Method for detecting manipulated cells

Abstract

This specification provides methods for detecting the expression of bicistronically expressed polypeptides, for example, in a population of immune cells, such as a population of CAR T cells, that have been manipulated into chimeric cytokine receptors. In one aspect, this disclosure provides an in vitro method for detecting bicistronically expressed polypeptides in chimeric antigen receptor (CAR) T cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 509,070, filed June 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The electronic document, created on June 14, 2024, is named "AT-061 / 02WO_ST26.xml" and has a size of 533,947 bytes. [Background technology]

[0003] Adoptive transplantation of immune cells genetically modified to recognize malignancy-associated antigens has been shown to be a promising new approach to treating cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs). CARs are fusion proteins composed of an antigen-recognition region and a T-cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993)). Immune cells containing CARs, such as CAR-T cells (CAR-Ts), have been engineered to confer antigen specificity to them while retaining or enhancing their ability to recognize and kill target cells.

[0004] T cell proliferation, cytotoxic efficacy, and persistence are driven by signaling pathways. Conventional CAR designs provide two signals: activation of CD3ζ (signal 1) and co-stimulation (signal 2, e.g., via the expression of 4-1BB, OX40, and / or CD28). In some situations, a third signal (signal 3), cytokine-induced cytokine receptor signaling (e.g., cytokine adjuvant for immune enhancement), may be desirable. However, several methods for providing signal 3, such as systemic infusion of recombinant cytokines / cytokine mimes, face significant limitations, including systemic toxicity in humans. Alternative approaches include manipulating immune cells (such as CAR T cells) to express chimeric cytokine receptors (CCRs), as described in U.S. Patents 2019-0292533A1, 2020-0291090A1, 2020-0276238A1, and 2021-0061881A1, each of which is incorporated herein by reference in whole. Improvements in CAR-T cell therapy, such as increased efficacy and / or precision of treatment, through the use of additional non-CAR polypeptides such as CCRs, benefit patient populations.

[0005] What is needed is a method for detecting the expression of chimeric cytokine receptors manipulated in different bisistronally expressed polypeptides, for example, in immune cell populations, for example, in CAR T cell populations. This specification provides compositions and methods to address this need. [Overview of the project]

[0006] In one embodiment, the present disclosure provides an in vitro method for detecting bicistronically expressed polypeptides in chimeric antigen receptor (CAR) T cells. In one embodiment, the method comprises the step of contacting CAR T cells with an exogenous agent. In another embodiment, the CAR T cells include polynucleotides, linked peptides, and additional polypeptides expressing bicistronically expressed polypeptides. In some embodiments, the linked peptide is located at the C-terminus of the bicistronically expressed polypeptide. In another embodiment, the method further comprises the step of detecting the linked peptide after the contact step. In one other embodiment, the detection of the linked peptide indicates the presence of bicistronically expressed polypeptides in CAR T cells.

[0007] In other embodiments, the bisistronally expressed polypeptide includes a transmembrane domain or is a membrane protein including a transmembrane domain. In one embodiment, the bisistronally expressed polypeptide further includes an intracellular domain. In another embodiment, the linking peptide is at the C-terminus of the bisistronally expressed polypeptide or the linking peptide is at the C-terminus of the intracellular domain. In one embodiment, the bisistronally expressed polypeptide further includes an intracellular domain. In one embodiment, the linking peptide is heterogeneous to the bisistronally expressed polypeptide. In one other embodiment, the linking peptide is contiguous with the intracellular domain of the bisistronally expressed polypeptide. In another embodiment, the intracellular domain is an intracellular signaling domain. In some embodiments, the additional polypeptide is a CAR.

[0008] In one additional embodiment, the exogenous agent used in the contact step is selected from a small molecule reagent and a target molecule, and the CAR specifically binds to the target molecule. In another embodiment, the small molecule reagent stimulates cytokine production in CAR T cells. In a further embodiment, the small molecule reagent comprises phorbol myristate acetate (PMA) and / or ionomycin. In one embodiment, the target molecule is a soluble target molecule, or the target molecule is expressed on the surface of the target cell. In another embodiment, the contact step comprises contacting CAR T cells with the target cell. In another embodiment, the target cell is an inactivated target cell. In a further embodiment, the contact step comprises contacting CAR T cells with a soluble target molecule. In one embodiment, the linked peptide is cleavable. In one embodiment, the linked peptide is enzymatically cleavable. In another embodiment, the linked peptide is a self-cleaving peptide. In one embodiment, the self-cleaving peptide is a P2A or T2A peptide. In another embodiment, the linked peptide comprises the amino acid sequence shown in any one of SEQ ID NOs. 305-320. In further embodiments, the bicistronically expressed polypeptide is a chimeric cytokine receptor (CCR). In one embodiment, the CCR includes a transmembrane domain and an intracellular domain. In another embodiment, the CCR further includes an extracellular domain, or the CCR does not include an extracellular domain. In one embodiment, the CCR is an inducible CCR, or the CCR is a constitutively active CCR (CACCR). In one embodiment, the CCR is a CACCR that does not include an extracellular domain.

[0009] In further embodiments, the detection step includes detecting the linked peptide by flow cytometry and / or protein immunoblotting assay.

[0010] In another embodiment, the disclosure provides an in vitro method for analyzing a population of CAR T cells, the CAR T cells expressing a bicistronically expressed polypeptide, a linked peptide, and a CAR. In one embodiment, the method includes the step of contacting a sample of the population of CAR T cells with an exogenous agent. In another embodiment, the method further includes the step of detecting the bicistronically expressed polypeptide in the sample after the contact step. In one embodiment, the linked peptide is bound to the C-terminus of the bicistronically expressed polypeptide. In another embodiment, the step of detecting the bicistronically expressed polypeptide includes detecting the linked peptide in the sample. In another embodiment, the method further includes the step of analyzing whether the bicistronically expressed polypeptide is detected in the sample at or above a predetermined level. In one embodiment, the predetermined level is that the linked peptide is present in at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the CAR T cells in the sample. In another embodiment, the population of CAR T cells includes a CAR T cell drug substance or a CAR T cell preparation. In one embodiment, the method further includes the step of formulating a population of CAR T cells to form a formulation if a bicistronically expressed polypeptide is detected in the sample at or above a predetermined level. In another embodiment, the method includes the step of freezing the formulation. [Brief explanation of the drawing]

[0011] [Figure 1] Figures A-C show schematic diagrams of the manipulated constitutively active chimeric cytokine receptor (CACCR) (A) and the vector for CACCR expression (BC) of the present disclosure. [Figure 2] A and B show the staining of cells using anti-idiotype antibodies against anti-CD19 antibodies at 24 hours (A) and 48 hours (B) by flow cytometry. [Figure 3]A to B show staining of cells using anti-idiotype antibodies against anti-CD19 antibody and anti-P2A antibody, incubated or not incubated with CD19-Fc (A) or irradiated cells (B) by flow cytometry. [Figure 4] Show detection of P2A in engineered immune cells using Western blot. [Figure 5] Show the manufacturing workflow of engineered immune cells, such as CAR T cells. [Figure 6] A to B show CCR detection in P+I stimulated samples and non-stimulated samples.

Mode for Carrying Out the Invention

[0012] The present disclosure provides a method for the measurement and / or detection of polypeptides expressed bicistronically in engineered immune cells, such as chimeric antigen receptor (CAR) cells. In one embodiment, the method includes contacting the engineered immune cells with an exogenous agent. In another embodiment, the engineered immune cells include a polynucleotide expressing a polypeptide expressed bicistronically, a linker peptide, and an additional polypeptide. In a further embodiment, the linker peptide is at the C-terminus or N-terminus of the polypeptide expressed bicistronically. In other embodiments, the method further includes detecting the linker peptide after the contacting step, thereby indicating the presence of the polypeptide expressed bicistronically in CAR T cells.

[0013] To implement this disclosure, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, within the scope of the skills of those skilled in the art, shall be used. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Culture(RIFreshney,ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-1998)J.Wiley and Sons;Methods in Enzymology(Academic Press, Inc.);Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.This is well explained in literature such as: Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995). For example, gene editing techniques using TALEN, CRISPR / Cas9, and megaTAL nucleases are within the scope of this technology, as described by T. Gaj et al. This is adequately explained in literature such as al., Genome-Editing Technologies: Principles and Applications, Cold Spring Harb Perspect Biol 2016;8:a023754, and in the citations therein.

[0014] definition As used herein, the terms "a" and "an" are used to mean one or more. For example, a reference to "a cell" or "an antibody" means "one or more cells" or "one or more antibodies."

[0015] As used herein, “self” means cells, cell lines, or populations of cells obtained from a subject and used to treat the aforementioned subject.

[0016] As used herein, “allogeneic” means cells or populations of cells used to treat the aforementioned subject, obtained not from the subject but from a donor instead.

[0017] As used herein, “bisistrically expressed polypeptide” means a polypeptide encoded by a first nucleic acid sequence present in a polynucleotide or expression vector / cassette together with at least a second nucleic acid sequence encoding at least a second polypeptide, where the first and second nucleic acid sequences are present as continuous nucleic acid sequences in the polynucleotide or vector. For bisistronic expression, the polynucleotide or expression vector / cassette includes, for example, nucleic acid sequences encoding ribosome skipping sequences, such as sequences encoding 2A peptides, including P2A, T2A, E2A, and F2A peptides, for example. This class of peptides, identified in the aftvirus subgroup of picornaviruses, causes a ribosome “skip” from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon. “Codon” means three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into a single amino acid residue. Therefore, two polypeptides can be synthesized from a single consecutive open reading frame within mRNA, provided that the polypeptides are separated by a 2A oligopeptide sequence in the in-frame. Such ribosome skipping mechanisms are well known in the art and are used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0018] As used herein, the term “labeling agent” generally refers to a drug that can interact with cellular components, including, but not limited to, cell membranes, molecules on and / or inside cells, and intracellular molecules. Interactions between a drug and cellular components may be covalent or non-covalent, reversible, or irreversible. Labeling agents may be specific to cellular components, including, but not limited to, cellular biomolecules (e.g., polypeptides, nucleic acids, lipids, etc.). In some embodiments, a labeling agent may be a drug specific to a biological target, such as an antibody or antibody fragment. In one embodiment, a labeling agent is a drug specific to cell surface molecules, such as cell surface or cell membrane proteins. In some cases, a labeling agent may comprise one or more detectable labels. In some embodiments, a labeling agent optionally comprises an antibody conjugated with a detectable label.

[0019] In some embodiments, the detectable label is selected from the group consisting of fluorescent labels, photochromic compounds, protein-based fluorescent labels, colorimetric molecules, magnetic labels, radioactive labels, oligonucleotide labels, and haptens.In some embodiments, the fluorescent label is Atto dye, Alexafluor dye, quantum dot, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine Lysamin Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 Conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira The labeling agent is selected from the group consisting of Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRED monomer, DsRED2 ("RFP"), mStrawberry, TurboFP602, AsRED2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusia, P3, peridinine chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In some embodiments, one or more labeling agents are used for flow cytometry.One or more labels may be directly or indirectly bound to the labeling agent, or conjugated to the labeling agent. In the indirect form, one or more labels may be bound to or conjugated to a molecule that can bind to the labeling agent. For example, a label may be conjugated to an oligonucleotide sequence that is complementary to another oligonucleotide sequence than the oligonucleotide conjugated to the labeling agent (e.g., an antibody conjugated to an oligonucleotide). The labels may also be used in conjunction with the methods and compositions of this disclosure in connection with a binder such as a secretion molecule binder, e.g., a secretion cytokine binder.

[0020] As used herein, “immune cells” refers to hematopoietic cells functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells, e.g., α / βT cells and γ / δT cells, regulatory T (Treg) cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.

[0021] As used herein, the term “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0022] As used herein, “expression vector” refers to a vector comprising a recombinant polynucleotide containing an expression control sequence operably ligated to the nucleotide sequence to be expressed. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0023] As used herein, “expression cassette” refers to an expression unit (may be more than one) of coding sequences operably linked to a promoter. In some embodiments, the expression of multiple coding sequences may be driven by a single promoter, and the multiple coding sequences are linked by sequences encoding 2A peptides, such as P2A or T2A. In some embodiments, the expression of multiple coding sequences occurs by ribosome skipping. As an example, a bisistronic expression cassette allows the expression of two proteins from the same RNA transcript driven by a single promoter. In some embodiments, the expression of multiple coding sequences may be driven by multiple promoters, each promoter operably linked to a coding sequence.

