 hours after treatment, but returned to baseline 24 hours later (Figure 5f). Instead, in PEN photoporation cells, the Ca 2+ levels remained constantly unchanged.

[0196] Next, the production of inflammatory cytokines (TNFα, IFNγ, IL-5, IL-6, IL-9, IL-10, IL-13, and IL-17A) was examined 24 and 48 hours after treatment. No significant increase in any cytokine was observed in response to PEN photoporation (Figure 5g). On the other hand, in electroporation, most inflammatory cytokines significantly increased 48 hours after treatment (compared to untreated T cells, TNFα: 7.2-fold increase, IFNγ: 7.4-fold increase, IL-6: 2.9-fold increase, IL-9: 6.3-fold increase, IL-13: 3.0-fold increase, and IL-17A: 4.7-fold increase). The degree of increase in several activation markers, including CD137 (4-1BB), CD154 (CD40L), and PD-1, was subsequently examined. All of these significantly increased in expression 24 and 48 hours after electroporation, while in PEN photoporation, only a slight increase in PD-1 was observed 48 hours after treatment (Figure 5h). Combining these results suggests phenotypic changes due to electroporation, which were not seen in PEN photoporation-treated T cells.

[0197] Next, the functionality of T cells after PEN photoporation and electroporation was verified in vitro. First, human T cells were subjected to PEN photoporation or electroporation (in a state without one or more (high) molecules that do not exist in native T cells), and then stimulated with CD3 / CD28 beads to examine T cell proliferation. After electroporation, the cell number decreased in the first 48 hours but began to grow again after 72 hours. This 2 - 3 day delay in growth after electroporation suggests an anergic state (Figure 5i). Interestingly, PEN photoporation completely maintained the proliferative capacity of human T cells without causing a significant delay in proliferation compared to untreated T cells.

[0198] Finally, the cytolytic ability of electroporated T cells and PEN - photoporated T cells pre - transfected with a tumor - targeting chimeric antibody receptor (CAR T cells) was compared. In the SKOV3 and H1650 cancer cell lines that are CD70 antigen - positive and express the PD1 ligand (PD - L1) at various levels, the tumor - killing ability of these CD70 - targeted CAR T cells was evaluated in vitro (Figure 10). PEN - photoporated cells showed efficient tumor cell killing, similar to untreated CAR T cells, especially when the effector - to - target ratio was high (Figure 5j). However, electroporation significantly reduced the cytolytic ability of CAR T cells. Taken together, these results confirm the presence of an anergic state in electroporated cells, which is the result of long - term adverse effects on T cell homeostasis, as previously reported. In clear contrast, PEN - photoporated T cells do not undergo changes in homeostasis and completely retain their cytolytic ability.

[0199] CAR - T cells transfected with siPD1 by PEN photoporation that exert therapeutic functions in vivo After confirming that PEN photoporation does not adversely affect the fitness of T cells or the cytolytic ability of CAR T cells, we finally evaluated its in vivo efficacy in SKOV3 tumor model mice (Figure 6a). We found that CAR T cells alone, CAR T cells PEN-photoporated with siPD1, and CAR T cells combined with PD1 antibody injection could suppress tumor growth over a one-month period (Figure 6b and Figure 11). Most importantly, we observed that CAR T cells treated with siPD1 were able to significantly reduce tumor volume even after 21 days, which was the same as the positive control using the PD-1 antibody (Figure 6b). In contrast, it took 25 days for CAR T cells alone to significantly suppress the tumor mass. These in vivo data support that PEN-photoporated T cells fully retain their therapeutic ability and that knockdown of the PD-1 receptor via siRNA is advantageous for the treatment of solid cancers.

[0200] Conclusion In the above example, the morphology, density, and distribution of IONPs embedded in electrospun nanofibers were characterized. Furthermore, it was shown that irradiating PEN with nanosecond laser pulses can safely and efficiently transfect various polymers into both adherent and suspended cells. Elemental analysis by inductively coupled plasma-tandem mass spectrometry (ICP-MS / MS) demonstrated that IONPs do not leak into the cell culture medium or cells after laser irradiation.

[0201] After demonstrating the potential of PEN photoporation for genetically manipulating difficult-to-transfect cells such as embryonic stem cells and human T cells, we transfected CAR-T cells with siPD1 using PEN photoporation to reduce the expression of the PD1 receptor and improve tumor killing ability in vivo. Additionally, PEN was shown to enable cell membrane permeabilization in various types of cells without contact with potentially toxic photothermal nanoparticles, thus opening the way for the use of photoporation in the safe and efficient generation of gene-modified cell therapies.