[0024] As used herein, “functionally expressed” means that a gene is expressed and that expression results in a functional gene end product. For example, if a gene codes for a protein, and the expression of the gene ultimately produces a properly functioning protein, then the cell functionally expresses the gene. Conversely, if a gene is not transcribed, or if the expression of the gene ultimately produces untranslated RNA, or if the translation only results in a protein that does not function, then, for example, the protein is not properly folded or transported to its site of action (e.g., a membrane, a membrane-bound protein), and thus the gene is not functionally expressed. Functional expression can be measured directly (e.g., by assaying the gene product itself) or indirectly (e.g., by assaying the effect of the gene product).

[0025] As used herein, the term “extracellular ligand-binding domain” refers to an oligo or polypeptide capable of binding to a ligand. Preferably, this domain is capable of interacting with cell surface molecules. For example, an extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular pathological condition. The term “stalk domain” is used herein to refer to any oligo or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, stalk domains are used to provide further flexibility and accessibility to the extracellular ligand-binding domain.

[0026] The term "intracellular signaling domain" refers to a portion of proteins that transduce effector signaling functional signals, instructing them to perform functions specific to the cell.

[0027] As used herein, the term “exogenous” refers to any material introduced from or produced outside of an organism, cell, tissue, or system. In some embodiments, the exogenous or recombinant sequence or exogenous or recombinant protein is not a naturally occurring sequence or protein and is not endogenous or natural to the cell, tissue, or organism.

[0028] The term "intracellular signaling domain" refers to a portion of proteins that transduce effector signaling functional signals, instructing them to perform functions specific to the cell.

[0029] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” of an antibody refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody may be performed by fragments of an intact antibody. Examples of binding fragments that fall within the scope of the term “antigen-binding fragment” of an antibody include the Fd fragment consisting of the Fab, Fab', F(ab')2, VH, and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single-domain antibody (dAb) fragments (see, e.g., Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs).

[0030] Antibodies, antibody conjugates, or polypeptides that "specifically bind" to a target are well understood terms in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts to or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with higher affinity than it does with alternative cells or substances. An antibody "specifically binds" to a target if it binds with higher affinity, binding activity, more rapidly, and / or for a longer duration than it binds to other substances. Under this definition, for example, an antibody (or partial or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but may include) exclusive binding.

[0031] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either individually or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held together in very close proximity by FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site. Several methods exist for determining CDRs. For example, methods based on interspecies sequence changes (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)), methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948), and the Chothia system (i.e., Chothia and Lesk, J. Mol. Biol. (1987) 196(4):901-917). As used herein, CDR may refer to a CDR defined by either method or a combination of both methods.

[0032] The antibodies (and CARs) of this disclosure can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthesis techniques, or combinations of techniques readily known in the art or other techniques (see, for example, jayasna, SD, Clin. Chem., 45:1628-50, 1999 and Fellouse, FA, et al, J. Mol. Biol., 373(4):924-40, 2007).

[0033] As is known in the art, when used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be conferred before or after chain assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Other types of modifications include, for example, "caps" that substitute one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those by non-charged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), such as those involving suspension portions of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those by insertors (e.g., acridine, psoralen, etc.), those by chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, those by modified bonds (e.g., α-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides. Furthermore, any of the hydroxyl groups normally present in the sugar may be substituted with, for example, a phosphonic acid group or a phosphate group, protected with a standard protecting group, or activated to prepare additional binding to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides may also include analogous forms of ribose or deoxyribose sugars generally known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azid-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars, such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and debasalized nucleoside analogs, such as methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) (optionally including an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl. Not all links in the polynucleotide need to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0034] In some embodiments, the antigen-binding domain is a recombinant antigen receptor. As used herein, the term “recombinant antigen receptor” broadly refers to a surface receptor that does not exist in nature and includes an extracellular antigen-binding domain or extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well known in the art. A CAR is a fusion protein that includes an antigen-recognition moiety, a transmembrane domain, and a T-cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993)).

[0035] As used herein, the terms “extracellular ligand-binding domain” or “extracellular antigen-binding domain” refer to polypeptides capable of binding to ligands or antigens, or polypeptides capable of interacting with cell surface molecules such as ligands or surface antigens. For example, an extracellular ligand-binding domain or antigen-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a specific pathological condition, e.g., a tumor-specific antigen. In some embodiments, the antigen-binding domain includes an antibody, or an antigen-binding fragment or antigen-binding moiety of an antibody. In some embodiments, the antigen-binding domain includes Fv or scFv, Fab or scFab, F(ab')2 or scF(ab')2, Fd, a monobody, an aphibody, a camelid antibody, a VHH antibody, a single-domain antibody, or darpin. In some embodiments, the ligand-binding domain includes a binding partner, e.g., a ligand that binds to a surface receptor, or the external domain of a surface receptor that binds to a ligand.

[0036] The term "intracellular signaling domain" refers to a portion of proteins that transduce effector signaling functional signals, instructing them to perform functions specific to the cell.

[0037] References to values ​​or parameters "about" in this specification include (and are described) embodiments that cover ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, or ±10% of the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X". Numerical ranges shall include the number that defines the range.

[0038] Wherever embodiments are described herein using the phrase “comprising,” it should be understood that similar embodiments are also provided, which are described using the terms “consisting of” and / or “consisting essentially of.”

[0039] An "antigen-binding protein" comprises one or more antigen-binding domains. As used herein, "antigen-binding domain" means any polypeptide that binds to a specific target antigen. In some embodiments, the antigen-binding domain binds to an antigen on tumor cells. In some embodiments, the antigen-binding domain binds to an antigen on cells involved in hyperproliferative disease, or to a viral or bacterial antigen.

[0040] Antigen-binding domains include, but are not limited to, antibody-binding regions, which are immunologically functional fragments. The term “immunologically functional fragment” (or “fragment”) of an antigen-binding domain is a type of antigen-binding domain that contains an antibody portion (regardless of how that portion is obtained or synthesized) and lacks at least some of the amino acids present in the full-length chain, but still possesses the ability to specifically bind to a target antigen. Such fragments are biologically active in that they bind to a target antigen and may compete with other antigen-binding domains, including intact antibodies, for binding to a given epitope.

[0041] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity-determining regions ("CDRs"), diabodies (where a heavy chain variable region on the same polypeptide as a light chain variable region is connected via short peptide linkers that are too short to allow pairing between two domains on the same chain), domain antibodies, bivalent antigen-binding domains (containing two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments may originate from any mammalian source, including, but are not limited to, humans, mice, rats, camelids, or rabbits. As those skilled in the art will understand, antigen-binding domains may contain non-protein components.

[0042] The variable regions typically exhibit the same general structure as the relatively conserved framework regions (FRs) linked by three hypervariable regions (CDRs). The CDRs from the two chains of each pair are usually aligned by the framework regions, potentially enabling binding to specific epitopes. From the N-terminus to the C-terminus, both the light-chain and heavy-chain variable regions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions of the heavy chain are usually called HC CDR1, CDR2, and CDR3. The CDR regions of the light chain are usually called LC CDR1, CDR2, and CDR3.

[0043] In some embodiments, the antigen-binding domain comprises one or more complementary binding regions (CDRs) present in the full-length light or heavy chain of the antibody, and in some embodiments, comprises a single heavy and / or light chain or a portion thereof. These fragments can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of the antigen-binding domain containing an intact antibody.

[0044] In some embodiments, the antigen-binding domain is an antibody or fragment thereof, comprising one or more of its complementarity-determining regions (CDRs). In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chain CDRs: CDR1, CDR2, and CDR3, and heavy chain CDRs: CDR1, CDR2, and CDR3.

[0045] The assignment of amino acids to the framework, CDR, and variable domains is typically done using Kabat numbering (see, e.g., Kabat et al. in Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lek, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82, and U.S. Patent No. 7,709,226), contact numbering schemes, AbM schemes (Antibody Modeling Program, Oxford). Follow the Molecular (Molecular) or AHo system (Honneger and Pluckthun, J Mol Biol (2001) 309(3):657-70).

[0046] In some embodiments, the antigen-binding domain is a recombinant antigen receptor. As used herein, the term “recombinant antigen receptor” broadly refers to a surface receptor that does not exist in nature and includes an extracellular antigen-binding domain or extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well known in the art. A CAR is a fusion protein that includes an antigen-recognition moiety, a transmembrane domain, and a T-cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993)).

[0047] Where any aspect or embodiment of this disclosure describes a group of Markush members or other alternative groups, this disclosure includes not only the entire group described as a whole, but also each individual member of the group, as well as any possible subgroups of the main group, and the main group with one or more members excluded. This disclosure also assumes the express exclusion of one or more members of any of the groups in the disclosed and / or claimed embodiments.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, this specification, including its definitions, shall prevail. Throughout this specification and the claims, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to mean the inclusion of the integer or group of integers described, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include the plural form, and plural terms shall include the singular form.

[0049] Similar or equivalent methods and materials described herein may also be used in the practice or testing of this disclosure, but appropriate methods and materials are described herein. Materials, methods, and examples are illustrative and not intended to be limiting.

[0050] Analysis of cell populations In one embodiment, the disclosure provides methods, assays, systems, compositions, and kits for detecting (and / or quantifying or counting) one or more engineered immune cells expressing a bicistronically expressed polypeptide from a population of engineered immune cells, such as CAR T cells. In one embodiment, one or more engineered immune cells include a polynucleotide sequence (or a vector containing the same) encoding the bicistronically expressed polypeptide. In another embodiment, the polynucleotide sequence (or a vector containing the same) further encodes a linked peptide and additional polypeptides. Generally, the bicistronically expressed polypeptide, linked peptide, and additional polypeptides (such as CAR polypeptides) are expressed in engineered immune cells and can be detected according to the methods described herein. In one embodiment, the methods described herein include the step of contacting engineered immune cells, e.g., CAR T cells, with an exogenous agent. In another embodiment, the method further includes detecting the linked peptide after the contact step. In another embodiment, the detection of the linked peptide indicates the presence of the bicistronically expressed polypeptide in the engineered immune cells.

[0051] The manipulated cells may be manipulated immune cells, such as CAR-T cells. Detection of bicistronally expressed polypeptides can provide important information about the manipulated immune cell population before or after formulation as a CAR T cell preparation, and / or before administration to a subject as part of a CAR T cell preparation. Furthermore, detection of bicistronally expressed polypeptides in manipulated immune cells can provide useful information about the cells during and / or after the manufacturing process. For example, the disclosed method enables the detection of bicistronally expressed polypeptides during and / or after the manufacturing process.

[0052] In additional embodiments, this disclosure relates to the analysis of different engineered immune cell populations. In one embodiment, the analysis includes a screening method for detecting and / or quantifying the number of engineered immune cells from a population of engineered immune cells that express a bisistronally expressed polypeptide. As discussed herein, a method for understanding the degree of bisistronally expressed polypeptide expression in an engineered immune cell population is valuable during the engineered cell manufacturing process. It is desirable not only to clarify whether a bisistronally expressed polypeptide is present in one or more cells of an engineered immune cell population, but it may also be important to quantify the frequency of bisistronally expressed polypeptide expression in the population with high precision and accuracy. The screening method of interest may include, but is not limited to, screening of engineered immune cells during or after the manufacturing process. In one embodiment, the screening includes the step of contacting an engineered immune cell population with an exogenous agent, followed by the step of detecting the bisistronally expressed polypeptide.

[0053] In one embodiment, the screening method is a quantitative and / or qualitative method. The screening methods described herein may be carried out using different methodologies, including, but are not limited to, flow cytometry, fluorescence-activated cell sorting (FACS) flow cytometry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), immunoblotting assay, immunofluorescence assay, immunochemistry (IHC) assay, Western blot analysis, and immunoprecipitation and molecular binding assays.

[0054] Detection using flow cytometry. Instruments for particle analysis, such as flow cytometers and scanning cytometers, allow individual particles or cells (e.g., a single engineered immune cell from an engineered immune cell population) to be characterized using optical parameters such as light scattering and fluorescence. For example, a flow cytometer allows an aqueous suspension containing individual particles (e.g., beads containing the analyte of interest) or cells to pass through a detection area where the suspension is exposed to one or more excitable light sources, such as lasers, thereby allowing the user to measure the light scattering and fluorescence properties of the particles or cells. Labeling particles or cells with one or more fluorescent dyes can facilitate detection. Multiple different particles or cells may be subjected to flow cytometry for simultaneous detection using spectrally different dyes to label different particles or cells. In other embodiments, multiple photodetectors can be arranged to measure different scattering parameters and / or different spectrally specific dyes. For example, one or more detectors may be configured to measure one or more sets of scattering parameters, and one or more additional detectors may be configured to measure one or more distinct dyes, thereby enabling the generation of data containing signals for each light scattering parameter and each fluorescence emission.

[0055] Commonly measured flow cytometry parameters include, but are not limited to, (i) excitation light scattered nearly along the forward direction by a particle or cell, or forward scattering (FSC); (ii) excitation light scattered nearly laterally by a particle or cell, or side scattering (SSC); and (iii) light emission from fluorescent molecules in one or more channels (frequency ranges) of the spectrum, or light emission from fluorescent dyes primarily detected in those channels. In one embodiment, different cell types from an engineered immune cell population can be identified by FSC, SSC, and fluorescence emission resulting from labeling various cell surface proteins on the cells with dye-labeled antibodies.

[0056] Data obtained from the analysis of particles or cells (e.g., a single engineered immune cell from an engineered immune cell population) by multicolor flow cytometry is multidimensional, and each cell corresponds to a point in a multidimensional space defined by the measured parameters. Populations of cells or particles are identified as clusters of points in the data space. Cluster identification, and thereby population identification, can be performed manually by drawing gates around populations displayed in one or more two-dimensional plots of data (referred to as “scatter plots” or “dot plots”). Alternatively, clusters can be identified and gates defining the boundaries of populations can be determined automatically. Flow cytometry is an important tool for the analysis and / or isolation of particles or cells (e.g., individual engineered immune cells from an engineered immune cell population) and cellular analytes or their components. Therefore, it can be used in connection with the analysis and / or isolation of engineered immune cells. In one embodiment, the disclosure provides a method for using fluid flow to linearly space donor cells from an engineered immune cell population so that they pass through a detection device individually. A single cell from an engineered immune cell population can be distinguished from other cells by their position in the fluid flow and the presence of detectable markers. As a result, a flow cytometer can be used to detect one or more bisistrically expressed polypeptides (as described herein) and / or to generate a bisistrically expressed polypeptide expression profile for the engineered immune cell population. Such a profile may include the proportion of cells expressing the bisistrically expressed polypeptide (as further described herein) of the total cell population being analyzed.

[0057] In one additional aspect, the disclosure provides a method for detecting engineered immune cells expressing a bicistronically expressed polypeptide from an engineered immune cell population by detecting the level of the bicistronically expressed polypeptide. In one embodiment, the detected level indicates that a subset of the engineered immune cells express the bicistronically expressed polypeptide. In another embodiment, the detected level of the bicistronically expressed polypeptide indicates the number of engineered immune cells expressing the bicistronically expressed polypeptide from the engineered immune cell population being analyzed.

[0058] In some embodiments, the method provides quantitative measurement of engineered immune cells from an engineered immune cell population that does not express a bicistronically expressed polypeptide. Flow cytometry can be used to quantify cells expressing or not expressing a bicistronically expressed polypeptide within a cell population, and / or to quantify cells of a specific cell type.

[0059] As described herein, flow cytometry is a method for quantifying the components or structural features of cells by optical means, primarily using specific labeling agents (as further described herein). Because different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture. Flow cytometry analysis includes two main steps: 1) labeling the selected cell types with one or more detectable labels or agents, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling cell types includes conjugating a labeled antibody to a marker expressed by a particular cell type. The antibody may be directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescently labeled second antibody that recognizes a first antibody.

[0060] In one other embodiment, the Disclosure provides a method for generating an expression profile of a bisistrically expressed polypeptide expressed in an engineered immune cell population based on the percentage of cells in which the bisistrically expressed polypeptide is detected, utilizing flow cytometry. In one embodiment, the method comprises providing an engineered immune cell population suspected to contain a bisistrically expressed polypeptide. In one embodiment, the method further comprises contacting the engineered immune cell population with an exogenous agent. In a further embodiment, the method comprises detecting the level of the bisistrically expressed polypeptide in the engineered immune cell population after the contact step, wherein the detected level of the bisistrically expressed polypeptide indicates that a certain percentage of the engineered immune cell population expresses the bisistrically expressed polypeptide. In another embodiment, the method comprises detecting the level of an additional polypeptide in the engineered immune cell population after the contact step, wherein the detected level of the additional polypeptide indicates that a certain percentage of the engineered immune cell population expresses the additional polypeptide. In some embodiments, the method includes detecting the levels of bicistronally expressed polypeptides and additional polypeptides, the detected levels indicating the expression rates of the bicistronally expressed polypeptides and additional polypeptides in the engineered immune cell population.

[0061] In one embodiment, the quantitative screening method is a flow cytometry assay. In one embodiment, the methods, assays, systems, compositions, and kits described herein may be used at different stages of the production of an engineered cell population. As shown in Figure 5, the immune cell population may undergo manufacturing steps 10-15 before being screened in 16 for the expression of bicistronically expressed polypeptides. The results of the screening in 16 inform a decision on whether to proceed with the use of the engineered immune cell population to formulate a cell therapy product, for example, a CAR T cell therapy product. If bicistronically expressed polypeptides are detected 18, the engineered immune cell population may be considered to have failed the screening assay. If bicistronically expressed polypeptides are detected 17, the bicistronically expressed polypeptides may be considered to have passed the screening assay and can proceed to the manufacturing process of the cell therapy product.

[0062] In one embodiment, a lot of the cell therapy formulation containing the manipulated immune cells that passed through 17 may originate from a subject who will ultimately receive the manipulated immune cell population. For example, the manipulated immune cells may include autoimmune cells obtained from the subject who will ultimately receive the manipulated immune cells, such as CAR T cells obtained from autoimmune cells. In another embodiment, the manipulated immune cells that passed through 17 may originate from a donor who is a different individual from the subject who will receive the manipulated immune cell population. For example, the manipulated immune cells may include alloimmune cells obtained from a healthy donor who is a different individual from the subject who will receive the manipulated immune cells, such as CAR T cells obtained from alloimmune cells.

[0063] In one embodiment, screening 16 can be performed after 13 or after 14, in addition to or as an alternative to screening 15 or later, according to the workflow in Figure 5. Furthermore, screening 16 may be delayed until after 19, when the manipulated immune cells have completed the manufacturing process, which ends in cryopreservation of 19 as a CAR T cell therapy preparation. The cryopreserved preparation can be thawed, and then screening 16 can be performed on a sample of the product to determine whether detection is pass or fail.

[0064] bicistronically expressed polypeptide In one embodiment, the disclosure relates to engineered immune cells and populations comprising them, as well as methods for detecting polypeptides expressed by the cells and populations. In one embodiment, the polypeptide expressed by the cells is a bisistronally expressed polypeptide. In one other embodiment, one or more bisistronally expressed polypeptides are expressed from a polynucleotide or a vector comprising the same, the polynucleotide comprising several different elements. As described herein, the polynucleotide comprises a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, the first and second nucleic acid sequences being separated by a third nucleic acid sequence encoding a linked peptide. In one embodiment, the third nucleic acid sequence encodes a set of amino acids, the first amino acid being added to the C-terminus of the bisistronally expressed polypeptide during translation. In another embodiment, the second amino acid translated after the translation of the first amino acid is the first N-terminal amino acid of the second polypeptide. In one other embodiment, the first and second amino acids are characterized by the absence of a peptide bond between them. In one embodiment, the first polypeptide contains a first amino acid at its C-terminus. In another embodiment, the second polypeptide contains a second amino acid at its N-terminus. In one embodiment, the first amino acid is glycine and / or the second amino acid is proline.

[0065] In other embodiments, the method described herein relates to the detection of a first polypeptide as a bisistronally expressed polypeptide. In another embodiment, the second polypeptide is a CAR. In a particular embodiment, the first polypeptide to be detected, i.e., the bisistronally expressed polypeptide to be detected, is a polypeptide different from the second polypeptide, i.e., a CAR. The first polypeptide, i.e., the bisistronally expressed polypeptide, may be any polypeptide, including but not limited to, a receptor polypeptide, a membrane polypeptide, a cytosolic polypeptide, and a secretory polypeptide.

[0066] In one embodiment, the first polypeptide is a membrane polypeptide containing an intracellular domain. In one embodiment, the intracellular domain is an intracellular signaling domain. In another embodiment, the intracellular domain is a cytokine signaling domain.

[0067] In one embodiment, the first polypeptide is a chimeric cytokine receptor (CCR). In another embodiment, the CCR is an inducible CCR. Generally, an inducible CCR is a receptor that is responsive to a ligand such as a small molecule (e.g., AP1903) or a protein (e.g., Epo, Tpo, or PD-L1). Inducible CCRs can be expressed in immune cells engineered to improve cytokine-inducible cytokine receptor signaling, such as CAR T cells (see U.S. Patent No. 2019-0292533A1, which is incorporated herein by reference in its entirety). In one embodiment, the present invention provides an inducible chimeric cytokine receptor comprising a dimerizing domain, a tyrosine kinase activating domain, and a tyrosine effector domain. In some embodiments, the tyrosine kinase activating domain comprises a Janus kinase (JAK) binding domain of or derived from a protein. In some of these embodiments, the tyrosine kinase activating domain further comprises a transmembrane domain. In some embodiments, the tyrosine kinase activating domain includes a tyrosine kinase domain of or derived from a receptor tyrosine kinase (RTK). In some of these embodiments, the tyrosine kinase activating domain further includes a transmembrane domain.

[0068] In another embodiment, the CCR is a CCR lacking an extracellular domain. In one other embodiment, the CCR is a constitutively active chimeric cytokine receptor (CACCR). The presence of a constitutively active and moduloable chimeric cytokine receptor allows signal 3 immunoenhancing to meet the need for immunoenhancing (see U.S. Patent No. 2020-0291090A1, which is incorporated herein by reference in whole). Generally, the CACCRs of this disclosure consist of two monomers, each monomer comprising (a) a transmembrane domain, (b) a JAK-binding domain, and (c) a mobilization domain, and these monomers are constitutively dimerized. In some embodiments, the CACCRs of this disclosure do not include an extracellular ligand-binding domain. In some embodiments, the monomers are identical, resulting in a constitutively active homodimer. In some embodiments, the monomers are not identical, resulting in a constitutively active heterodimer, which may be desirable in certain circumstances. The CACCR monomers of this disclosure can spontaneously dimerize and activate signal transduction in the absence of any exogenous stimuli or ligands (ligand-independent dimerization).

[0069] Table 1A shows exemplary amino acid sequences useful in CACCR. [Table 1]

[0070] Table 1B shows exemplary transmembrane sequences and JAK binding sequences useful in CACCR provided herein. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0071] Table 1C provides exemplary amino acid sequences of the recruitment domains of the CACCRs of this disclosure. In some embodiments, the CACCRs of this disclosure include a recruitment domain comprising an amino acid sequence selected from one or more receptor sequences in Table 1C. In some embodiments, the CACCRs of this disclosure include a recruitment domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences in Table 1C. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0072] Table 1D shows exemplary CACCR sequences of the present disclosure (e.g., full-length CACCR sequences or their components). CACCR may be expressed in CD8SS of a signal sequence, for example, the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10

[0073] In another embodiment, the CCR is an inducible PD-1 CCR, which is active when bound to a PD-1 ligand or activated with an anti-PD-1 antibody (see U.S. Patent No. 2020-0276238A1, which is incorporated herein in its entirety by reference). Similarly, constitutively active PD-1 chimeric cytokine receptors are provided herein. Engineered immune cells expressing the PD-1 CCR of the Disclosure, e.g., CAR T cells, are provided herein. In one embodiment, the inducible PD-1 CCR of the Disclosure activates signaling when bound to, for example, a PD-1 ligand (e.g., PD-L1, PD-L2) or a PD-1 antibody. These receptors activate signaling when the monomers of the receptor cluster and / or dimerize. In some embodiments, the monomer of the PD-1 CCR of the Disclosure comprises (a) a PD-1 external domain, (b) a transmembrane domain, (c) a Janus kinase (JAK) binding domain, and (d) a STAT recruitment domain (e.g., from the cytoplasmic domain of a receptor, e.g., from a cytokine receptor). The recruitment domain may be a STAT recruitment domain, an AP1 recruitment domain, a Myc / Max recruitment domain, or an NFκB recruitment domain. The PD-1 chimeric cytokine receptor can bind to a PD-1 ligand and / or be clustered and activated with an anti-PD-1 antibody. PD-1 chimeric cytokine receptors activate signaling when they bind to, for example, PD-L1 ligands, PD-L2 ligands, and / or PD-1 antibodies.