[0202] As a specific example, in the above example, T cells containing siRNA that does not exist in native T cells are described, and this siRNA is introduced into T cells by photoporation. Further, the homeostasis of the T cells within 24 hours after photoporation is not affected and is equivalent to the homeostasis before photoporation or is equivalent compared to non-photoporated T cells. Thereafter, the PEN photoporated T cells do not change in homeostasis and completely retain their cytolytic function. Further, this example has confirmed that the PEN photoporated T cells completely retain their therapeutic ability and that knockdown of the PD-1 receptor via siRNA provides a therapeutic advantage in the treatment of solid tumors. This example further shows the potential for clinical applications, such as the production of engineered cells for cell therapy including adoptive T cell therapy.

[0203] The present invention is not limited to the embodiments described above, and it is considered that several changes can be made to the presented examples without re-evaluating the appended claims. For example, although the present invention has been described with reference to the delivery of siRNA to T cells, it is clear that the present invention can also be applied to other macromolecules such as other types of nucleic acids, proteins, peptides, chemical substances, polysaccharides, and combinations thereof.

Claims

**Claim 1** A photo-porated T cell, wherein the homeostasis of the T cell is not affected within at least 24 hours after photo-poration and is equivalent to that before photo-poration or equivalent to that of a non-photo-porated T cell. **Claim 2** The T cell according to claim 1, wherein one or more polymers are introduced into the T cell by the photo-poration. **Claim 3** The T cell according to claim 1 or 2, wherein the cell size of the T cell within at least 24 hours after photo-poration is at most 3% different compared to the cell size of the T cell before photo-poration or compared to a non-photo-porated T cell. **Claim 4** The T cell according to any one of claims 1 to 3, wherein the calcium level of the T cell within at least 24 hours after photo-poration is at most 2% different compared to the calcium level of the T cell before photo-poration or compared to a non-photo-porated T cell. **Claim 5** The T cell according to any one of claims 1 to 4, wherein the photo-poration does not result in an increase in CD137, PD1 or CD154 within at least 24 hours after photo-poration compared to the level before photo-poration. **Claim 6** The proliferation of T cells N / N at time intervals from 0 to 72 hours after photoporation ０ The T cell according to any one of claims 1 to 5, wherein the increase is from at least 1 to at least 2. **Claim 7** The T cell according to any one of claims 1 to 6, wherein the T cell is a CAR T cell. **Claim 8** The T cell according to claim 7, wherein the CAR T cell after photo-poration maintains a tumor cell lysis ability similar to that of its non-photo-porated counterpart. **Claim 9** The T cell according to claim 8, wherein the tumor cell lysis ability is similar when the ratio of effector to target is at least 5 / 1. **Claim 10** The target of the CAR T cells is at least one of the following targets: CD70, TNFRSF17, ILR3A, SDC1, EGFRvIII, MUC1, FAP, CD44, CD19, MS4A1, CD22, EP-CAM, PDCD1, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD274, FOLR1, SLAMF7, CD5, NCAM1, ERBB2, KDR, L1CAM, GD2, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEA-CAM5, MET, EGFR, MSLN, FOLH1, CD23, CD276, CSPG4, CD133, TEM1, GPNMB, PSCA, and the T cells according to any one of claims 7 to 9.

11. The T cells according to any one of claims 1 to 10, wherein photoporation is performed by photo-responsive organic nanoparticles.

12. The T cells according to claim 11, wherein the photo-responsive organic nanoparticles are embedded in a solid structure such as a fiber or a combination of fibers.

13. The T cells according to any one of claims 1 to 12, wherein the one or more (high) molecules are selected from the group consisting of nucleic acids, proteins, peptides, chemical substances, polysaccharides, and combinations thereof.

14. A population of T cells according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising a therapeutically effective amount of the T cells according to any one of claims 1 to 13 and an excipient.

16. The T cells according to any one of claims 1 to 13, the population of T cells according to claim 14, or the pharmaceutical composition according to claim 16 for therapeutic use.

17. The T cells, the population of T cells, or the pharmaceutical composition for use according to claim 16, which is administered to a subject by intravenous, subcutaneous, or transdermal administration.

18. The T cells, the population of T cells, or the pharmaceutical composition for use according to any one of claims 16 to 17, which is administered to a patient.

19. The T cells, the population of T cells, or the pharmaceutical composition for use according to any one of claims 16 to 18, wherein the T cells of the T cells, the population, or the composition are allogeneic to the patient.

20. The T cells, the population of T cells, or the pharmaceutical composition for use according to any one of claims 16 to 18, wherein the T cells of the T cells, the population, or the composition are autologous to the patient.

21. T cells, a population of T cells, or a pharmaceutical composition for use according to any one of claims 18 to 20, wherein the patient has a cell proliferative disorder.

22. T cells, a population of T cells, or a pharmaceutical composition for use according to claim 21, wherein the cell proliferative disorder is an autoimmune disease and the T cells target autoimmune cells.

23. T cells, a population of T cells, or a pharmaceutical composition for use according to claim 21, wherein the cell proliferative disorder is cancer and the T cells target cancer cell antigens.

24. T cells, a population of T cells, or a pharmaceutical composition for use according to any one of claims 18 to 23, wherein the patient is human.

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