[0074] The constitutively active PD-1 CCRs of this disclosure are active regardless of the availability of PD-1 ligands, but their activity may be increased in the presence of PD-ligands. In some embodiments, the monomer of the PD-1 CCR of this disclosure comprises (a) a PD-1 external domain, (b) a transmembrane domain, (c) a Janus kinase (JAK) binding domain, and (d) a STAT recruitment domain (e.g., from the cytoplasmic domain of a receptor, e.g., from a cytokine receptor). In some embodiments, the monomer of the PD-1 CCR of this disclosure comprises (a) a PD-1 external domain, (b) a transmembrane domain, (c) a Janus kinase (JAK) binding domain, and (d) a recruitment domain (e.g., from the cytoplasmic domain of a receptor, e.g., from a cytokine receptor). The recruitment domain may be a STAT recruitment domain, an AP1 recruitment domain, a Myc / Max recruitment domain, or an NFκB recruitment domain. In some embodiments, constitutively active PD-1 CCRs can bind to a PD-1 ligand or an anti-PD-1 antibody and / or are clustered, and the receptor activity can be further increased by binding to a PD-1 ligand or an anti-PD-1 antibody.

[0075] Table 1E shows exemplary PD1 amino acid extracellular domain sequences of this disclosure. Note that the expression and extracellular location of exemplary PD1 amino acid sequences can be achieved using signal sequences. In exemplary embodiments, the CD8 signal sequence (CD8SS)MALPVTALLLPLALLLHAARP (SEQ ID NO: 127) is used. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0076] In some embodiments, the extradomain of PD-1 is dominant-negative. Table 1F shows exemplary PD-1 dominant-negative (DN) sequences of this disclosure. The DN sequences in Table 1E may be expressed with the assistance of a signal sequence, for example, the CD8SS signal sequence of sequence number 127. [Table 6]

[0077] Table 1G shows exemplary transmembrane amino acid sequences bound to the intracellular JAK2-binding domain sequence for use in PD-1 CCR. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0078] In some embodiments, the PD-1 chimeric cytokine receptor of this disclosure comprises an amino acid sequence of a STAT recruitment domain that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one amino acid sequence among those shown in Table 1H. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0079] Table 1I shows exemplary PD-1 CCR sequences of this disclosure. The receptor may be expressed at a signal sequence, e.g., CD8SS of SEQ ID NO: 127. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11] [Table 9-12] [Table 9-13] [Table 9-14] [Table 9-15] Table 9-16 Table 9-17 Table 9-18 Table 9-19 Table 9-20 Table 9-21 Table 9-22 Table 9-23 Table 9-24 Table 9-25 Table 9-26 Table 9-27 Table 9-28

[0080] In another embodiment, the CCR is a CCR comprising a TGF-β binding domain. In one embodiment, the CCR is an inducible CCR which is active when bound to a TGF-β ligand (e.g., TGF-β1, TGF-β2, and / or TGF-β3) or when activated with an anti-TGF-β antibody (see U.S. Patent No. 2021-0061881A1, which is incorporated herein by reference in its entirety). This specification provides engineered immune cells expressing the TGF-β CCR of the Disclosure, e.g., CAR T cells. The CCR of the Disclosure activates signaling upon binding of a TGF-β ligand (e.g., TGF-β1, TGF-β2, and / or TGF-β3) or an anti-TGF-β receptor antibody. These receptors activate signaling when the monomers of the receptor cluster and / or dimerize. The chimeric cytokine receptors of this disclosure are dual-function chimeric cytokine receptors capable of neutralizing the immunosuppressive effect of TGF-β ligands while simultaneously mimicking the transmission of immunoenhancing cytokine signals. In some embodiments, the monomer of the chimeric cytokine receptor of this disclosure comprises (a) a binding domain capable of binding to a TGF-β ligand or an anti-TGF-β receptor antibody, (b) a transmembrane domain, (c) a Janus kinase (JAK) binding domain, and (d) a STAT mobilization domain (e.g., from the cytoplasmic domain of the receptor, e.g., from the cytokine receptor). Each domain may be linked either directly or via one or more peptide linkers. In some embodiments, the monomer of the chimeric cytokine receptor of this disclosure comprises (a) a binding domain capable of binding to a TGF-β ligand or an anti-TGF-β receptor antibody, (b) a transmembrane domain, (c) a Janus kinase (JAK) binding domain, and (d) a mobilization domain (e.g., from the cytoplasmic domain of the receptor, e.g., from the cytokine receptor). The mobilization domain may be a STAT mobilization domain, an AP1 mobilization domain, a Myc / Max mobilization domain, or an NFκB mobilization domain. In some embodiments, the chimeric cytokine receptor is clustered and activated upon binding to a TGF-β ligand and / or clustered and activated by an anti-TGF-β receptor antibody.Chimeric cytokine receptors activate signaling when they bind to, for example, TGF-β ligands and / or TGF-β receptor antibodies. In some embodiments, the TGF-β receptor antibody is, but is not limited to, PF-03446962 or LY3022859. In some embodiments, the chimeric cytokine receptor is constitutively clustered or dimerized. Linked peptides

[0081] In one embodiment, the in vitro method described herein utilizes a linked peptide. In one embodiment, the linked peptide is part of a bicistronically expressed polypeptide. In other embodiments, the engineered immune cells, such as CAR T cells, include a polynucleotide sequence encoding a bicistronically expressed polypeptide, a linked peptide, and additional polypeptides. Table 1J provides a list of exemplary linked peptides and their amino acid sequences. [Table 10]

[0082] In one embodiment, the amino acid sequence of the linked peptide comprises D-(V or I)-ExNPGP, where x is any amino acid. Post-translation, the amino acid sequence attached to the C-terminus of the first polypeptide, i.e., the bicistronically expressed polypeptide, comprises D-(V or I)-ExNPG, where x is any amino acid.

[0083] This disclosure includes modifications to bicistronically expressed polypeptides and polypeptides containing CARs, as well as modifications to linked peptides, including sequences provided herein, for example in Tables 1J and 2, which include functionally equivalent bicistronically expressed polypeptides, CARs, or linked peptides having modifications that do not significantly affect their properties, and variants with enhanced or reduced activity. For example, by altering the amino acid sequence of a linked peptide (e.g., by deletion, insertion, and / or substitution), a linked peptide with desired properties can be obtained. Modification of peptides and polypeptides is a routine practice in the art and therefore does not need to be described in detail herein. Examples of modified polypeptides or peptides include polypeptides or peptides having conservative substitutions of amino acid residues, deletions or additions of one or more amino acids that do not significantly and detrimentally alter functional activity, or enhance the functionality of the linked peptide. A bicistronically expressed polypeptide hat contains a linked peptide and / or CAR. Deletions may include one or more terminal deletions in the linked peptide.

[0084] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions, as well as intrasequence insertions of single or multiple amino acid residues, ranging in length from one to several residues.

[0085] Substitutional variants involve the removal of at least one amino acid residue in a polypeptide or peptide, with a different residue inserted in its place. Conservative substitutions are shown in Table 2 under the heading "Conservative Substitutions." If such substitutions result in a change in biological activity, they are indicated as "Exemplary Substitutions" in Table 2, or more substantial changes are introduced, as further described below with reference to amino acid classes, and the product may be screened. [Table 11]

[0086] immune cells Manipulated immune cells are obtained from donor cells. Manipulated cells derived from donor cells, which are suitable for use with the methods and / or reagents described herein, include immune cells.

[0087] Prior to in vitro manipulation or genetic modification (e.g., as described herein), donor cells (e.g., immune cells) for use in the methods herein can be obtained from the subject. Donor cells can be obtained from several non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, immune cells derived from stem cells or iPS cells, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In some embodiments, any number of T cell lines known and available to those skilled in the art may be used. In some embodiments, donor cells may originate from a healthy donor, a patient diagnosed with cancer, a patient diagnosed with an autoimmune disorder, or a patient diagnosed with an infection. In some embodiments, donor cells may be part of a mixed population of cells exhibiting different phenotypic features.

[0088] In some embodiments, the immune cells are autoimmune cells obtained from the target ultimately receiving the manipulated immune cells. In some embodiments, the immune cells are alloimmune cells obtained from a donor, which is a different individual from the target ultimately receiving the manipulated immune cells.

[0089] In some embodiments, immune cells include T cells. T cells can be obtained from several non-limiting sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, T cells derived from stem cells or iPS cells, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from blood volumes collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL® isolation.

[0090] Donor cells can be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and placed in a buffer or culture medium suitable for subsequent processing.

[0091] Donor PBMCs can be used directly for genetic modification in immune cells (such as CARs or TCRs) using the methods described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be classified into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or proliferation.

[0092] In certain embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes using, for example, centrifugation with a PERCOLL® gradient. Specific subpopulations of T cells, such as CCR7+, CD95+, CD122, CD27+, CD69+, CD127+, CD28+, CD3+, CD4+, CD8+, CD25+, CD62L+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the desired cell population for use in this disclosure.

[0093] In some embodiments, a population of donor cells, such as T cells or other immune cells, is enriched for CD4+ cells.

[0094] In some embodiments, a population of donor cells, such as immune cells like T cells, is enriched for CD8+ cells.

[0095] In some embodiments, CD8+ cells are further classified by identifying cell surface antigens associated with each of these cell types: naive cells, central memory cells, and effector cells. In some embodiments, phenotypic markers expressed in naive T cells include CD45RA+, CD95-, IL2Rb-, CCR7+, and CD62L+. In some embodiments, phenotypic markers expressed in stem cell memory T cells include CD45RA+, CD95+, IL2Rb+, CCR7+, and CD62L+. In some embodiments, phenotypic markers expressed in central memory T cells include CD45RO+, CD95+, IL2Rb+, CCR7+, and CD62L+. In some embodiments, phenotypic markers expressed in effector memory T cells include CD45RO+, CD95+, IL2Rb+, CCR7+, and CD62L-. In some embodiments, the expression of phenotypic markers in T effector cells includes CD45RA+, CD95+, IL2Rb+, CCR7-, and CD62L-. Thus, CD4+ and / or CD8+ T helper cells can be classified into naive cells, stem cell memory cells, central memory cells, effector memory cells, and T effector cells by identifying the cell population possessing cell surface antigens.

[0096] It will be understood that the donor PBMC may further contain other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors having the coding sequence of the chimeric receptor disclosed herein may be introduced into a population of human donor T cells, NK cells, or NKT cells. Standard procedures are used for the cryopreservation of CAR-expressing T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or proliferation of T cells are carried out in the absence of non-human animal-derived products such as fetal calf serum and fetal bovine serum. In various embodiments, the cryopreservation medium may include, for example, CryoStor® CS2, CS5, or CS10, or other media containing DMSO, or media without DMSO.

[0097] Manipulated immune cells This specification provides bicistronically expressed polypeptides and additional polypeptides, such as engineered immune cells (e.g., CAR-T cells) expressing the CARs of this disclosure obtained from donor cells as described herein.

[0098] In some embodiments, the engineered immune cells comprise a polynucleotide sequence encoding a bicistronically expressed polypeptide, a linked peptide, and additional polypeptides, such as CARs. In one embodiment, a CAR comprises one or more extracellular antigen-binding domains. In some embodiments, the engineered immune cells comprise a population of CARs, each containing a different extracellular antigen-binding domain. In some embodiments, the immune cells comprise a population of CARs, each containing the same extracellular antigen-binding domain.

[0099] Manipulated immune cells can be allogeneic or autologous.

[0100] In some embodiments, the manipulated immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), NK cells, NK-T cells, TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells.

[0101] In some embodiments, the manipulated immune cells may be obtained from or derived from a group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the manipulated immune cells are T cells. In some exemplary embodiments, the manipulated immune cells are αβ T cells. In some exemplary embodiments, the manipulated immune cells are γδ T cells. In some exemplary embodiments, the manipulated immune cells are macrophages.

[0102] In some embodiments, the manipulated immune cells may be derived from stem cells, for example, but not limited to, adult stem cells, non-human embryonic stem cells, more specifically, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells. The stem cells may be CD34+ or CD34-.

[0103] In some embodiments, donor cells are derived from or prepared from peripheral blood. In some embodiments, donor cells are derived from or prepared from peripheral blood mononuclear cells. In some embodiments, donor cells are derived from or prepared from bone marrow. In some embodiments, donor cells are derived from or prepared from umbilical cord blood. In some embodiments, donor cells are human cells. In some embodiments, they are transfected or transduced by a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, gene gun (e.g., Gene Gun), transfection, lipid transfection, polymer transfection, nanoparticles, viral transfection, or viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplex. In some embodiments, donor cells are T cells reprogrammed from non-T cells. In some embodiments, donor cells are T cells reprogrammed from T cells.

[0104] Detection agent (including antibodies and their fragments) In various embodiments, the disclosed method for detecting bicistronally expressed polypeptides in engineered immune cells involves the use of an antibody or antigen-binding agent (e.g., one comprising an antigen-binding domain, or one comprising an antibody or a fragment thereof). As discussed below, in various embodiments, engineered immune cells derived from donor cells of a donor cell population may also include a binding agent.

[0105] As used herein, the term “antibody” refers to a polypeptide containing standard immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies, such as those produced naturally, are approximately 150 kD tetrameric drugs consisting of two identical heavy-chain polypeptides (each approximately 50 kD) and two identical light-chain polypeptides (each approximately 25 kD), which associate with each other and are commonly referred to as a “Y-shaped” structure. Each heavy chain consists of at least four domains (each approximately 110 amino acids long), an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and carboxy-terminal CH3 (located at the base of the Y stem). A short region known as the “switch” joins the heavy-chain variable region to the constant region. The “hinge” joins the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region conjugate two heavy chain polypeptides together in the intact antibody. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain. Those skilled in the art are familiar with the structure and sequence elements of antibodies and recognize the “variable” and “constant” regions in the provided sequence, and understand that there is some flexibility in defining the “boundaries” between such domains, and that as a result, different presentations of the same antibody chain sequence may exhibit such boundaries at positions shifted by one or a few residues, for example, to different presentations of the same antibody chain sequence.

[0106] An intact antibody tetramer consists of two heavy-light tetramers, where the heavy and light chains are linked to each other by a single disulfide bond. Two other disulfide bonds connect the hinge regions of the heavy chains, resulting in the dimers being linked to each other to form a tetramer. Furthermore, naturally produced antibodies are glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two β-sheets (e.g., 3-stranded, 4-stranded, or 5-stranded sheets) packed in contact with each other in a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops known as "complementarity-determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR region forms a β-sheet that provides a structural framework to the domain, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space, so that they create a single hypervariable antigen-binding site located at the tip of the Y structure. The Fc region of naturally occurring antibodies binds to complement system elements and also to receptors on effector cells, these effector cells include, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding attributes of the Fc region for Fc receptors can be modified through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present invention include a glycosylated Fc domain, which includes a modified or manipulated Fc domain, such as glycosylation.

[0107] For the purposes of this disclosure, in certain embodiments, any polypeptide, or complex of polypeptides, containing a sufficient immunoglobulin domain sequence as found in natural antibodies, is referred to as an “antibody” and / or can be used as an “antibody,” regardless of whether such polypeptides are produced naturally (e.g., by animals responding to an antigen) or by genetic engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal, and in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, the antibody sequence elements are humanized, primated, chimeric, etc.

[0108] Furthermore, as used herein, the term “antibody” refers to any construct or format known or developed in the art to utilize the structural and functional characteristics of an antibody in an alternative presentation. For example, in some embodiments, the antibodies used in the methods of this disclosure include: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain variable fragments (scFV); polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies (also referred herein as nanobodies or VHH); shark antibodies, masked antibodies (e.g., Probodies®); and small modular antibodies. ImmunoPharmaceuticals (SMIP®), single-chain or tandem diabolic (TandAb®); VHH; Anticalin®; Nanobody® minibody; BiTE®; Ankyrin repeat protein or DARPIN®; Avmer®; DART; TCR-like antibody; Adnectin®; Affilin®; Trans-body®; Affibody®; TrimerX®; microprotein; Fynomer®; Centyrin®; and KALBITOR® are in a format selected from these. In some embodiments, antibodies may lack covalent modifications (e.g., glycan attachments) that they have when naturally produced. In some embodiments, antibodies may include covalent modifications (e.g., glycan attachments), a payload (e.g., a detectable portion, a treatment portion, a catalytic portion, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0109] As used herein, the term “antibody agent” generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may comprise one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may comprise one or more sequence elements such as humanized, primated, or chimeric, as known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or formats known or developed in the art to utilize the structural and functional characteristics of an antibody in an alternative presentation. For example, the antibody samples used in accordance with the methods of this disclosure include intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain FV; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (SMIP®), single-chain or tandem-type diabodies (TandAb®); VHH; Anticalin®; Nanobody® minibodies; and BiTE (Registered Trademark); Ankyrin Repeat Protein or DARPIN®; Avmer®; DART; TCR-like antibody; Adnectin®; Affilin®; Trans-body®; Affibody®; TrimerX®; Microprotein; Fynomer®, Centyrin®; and KALBITOR®, in a format selected from these.

[0110] The antibody or antibody agent used in carrying out the methods of this disclosure may be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In certain embodiments, the antigen-binding molecule is selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof.

[0111] Antibodies and antibody preparations include antibody fragments. “Antibody fragment” refers to a portion of an intact antibody, e.g., the antigen-binding region and / or variable region of an intact antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, linear antibodies, antibody fragment antibodies, and polyspecific antibodies formed from scFv fragments, as well as other fragments. Antibodies also include, but are not limited to, polyclonal monoclonal antibodies, chimeric dAb (domain antibodies), single-chain antibodies, Fab, Fa, F(ab')2 fragments, and scFv. Antibodies can be whole antibodies, immunoglobulins, or antibody fragments. Antibody fragments can be produced by a variety of techniques, including, but are not limited to, proteolysis of intact antibodies known in the art, and the production of recombinant host cells (e.g., E. coli, Chinese hamster ovary (CHO) cells, or phages).

[0112] In some embodiments, the antibody or antibody agent may be a chimeric antibody (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, while the rest of the heavy chain and / or light chain originates from a different source or species. In one example, a chimeric antibody may include a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody may be "class-switched," meaning that its class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0113] In some embodiments, the chimeric antibody may be a humanized antibody (e.g., Almagro and Francsson, Front. Biosci., 13:1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321 and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et al. See also al., Methods, 36:43-60 (2005); Osbourn et al., Methods, 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000). Humanized antibodies are chimeric antibodies containing amino acid residues derived from non-human hypervariable regions (HVRs) and amino acid residues derived from human framework regions (FRs). In certain embodiments, a humanized antibody will contain substantially all of at least one, typically two, variable domains, with all or substantially all of its hypervariable regions (e.g., CDRs) corresponding to those of a non-human antibody, and all or substantially all of its framework regions (FRs) corresponding to those of a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody.

[0114] In some embodiments, the antibodies or antibody preparations provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art (see, for example, van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5:368-74 (2001); and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008)). Human antibodies may be antibodies produced by humans or human cells, or antibodies having an amino acid sequence corresponding to the amino acid sequence of an antibody obtained from a non-human source utilizing the human antibody repertoire or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies may be prepared using methods well known in the art.

[0115] As used herein, the term “antibody agent” generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may comprise one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may comprise one or more sequence elements such as humanized, primated, or chimeric, as known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or formats known or developed in the art to utilize the structural and functional characteristics of an antibody in an alternative presentation. For example, the antibody samples used in accordance with the methods of this disclosure include: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain FVs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); and small modular antibodies. ImmunoPharmaceuticals (SMIP®), single-chain or tandem diabolic proteins (TandAb®); VHH; Anticalin®; Nanobody® minibodies; BiTE®; Ankyrin repeat protein or DARPIN®; Avmer®; DART; TCR-like antibodies; Adnectin®; Affilin®; Trans-body®; Affibody®; TrimerX®; microproteins; Fynomer®; Centyrin®; and KALBITOR® are in a format selected from, but are not limited to, these.

[0116] Chimeric antigen receptor As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on cancer cells). Upon binding to a target antigen, the CAR can activate immune cells to attack and destroy cells possessing that antigen (e.g., cancer cells). CARs may also incorporate co-stimulatory or signaling domains to enhance their potency. See Krause et al., J.Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci.Transl.Med. 3:95 (2011); Porter et al., N.Engl.J.Med. 365:725-33 (2011), and Gross et al., Annu.Rev.Pharmacol.Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0117] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an antigen-binding domain that specifically binds to a target.

[0118] In some embodiments, the antigen-specific CAR further comprises a safety switch and / or one or more monoclonal antibody-specific epitopes.

[0119] 1. Antigen-binding domain As described herein, the CARs include an antigen-binding domain. As used herein, “antigen-binding domain” means any polypeptide that binds to a specific target antigen. In some embodiments, the antigen-binding domain binds to an antigen on tumor cells. In some embodiments, the antigen-binding domain binds to an antigen on cells involved in hyperproliferative disease.

[0120] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs as described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3.

[0121] An antigen-binding domain is said to be "selective" if it binds to one target more tightly or with higher affinity than it binds to a second target.

[0122] The antigen-binding domain of the CAR selectively targets cancer antigens. In some embodiments, the cancer antigen is selected from EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin 18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34. In some embodiments, the CAR includes an antigen-binding domain that targets EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin 18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34.

[0123] In some embodiments, cancer antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein (EGP2), epithelial glycoprotein 40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AchR), folate receptor, ganglioside G2 (GD2), ganglioside G3 (GD3), and human The group is selected from the following: skin growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit α2 (IL-13Ra2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), LI cell adhesion molecule (LICAM), melanoma antigen family A and 1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligand, cancer testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Welms oncoprotein (WT-1).

[0124] Variants of the antigen-binding domain (e.g., VH and / or VL variants of the CDR), for example, variable light chains and / or variable heavy chains, each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity with respect to the amino acid sequence of the antigen-binding domain sequence, are also included. In some cases within the scope of this disclosure, such molecules include at least one heavy chain and one light chain, while in other cases, the variant form includes two variable light chains and two variable heavy chains (or their subparts). Those skilled in the art can determine preferred variants of the antigen-binding domain described herein using well-known techniques. In certain embodiments, those skilled in the art can identify preferred regions of the molecule that can be modified without disrupting activity by targeting regions not considered important to activity.

[0125] In certain embodiments, the polypeptide structure of the antigen-binding domain is antibody-based, but is not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof. In some embodiments, the antigen-binding domain comprises or consists of an avimer.

[0126] In some embodiments, the antigen-binding domain is scFv.

[0127] In some embodiments, the antigen-selective CAR includes a leader or signal peptide.

[0128] In other embodiments, this disclosure relates to isolated polynucleotides encoding any one of the antigen-binding domains described herein. In some embodiments, this disclosure relates to isolated polynucleotides encoding CARs. Also provided herein are vectors comprising polynucleotides and methods for constructing them.

[0129] In other embodiments, this disclosure relates to isolated polynucleotides encoding any one of the antigen-binding domains described herein. In some embodiments, this disclosure relates to isolated polynucleotides encoding CARs. Also provided herein are vectors comprising polynucleotides and methods for constructing them.

[0130] In some embodiments, CAR immune cells (e.g., CAR-T cells) that can form components of an engineered immune cell population (derived from donor cells of the donor cell population described herein) generated by carrying out the methods of this disclosure contain polynucleotides encoding a safety switch polypeptide, such as RQR8. See, for example, WO2013153391A (the whole of which is incorporated herein by reference). In CAR immune cells (e.g., CAR-T cells) containing polynucleotides, the safety switch polypeptide may be expressed on the surface of the CAR immune cells (e.g., CAR-T cells).

[0131] 2. Hinged Domain The extracellular domain of the CARs of this disclosure may include a “hinge” domain (or hinge region). This term generally refers to any polypeptide that functions to link the transmembrane domain in the CAR to the extracellular antigen-binding domain in the CAR. In particular, hinge domains can be used to provide further flexibility and accessibility to the extracellular antigen-binding domain.

[0132] The hinge domain may contain up to 300 amino acids, 10 to 100 amino acids in some embodiments, or 25 to 50 amino acids in some embodiments. The hinge domain may be derived from all or part of naturally occurring molecules, e.g., CD8, CD4, CD28, 4-1BB, or all or part of the extracellular region of IgG (in particular, the hinge region of IgG; it will be understood that the hinge region includes some or all of molecules belonging to the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or from all or part of the constant region of the antibody heavy chain. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a completely synthetic hinge sequence. In some embodiments, the hinge domain is part of a human CD8α chain (e.g., NP_001139345.1). In other embodiments, the hinge and transmembrane domains include a portion of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein includes a subsequence of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα, particularly a hinge region of any of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain includes a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein includes scFv, a human CD8α hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain.

[0133] In some embodiments, the hinge domain in the CAR of the Disclosure is the transmembrane domain of CD8α. In some embodiments, the hinge domain in the CAR of the Disclosure is a CD8α hinge domain comprising the amino acid sequence PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 332) or TTTPAPRPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 340). In some embodiments, the CD8α hinge domain comprises a nucleic acid sequence encoding the hinge amino acid sequence of SEQ ID NO: 332. In some embodiments, the hinge domain in the CAR of the Disclosure is a CD28 hinge domain.

[0134] 3. Transmembrane domain The CARs of this disclosure are designed with a transmembrane domain fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex, by avoiding such domain binding to the transmembrane domains of the same or different surface membrane proteins. In some embodiments, a short linker may form a bond between one or more of the extracellular, transmembrane, and intracellular domains of the CAR.

[0135] Transmembrane domains suitable for CARs disclosed herein include, for example, but not limited to, lymphocytes, such as T helper (T) cells. h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg (b) having the ability to be expressed on the surface of immune cells such as lymphocytes, including (b) natural killer (NK) cells, and / or (b) having the ability to interact with extracellular antigen-binding domains and intracellular signaling domains to guide the cellular response of immune cells against target cells.

[0136] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein.

[0137] Transmembrane regions particularly used in this disclosure include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, and Tegrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT Ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof may be derived from (or include or correspond to) these ligands.

[0138] As a non-limiting example, the transmembrane domain may be derived from or be a part of T cell receptors such as α, β, γ, or δ, polypeptides constituting the CD3 complex, IL-2 receptor p55(chain), p75(β or γ chain), Fc receptor subunit chains, particularly Fcγ receptor III or CD protein. Alternatively, the transmembrane domain may be synthetic and may primarily contain hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0139] In some embodiments, the transmembrane domain in the CAR of the Disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the Disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT or IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 333). In some embodiments, the CD8α transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence IYIWAPLAGTCGVLLLSLVIT or SEQ ID NO: 333. In some embodiments, the hinge and transmembrane domain in the CAR of the Disclosure is a CD8α hinge and transmembrane domain comprising the amino acid sequence SEQ ID NO: 323 or TTTPARPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITCD.

[0140] In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD28 transmembrane domain.

[0141] 4. Intracellular domains The intracellular (cytoplasmic) domain of the CARs of this disclosure can provide activation of at least one of the normal effector functions of immune cells containing the CARs. For example, the effector function of a T cell may refer to cytolytic activity or helper activity including cytokine secretion.

[0142] In some embodiments, the activated intracellular signaling domain for use in CAR may be, for example, but not limited to, cytoplasmic sequences of T cell receptors and co-receptors that act in coordination to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0143] Suitable (e.g., activated) intracellular domains include, but are not limited to, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin proteins, and cytokines. Receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT It should be understood that these include signaling domains derived from (or corresponding to) ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83, or any combination thereof.

[0144] The intracellular domains of the CARs of this disclosure can incorporate a co-stimulatory signaling domain (referred to herein as a co-stimulatory molecule) in addition to the activation domain described above to enhance their potency. The co-stimulatory domain can provide a signal in addition to the primary signal provided by the activation molecule described herein.

[0145] Preferred co-stimulatory domains within the scope of this disclosure include, for example, CD28, OX40, 4-1BB / CD137, CD2, CD3(α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, Lymphocyte Function-Associated Antigen-1 (LFA-1 (CD1 1a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNFR, integrin, signal transduction lymphocyte activating molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α , CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1 -1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(tactile), CEACAM1, CRT It should be understood that AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligands, or fragments, or combinations thereof, may be derived from (or correspond to). It should be understood that additional co-stimulatory molecules or fragments not listed above are within the scope of this disclosure.

[0146] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the 4-1BB / CD137 domain either by itself or in combination with any other desired intracellular domain useful for the CAR of this disclosure. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native nucleic acid sequence of 4-1BB / CD137 is described in NCBI reference sequence: NM_001561.5.

[0147] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD28 domain either by itself or in combination with any other desired intracellular domain(s) useful for the CARs of this disclosure. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native nucleic acid sequence of CD28 is described in NCBI reference sequence: NM_006139.1.

[0148] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD3ζ domain either by itself or in combination with any other desired intracellular domain(s) useful with respect to the CAR of this disclosure.

[0149] For example, the intracellular domain of a CAR may include a CD3ζ chain portion and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of this disclosure may be linked to each other randomly or in a specific order. In some embodiments, the intracellular domain is designed to include an activating domain of CD3ζ and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to include an activating domain of CD3ζ and a signaling domain of 4-1BB.

[0150] In some embodiments, the intracellular signaling domain of the CAR of the Disclosure includes a domain of a co-stimulatory signaling molecule. In some embodiments, the intracellular signaling domain of the CAR of the Disclosure includes a portion of a co-stimulatory molecule selected from the group consisting of fragments 4-1BB (GenBank:AAA53133.) and CD28 (NP_006130.1).

[0151] Table 3 provides exemplary sequences of CAR components that can be used with the CARs disclosed herein, as well as the antibodies and / or CAR sequences exemplified herein. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0152] Table 4 provides exemplary sequences of chimeric cytokine receptor (CCR) components that may be used in the CCRs disclosed herein. The CCR may be expressed in CD8SS of a signal sequence, for example, the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127). [Table 13]

[0153] Table 5 provides exemplary CACCR sequences showing the P2A ligation sequence-GSGATNFSLLKQAGDVEENPG (SEQ ID NO: 307), with or without the anti-BCMA CAR. [Table 14-1] [Table 14-2]

[0154] Engineered immune cells containing CAR Furthermore, the Specified Provision provides engineered immune cells and populations of engineered immune cells which include i) polynucleotides, linked peptides, and additional polypeptides (e.g., CARs) encoding bicistronically expressed polypeptides, linked peptides, and additional polypeptides, e.g., CARs (e.g., CAR-T cells or CAR+ cells).

[0155] In some embodiments, the engineered immune cells include CAR T cells, each CAR T cell containing an extracellular antigen-binding domain that reduces or eliminates the expression of endogenous TCRs. In some embodiments, the population of engineered immune cells includes a population of CAR T cells, each CAR T cell containing two or more different extracellular antigen-binding domains that reduce or eliminate the expression of endogenous TCRs. In some embodiments, the engineered immune cells include a population of CARs, each CAR T cell containing the same extracellular antigen-binding domain that reduces or eliminates the expression of one or more desired biomarkers (as described herein) and / or endogenous TCRs.

[0156] Manipulated immune cells can be allogeneic or autologous.

[0157] In some embodiments, the manipulated immune cells or population of manipulated immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), NK cells, NK-T cells, TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells that express CARs. In some embodiments, the T cells may be derived from a group consisting of CD4+ T lymphocytes, CD8+ T lymphocytes, or a population including a combination of CD4+ and CD8+ T cells.

[0158] In some embodiments, the engineered immune cells or populations of engineered immune cells generated using the methods of the present disclosure may be derived from, for example, stem cells, without limitation. Stem cells may be adult stem cells, non-human embryonic stem cells, more specifically, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0159] In some embodiments, the engineered immune cells or populations of engineered immune cells produced using the methods of the present disclosure are obtained from or prepared from peripheral blood. In some embodiments, the engineered immune cells are obtained from or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the engineered immune cells are obtained from or prepared from bone marrow. In some embodiments, the engineered immune cells are obtained from or prepared from umbilical cord blood. In some embodiments, the donor cells are human cells. In some embodiments, the donor cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, gene gun (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplex.

[0160] In some embodiments, the engineered immune cells expressing an antigen-specific CAR on their cell surface membranes contain stem cell memory and central memory cells at a percentage of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100%.

[0161] In some embodiments, the engineered immune cells expressing an antigen-specific CAR on their cell surface membranes contain stem cell memory cells and central memory cells at a percentage of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70%.

[0162] In some embodiments, the engineered immune cells express an antigen-specific CAR enriched in T CM and / or T SCM cells on their cell surface membranes, and as a result, the engineered immune cells contain at least about 60%, 65%, 70%, 75%, or 80% of a combination of T CM cells and T SCM cells. In some embodiments, the engineered immune cells express an antigen-specific CAR enriched in T CM and / or T SCM cells on their cell surface membranes, and as a result, the engineered immune cells contain at least about 70% of a combination of T CM cells and T SCM cells. In some embodiments, the engineered immune cells express an antigen-specific CAR enriched in T CM and / or T SCM cells on their cell surface membranes, and as a result, the engineered immune cells contain at least about 75% of a combination of T CM cells and / or T SCM cells.

[0163] In some embodiments, the manipulated immune cells are inflammatory T lymphocytes expressing CAR. In some embodiments, the manipulated immune cells are cytotoxic T lymphocytes expressing CAR. In some embodiments, the manipulated immune cells are regulatory T lymphocytes expressing CAR. In some embodiments, the manipulated immune cells are helper T lymphocytes expressing CAR.

[0164] Genetic modification of CAR T cells In some embodiments, engineered immune cells derived from donor cells having a specific biomarker profile according to this disclosure may contain one or more disrupted or inactivated genes. In some embodiments, target antigens (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD7) may be included. By knocking out (0) and introducing a CAR targeting the same antigen (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 CAR), the induced CAR activation can be avoided. As described herein, in some embodiments, the immune cells manipulated according to this disclosure contain one disrupted or inactivated gene selected from the group consisting of MHC1(β2M), MHC2(CIITA), EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70, TCRα, and TCRβ, and / or express a CAR or a multichain CAR. In some embodiments, the cells contain a multichain CAR.In some embodiments, isolated cells contain two disrupted or inactivated genes selected from the group consisting of CD52 and TCRα, CDR52 and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, MHC-1 and TCRα, MHC-1 and TCRβ, MHC2 and TCRα, and MHC2 and TCRβ, and / or express CAR or multi-chain CAR.

[0165] In some embodiments, the isolated cells contain polynucleotides encoding polynucleotides containing multi-chain CARs. In some embodiments, the isolated cells according to this disclosure include CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, DLL3 and CD52, DLL3 and TCRα, DLL3 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, B The expression of a CAR and / or pTα transgene comprising two disrupted or inactivated genes selected from the group consisting of TLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or a CAR and / or pTα transgene comprising a multichain CAR. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing an endonuclease into donor cells that can selectively inactivate genes by selective DNA cleavage. In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE-nuclease, or TALEN®), or a CRISPR (e.g., Cas9 or Cas12) endonuclease.

[0166] In some embodiments, the TCR is rendered non-functional in the cells according to this disclosure by disrupting or inactivating the TCRα gene and / or TCRβ gene(s). In some embodiments, methods are provided for obtaining modified cells derived from an individual, the cells being able to grow independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can grow independently of the MHC signaling pathway, which are readily obtained by this method, are included within the scope of this disclosure. Modified cells disclosed herein can be used to treat patients in need of host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Accordingly, the scope of this disclosure is a method for treating patients in need of host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), the method comprising treating the aforementioned patients by administering to them an effective amount of modified cells containing disrupted or inactivated TCRα and / or TCRβ genes.

[0167] This disclosure provides a method for determining the purity of a population of engineered immune cells that lack or have reduced endogenous TCR expression. In some embodiments, the engineered immune cells include TCR+ cells with a concentration of less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%. Such populations may be products of the methods of this disclosure.

[0168] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agent can be, for example, a purine nucleotide analog (PNA), and thus renders the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, cladribine, fludarabine, cyclophosphamide, and cytarabine, which can be used alone or in combination. The PNA is metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate forms of PNA. Its triphosphate form competes with ATP for DNA synthesis, acts as an apoptosis promoter, and is a potent inhibitor of ribonucleotide reductase (RNR) involved in trinucleotide production.

[0169] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines can be further genetically modified by disrupting or inactivating the TCRα gene.

[0170] The present disclosure also provides engineered immune cells comprising a polynucleotide encoding any of the bicistronically expressed polypeptide, the linker polypeptide, and the CAR polypeptide described herein. In some embodiments, the CAR can be introduced into the immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also include, for example, a selectable marker that provides for the identification and / or selection of cells that receive the vector.

[0171] CAR polypeptides can be synthesized in situ within cells after the polynucleotide encoding the CAR polypeptide has been introduced into the cells. Alternatively, the CAR polypeptide can be produced outside the cells and then introduced into the cells. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods (e.g., methods using lentiviral vectors) can be used to integrate the polynucleotide construct into the cell genome. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and polynucleotide constructs not integrated into the cell genome. In other embodiments, viral methods may be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (e.g., retroviruses, adenoviruses) or liposomes. Transient transformation methods include, but are not limited to, microinjection, electroporation, or particle collision. Polynucleotides can be contained in vectors, such as plasmid vectors or viral vectors.

[0172] In some embodiments, an isolated nucleic acid is provided, comprising a promoter operably ligated to a first polynucleotide encoding an antigen-binding domain, at least one co-stimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vectors, mouse leukemia virus vectors, SFG vectors, adenovirus vectors, lentivirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained within a plasmid.

[0173] In some embodiments, the isolated nuclear construct is contained within a viral vector and introduced into the genome of engineered immune cells by random integration, such as lentivirus- or retrovirus-mediated random integration. In some embodiments, the isolated nucleic acid construct is contained within a viral or non-viral vector and introduced into the genome of engineered immune cells by site-specific integration, such as adenovirus-mediated site-specific integration.

[0174] Production of Engineered Immune Cells (Including CAR T Cells) Methods are provided herein for analyzing or determining various characteristics of donor cells from a donor cell population and / or engineered immune cells (including engineered immune cells such as CAR-expressing or CAR+ cells) from a population of immune cells. As described herein, engineered immune cells such as CAR T cells can be modified to reduce or eliminate the expression or activity of the endogenous TCR, and the remaining TCR+ engineered immune cells can be depleted at the end of production according to the methods described herein. The present disclosure provides methods for characterizing a population of engineered immune cells or analyzing them, either to characterize a formulation or as part of a manufacturing process. The present disclosure provides methods for characterizing or analyzing other characteristics such as the potency or multifunctionality of engineered immune cells, either to characterize a formulation or as part of a manufacturing process. In some embodiments, engineered immune cells such as CAR T cells are manufactured according to Good Manufacturing Practice (GMP).

[0175] In producing polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc. according to the present disclosure, various known techniques can be utilized.

[0176] Prior to in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. Cells expressing a CAR can be derived from an allogeneic or autologous source and the endogenous TCR described herein can be depleted.

[0177] 1. Raw materials In some embodiments, immune cells include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, T cells can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll® isolation.

[0178] Cells can be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and then placed in a buffer or culture medium suitable for subsequent processing.

[0179] In certain embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes using, for example, centrifugation with a PERCOLL® gradient. Specific subpopulations of T cells (e.g., CD28+, CD4+, CD45RA-, and CD45RO+ T cells, or CD28+, CD4+, CDDS+, CD45RA-, CD45RO+, and CD62L+ T cells) can be further isolated by positive or negative selection techniques known in the Art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the desired cell population for use in this disclosure.

[0180] PBMCs can be used directly for genetic modification in immune cells (such as CARs or TCRs) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be classified into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or proliferation. In some embodiments, CD8+ cells are further classified by identifying cell surface antigens associated with each of these types of CD8+ cells: naive cells, stem cell memory cells, central memory cells, and effector cells. In some embodiments, the expression of phenotypic markers for central memory T cells includes CD27, CD45RA, CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4+ T cells are further sorted into subpopulations. Thus, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations that possess cell surface antigens.

[0181] 2. Stem cell-derived immune cells In some embodiments, immune cells may be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSCs, mesenchymal cells, iPS cells, and other types of stem cells can be cultured in immortal cell lines or isolated directly from patients. Various methods for isolating, developing, and / or culturing stem cells are known in the art and can be used to carry out this disclosure.

[0182] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the raw material may be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The raw material may be embryonic stem cells. The raw material may be B cells, or any other cells from peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0183] 3. Genetic modification of isolated cells Donor immune cells from a donor immune cell population, such as T cells, may be genetically modified after isolation using known methods, or the donor immune cells may be activated and proliferated (or differentiated in the case of precursors) in vitro before genetic modification. In some embodiments, isolated donor immune cells are genetically modified to reduce or eliminate the expression or activity of endogenous TCRα and / or CD52. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / Cas12a, zinc finger nucleases (ZFNs), TALENs, MegaTAL, meganucleases, base editing, or prime editing) to reduce or eliminate the expression or activity of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells such as T cells are genetically modified with the chimeric antigen receptor described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding CARs), and then activated and / or proliferated in vitro.

[0184] Specific methods for constructing the constructs and manipulated immune cells described herein are described in PCT application PCT / US15 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0185] It will be understood that PBMCs may further contain other cytotoxic lymphocytes such as NK cells or NKT cells. Expression vectors having the coding sequence of the chimeric receptor disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Transduced T cells having the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, which can then be further augmented to increase the number of these CAR-expressing T cells, in addition to cell activation, using anti-CD3 antibodies and IL-2, or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of CAR-expressing T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or proliferation of T cells are carried out in the absence of non-human animal-derived products such as fetal calf serum and fetal bovine serum.

[0186] In the case of polynucleotide cloning, the vector is introduced into host cells (isolated host cells) to enable the replication of the vector itself, thereby amplifying copies of the polynucleotides it contains. Cloning vectors may, but are not limited to, generally include sequence components, including an origin of replication, a promoter sequence, a transcription start sequence, an enhancer sequence, and selectable markers. These components can be appropriately selected by those skilled in the art. For example, the origin of replication may be selected to promote the self-replication of the vector in host cells.

[0187] In some embodiments, this disclosure provides isolated host cells containing the vectors provided herein. These vector-containing host cells may be useful for the expression or cloning of polynucleotides contained in the vectors. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells, particularly human cells.

[0188] Vectors can be introduced into a host using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microprojectile collision, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of the vector of interest are well known in the art. In further embodiments, a mixture of different expression vectors can be used when genetically modifying a donor population of immunoeffector cells, where each vector encodes a different CAR as disclosed herein. The resulting transduced immunoeffector cells form a mixed population of engineered cells, the proportion of which express multiple different CARs.

[0189] In one embodiment, the present disclosure provides a method for preserving genetically engineered cells expressing CARs or TCRs. This involves cryopreserving immune cells so that they remain viable upon thawing. A percentage of immune cells expressing CARs may be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignant tumors. If necessary, the cryopreserved transformed immune cells can be thawed, grown, and increased for more such cells.

[0190] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing the cells, and concentrating them in a medium and container system ("pharmaceutically acceptable") suitable for administration in therapeutically effective doses. Suitable infusion media may be any isotonic medium formulation, typically ordinary saline, Normosol® (Abbott), or Plasma-Lyte® A (Baxter), but 5% dextrose aqueous solution or Ringer's lactate may also be used. Human serum albumin may be supplemented in the infusion medium.

[0191] 4. Allogeneic CAR T cells The process for manufacturing allogeneic CAR T therapy involves collecting selected, screened, and tested healthy donor immune cells, including T cells from healthy donors. Next, the T cells of the donor immune cells are engineered to express a chimeric antigen receptor (CAR) that recognizes a specific cell surface protein expressed in hematological malignancies or solid tumors. The allogeneic T cells are genetically edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. The T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can be knocked out to make the CAR T product resistant to anti-CD52 antibody therapy. Thus, by using anti-CD52 antibody therapy to suppress the host immune system and keep the CAR T engrafted, a complete therapeutic effect can be achieved. Subsequently, the engineered T cells are subjected to further processing, which optionally includes a depletion step to remove unwanted T cells (e.g., unwanted T cells expressing the TCR gene) that express the biomarkers described herein, and a purification step, and are finally cryopreserved in vials for delivery to patients.

[0192] 5. Autologous CAR T cells Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting the patient's own cells (e.g., white blood cells including T cells) and genetically engineering the T cells to express a CAR that recognizes a target expressed on the cell surface of one or more specific cancer cells to kill the cancer cells. Then, the engineered cells are cryopreserved and subsequently administered to the patient.

[0193] Methods of in vitro selection In some embodiments, methods are provided for in vitro sorting of a population of immune cells, wherein a subset of the immune cell population includes engineered immune cells expressing antigen-specific CARs containing an epitope specific to a monoclonal antibody (e.g., an exemplary mimotope sequence). The method includes contacting the immune cell population with an epitope-specific monoclonal antibody and selecting immune cells that bind to the monoclonal antibody to obtain a population of cells enriched with engineered immune cells expressing antigen-specific CARs.

[0194] In some embodiments, the aforementioned monoclonal antibody specific to the aforementioned epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0195] In some embodiments, the aforementioned monoclonal antibodies specific to the aforementioned epitopes are optionally conjugated to magnetic particles. In these embodiments, the step of selecting cells that bind to the monoclonal antibodies can be performed by magnetically activated cell sorting (MACS).

[0196] In some embodiments, the mAb used in the method for sorting immune cells expressing CAR is selected from alemtuzumab, ibritumomab tiusetan, muromonab-CD3, tocitumomab, abisiximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the mAb is rituximab. In another embodiment, the aforementioned mAb is QBEND-10. In yet another embodiment, the mAb binds to TCRa or TCRb.

[0197] In some embodiments, the population of CAR-expressing immune cells obtained when the above-described method for in vitro selection of CAR-expressing immune cells is used contains at least 70%, 75%, 80%, 85%, 90%, and 95% of the CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained when the above-described method for in vitro selection of CAR-expressing immune cells is used contains at least 85% of the CAR-expressing immune cells.

[0198] In some embodiments, the population of CAR-expressing immune cells obtained using the above-described method for in vitro sorting of CAR-expressing immune cells shows increased in vitro cytotoxic activity compared to the initial (unsorted) cell population. In some embodiments, the aforementioned in vitro cytotoxic activity is increased by 10%, 20%, 30%, 40%, or 50%. In some embodiments, the immune cells are T cells.

[0199] In some embodiments, the mAbs are pre-bonded to a support or surface. Non-limiting examples of solid supports include beads, agarose beads, magnetic beads, plastic weld plates, glass weld plates, ceramic weld plates, columns, or cell culture bags.

[0200] CAR-expressing immune cells to be administered to recipients can be enriched in vitro from a source population. Methods for growing the source population may include selecting cells expressing antigens such as the CD34 antigen using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0201] Flow cytometry can be used to quantify specific cell types within a population of cells. Generally, flow cytometry is a method for quantifying the components or structural characteristics of cells using optical means. Because different cell types can be distinguished by quantifying structural characteristics, flow cytometry and cell sorting can be used to count and sort cells with different phenotypes in a mixture.

[0202] In some embodiments, the method used to sort T cells expressing CAR is magnetically activated cell sorting (MACS). Magnetically activated cell sorting (MACS) is a method for separating different cell populations in response to surface antigens (e.g., CD molecules) by using superparamagnetic nanoparticles and a column. Using MACS, pure cell populations can be obtained. Cells in a single-cell suspension can be magnetically labeled with microbeads. The sample is added to a column consisting of ferromagnetic spheres covered with a cell-friendly coating, allowing for rapid and gentle separation of cells. Unlabeled cells pass through, while magnetically labeled cells are retained within the column. The flow-through can be collected as an unlabeled cell fraction. After a washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0203] Detailed protocols for purifying specific cell populations, such as T cells, can be found in Basu S et al. (2010) (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41):1546).

[0204] Pharmaceutical compositions and therapies In some embodiments, the manipulated immune cells described herein are formulated by first recovering them from their culture medium, then washing the cells, and concentrating them in a medium and container system ("pharmaceutically acceptable" carrier) suitable for administration in therapeutically effective doses. Suitable infusion media may be any isotonic medium formulation, typically ordinary saline, Normosol® (Abbott), or Plasma-Lyte® A (Baxter), but 5% dextrose aqueous solution or Ringer's lactate may also be used. Human serum albumin may be supplemented in the infusion medium.

[0205] In various embodiments, the desired therapeutic amount of cells in the composition is typically at least two cells (e.g., at least one CD8+ central or stem cell memory T cell and at least one CD4+ helper T cell subset, or two or more CD8+ central or stem cell memory T cells, or two or more CD4+ helper T cell subsets), or more typically 10 2 It is a super cell, 10 6 The following 10 7 The following 10 8 The following, or 10 9 The following cells, 10 10 It may be fewer cells. The number of cells will depend on the intended use of the composition and the type of cells contained therein. The desired cell density is typically 10 6 Larger than cells / ml, generally 10 7Larger than cells / ml, generally 10 8 The number of cells / ml is greater than 10. A clinically relevant number of immune cells is cumulatively 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The above cells can be distributed for multiple injections. In some aspects of this disclosure, in particular, all injected cells are redirected to a specific target antigen, so that approximately 10 5 / kilogram or about 10 6 / kilogram (10 per patient) 6 ~10 11 A smaller number of cells within the range of ) can be administered. CAR therapy can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic, or heterogeneic to the patient being treated.

[0206] The CAR-expressing cell populations of this disclosure can be administered alone, in combination with diluents, and / or in combination with other components such as IL-2 or other cytokines or cell populations as pharmaceutical compositions. The pharmaceutical compositions of this disclosure may contain, as described herein, a TCR-expressing cell population such as CARs or T cells in combination with one or more pharmaceutically acceptable or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure are preferably formulated for intravenous administration.

[0207] The pharmaceutical composition (solution, suspension, etc.) may contain one or more of the following: a sterile diluent (water for injection, physiological saline (preferably physiological saline), Ringer's solution, isotonic sodium chloride solution, etc.), a fixing oil such as a synthetic monoglyceride or diglyceride that functions as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol, or other solvents; an antimicrobial agent such as benzyl alcohol and methylparaben; an antioxidant such as ascorbic acid and sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, and phosphate; and an agent for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

[0208] Methods are provided for treating diseases or disorders, including cancer and autoimmune diseases. In some embodiments, the disclosure relates to the creation of a T cell-mediated immune response in a subject, which includes administering an effective amount of the engineered immune cells of the present invention to the subject. In some embodiments, the T cell-mediated immune response is against target cells(s). In some embodiments, the engineered immune cells include chimeric antigen receptors (CARs). Using CARs including the immune cells of the present disclosure, CD19-related diseases or disorders and / or BCMA-related diseases or disorders can be treated. In some embodiments, the diseases or disorders may include, but are not limited to, autoimmune diseases, including lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, and myositis.

[0209] All references cited herein, including patents, patent applications, articles, textbooks, etc., and references cited therein, are incorporated herein by reference only if they do not already exist.

[0210] The following embodiments are provided for illustrative purposes only. Therefore, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. [Examples]

[0211] Example 1 - Immune cells engineered to express chimeric antigen receptors (CARs) and chimeric cytokine receptors (CCRs) In connection with adoptive immune cell transplantation, the beneficial activity of CAR T cells, e.g., activation, proliferation, persistence, and / or potency, can be enhanced via cytokine-induced cytokine receptor signaling, also known as signal 3. Current approaches to signal 3 include combining CAR T cell therapy with systemic infusion of recombinant cytokines / cytokine mimes and manipulating CAR T cells to secrete / express cytokines extracellularly, but these have drawbacks such as the risk of systemic toxicity. In an alternative approach, signal 3 can be enhanced using a constitutively active chimeric cytokine receptor (CACCR) (see U.S. Patent No. 20200291090A1, which is incorporated herein by reference in its entirety). An exemplary CACCR was designed using a sequence derived from the thrombopoietin receptor (TpoR). TpoR can activate the JAK-Stat signaling pathway and signaling as a homodimeric receptor. In this embodiment, immune cells were manipulated to express a CD19-specific CAR and a constitutively active chimeric cytokine receptor (CACCR).

[0212] CAR-T cells containing an exemplary CD19 CAR having a 4G7 scFv directed to CD19 bound to a 4-1BB and CD3z signaling domain (see, for example, U.S. Patent No. 10,874,693, which is incorporated herein by reference in whole) were selected for further manipulation of the CACCR. CD19-specific CAR-T cells co-expressing a CACCR consisting of a cleaved IL2Rb tail were generated. CAR-T cells containing an exemplary BCMA CAR having a P5A2 scFv directed to BCMA bound to a 4-1BB and CD3z signaling domain (see, for example, U.S. Patent Application No. US20210260118A1, which is incorporated herein by reference in whole) were also selected for testing.

[0213] Figure 1A shows a schematic diagram of the engineered constitutively active chimeric cytokine receptor. Figure 1B shows a schematic diagram of the lentiviral vector used to co-express the constitutively active chimeric cytokine receptor and the anti-CD19 CAR. Constructs encoding the anti-CD19 CAR containing SEQ ID NO: 335 (see Table 3) and CACCR containing SEQ ID NO: 343 (see Table 4) were used.

[0214] Figure 1C shows a schematic diagram of the lentiviral vector used to co-express a constitutively active chimeric cytokine receptor and an anti-BCMA CAR. Constructs encoding an anti-BCMA CAR containing SEQ ID NO: 342 (Table 3) and / or CACCR containing SEQ ID NO: 343 (Table 4) were used.

[0215] In an exemplary protocol for producing lentiviruses encoding CACCR and CD19 CAR, HEK293T cells were plated at a rate of 450,000 cells per mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentiviruses were prepared by mixing 1.5 μg of the lentiviral packaging vector psPAX2, 0.5 μg of pMD2G, and 0.5 μg of a suitable transfer CAR vector in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate ("DNA mix"). 10 μL of lipofectamine 2000 (Invitrogen) in 250 μL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes, and a total volume of 500 μL was slowly added to the side of the wells containing HEK293T cells. One day after transfection, the culture medium from each well of HEK293 T cells in a 6-well plate was replaced with 2 mL of T cell transduction medium per well, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, the lentiviral supernatant from the HEK293 T cells was collected, passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and the crude lentiviral supernatant was used directly for T cell transduction. On day 0, purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL of human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat#130-111-160, 1:100 dilution) in a Grex-24 plate (Wilson Wolf, cat#80192M). On the second day, T cells were resuspended in T cell transduction medium at a concentration of 500,000 cells / mL and transduced with 100 IU / mL of human IL-2 in a Grex-24 plate along with an equal volume of crude lentivirus supernatant.On day 5, CACCR-expressing CAR-T cells were cultured by replacing the used medium with T cell proliferation medium, i.e., 100 IU / mL human IL-2 along with X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). At this point, control CAR-T cells lacking CACCR were grown either with 100 U / mL human IL-2 alone, or with 100 U / mL human IL-2 and 10 ng / mL human IL-15 (Miltenyi Biotec). If necessary, cells were grown in larger G-Rex containers (Wilson Wolf) using T cell proliferation medium and recombinant cytokines at their respective concentrations. On day 13, cells were stained with the Zombie NIR Fixable Viability Kit (Biolegend), labeled with BUV395 conjugate CD3 antibody (Biolegend) and P5A2 scFv-specific anti-idiotype antibodies, and then FACS sorting was performed to enrich CAR+ T cells. Next, the selected CAR+ T cells were cultured for a further 2 days in T cell proliferation medium in a Grex-24 plate. CACCR CD19 CAR+ T cells were left in the absence of exogenous cytokines and treated with either 100 U / mL human IL-2 or 10 ng / mL human IL-15. Selected control CAR+ T cells were also left in the absence of exogenous cytokines. On day 15, the live CAR+ T cells were enriched using the Easy Sep Dead Cell Removal Kit (StemCell Technologies), and the cell pellet was rapidly frozen for subsequent RNA extraction and NanoString gene expression analysis (human CAR-T panel; NanoString Technologies).

[0216] Example 2 - Detection of CACCR in stimulated CAR T cells Co-culture setup for CAR T cell stimulation. In this experiment, two different CAR T cell preparation lots were used as effector cells: a) PBMCs used as a starting material to provide cells engineered to express CD19-specific CAR and CACCR (lot 02), and b) CD4 / CD8+ T cells used as a starting material to provide cells engineered to express CD19-specific CAR and CACCR (lot 04). Both CAR T cell preparation lots were engineered from the same donor. Cells from the two CAR T preparation lots were stimulated using irradiated DGL (Daudi cells purchased from ATCC (CCL-213™), engineered with GFP-luciferase, and irradiated for the assay) or CD19-Fc (Acro bio, USA) at 2.5 ug / ml. Both effector cells were thawed and cultured at 1E6 cells / ml in X-vivo medium containing 10% FBS. Irradiated DGL was added to the culture in a 2:1 ratio of effector cells to target cells. After setup, co-cultures were sampled at 20, 24, and 48 hours for Western blotting and flow cytometry.

[0217] Intracellular staining of P2A using flow cytometry. Collected cells were stained with CD19-specific CAR anti-idiotype antibody PE (Acro Biosystems) and FVS780 (BD Bioscience) in flow staining buffer (554656, BD). Cells were fixed and permeabilized using the FOXP3 transcription factor staining buffer set (00-5523-00, eBioscience) according to the protocol. Generally, cells were fixed at room temperature (RT) for 20 minutes using the fixation / permeabilization buffer and stained with P2A-APC (NBP2-59627AF647, Novus Bio) in the permeabilization buffer for 30 minutes at room temperature. Cells were resuspended in the staining buffer and analyzed using the LSRFortessa® cell analyzer (BD Bioscience0). Data were analyzed using FlowJo9.2 software (Tree Star, Inc.).

[0218] As described above, CAR T cell preparation cells engineered to express CD19-specific CAR and CACCR were incubated with or without IR-DGL. As shown in Figure 2, the cells were harvested and stained with CD19-specific CAR anti-idiotype antibody PE (Acro Biosystems) and FVS780 (Live / Dead survival staining) for 24 hours (Figure 2A) or 48 hours (Figure 2B), and then fixed for intracellular staining of P2A using a transcription factor staining buffer set. P2A and CAR+ double-positive cell populations were gated from single live cells under different culture conditions.

[0219] Figure 3 shows the results of additional experiments in which cells were incubated with or without CD19Fc or IR-DGL (Figure 3A). Cells were harvested after 24 hours and stained with CD19-specific CAR anti-idiotype antibody PE (the entire antibody is incorporated by reference, see U.S. Patent No. 20200331998A1) (custom-made by Biosystems) and FVS780 (Live / Dead survival staining), and then fixed for intracellular staining of P2A using a transcription factor staining buffer set. P2A+ and CAR+ double-positive cell populations were gated from single live cells under different culture conditions.

[0220] P2A detection was performed using Western blotting. HEK293T cells transfected with BCMA CAR+CACCR were used as a positive control for P2A protein detection. Cell lysates of all samples at a concentration of 0.2E6 cells / µl were prepared using a mixture of M-PER buffer, a protease inhibitor, and a reducing agent. 0.26E6 cells for the positive control and 2.6E6 cells for the other samples were placed in a 10% Bis-tris gel. A mixture of 1:200 2A peptide (3H4) antibody (Novsubio, Cat#NBP2-59627) and 1:1000 β-tubulin (Cell Signaling, Cat#2128L) was used as the primary antibody. A 1:5000 mixture of IRD-800CW goat anti-rabbit IgG (LI-COR, Cat#926-32211) and IRD-680CW goat anti-mouse IgG (LI-COR, Cat#926-68070) was used as the secondary antibody. The blot was visualized using Odyssey CLx. Table 6 lists the samples for each lane of the Western blot. [Table 15]

[0221] As shown in Figure 4, tubulin was shown as a band at 50 kDa (see up arrow) and served as a technical control. The P2A protein was shown as a band at 24.3 kDa (see down arrow). The P2A protein was detected in the positive control and stimulated samples, shown in lanes 1, 2, 3, 4, 6, 7, and 8. The P2A protein was not detected in the unstimulated samples, shown in lanes 5 and 9.

[0222] CAR T cells containing anti-BCMA CARs with P5A2 scFv and CACCR expression (see Example 1 and Table 5 above) were treated with PMA + ionomycin (P+I), and the detection level of CACCR was observed. CAR T cells containing anti-BCMA CARs with P5A2 scFv but without CACCR expression (see Example 1 and Table 5 above) were included as a negative control.

[0223] The samples listed in Table 7 below were tested. [Table 16]

[0224] Frozen whole blood containing EDTA as an anticoagulant (Stem Cell Technologies) was used. Frozen CAR T cell lots (anti-BCMA: 14e6 CAR+ cells / ml per 1ml; anti-BCMA+CACCR: 40e6 CAR+ cells / 2ml - see Table 8) were used. Frozen-thawed CAR T cells were spiked into frozen-thawed whole blood (WB). 10% of viable whole blood cells were CAR+ cells. [Table 17]

[0225] Anti-BCMA CARs possessing P5A2 scFv were detected using an anti-idiotype antibody against CARs (see published U.S. Patent Application No. 20240101710A1, which is incorporated herein by reference in its entirety).

[0226] Anti-BCMA CAR was detected in all samples (data not shown). CACCR was detected via P2A in either a) WB / CAR mixtures stained without stimulation after cell mixing, or b) WB / CAR mixtures stimulated with PMA + ionomycin for 24 hours before staining. After staining cells for viability and surface markers, fix / perm was performed for intracellular P2A staining. As shown in Figures 6A-6B, CCR was detected in P+I stimulated samples (right panel) using the anti-P2A antibody P2A-APC (NBP2-59627AF647, Novus Bio), but not in unstimulated samples (left panel). As expected, CACCR was not detected in any sample (data not shown).

Claims

1. An in vitro method for detecting bicistronally expressed polypeptides in chimeric antigen receptor (CAR) T cells, a) Contacting the CAR T cells with an exogenous drug, wherein the CAR T cells comprise a polynucleotide expressing the bicistron-expressed polypeptide, a linked peptide, and an additional polypeptide, the linked peptide being at the C-terminus of the bicistron-expressed polypeptide, and the contact, b) The method comprising detecting the linked peptide after step a) to indicate the presence of the bicistronically expressed polypeptide in the CAR T cells.

2. The method according to claim 1, wherein the bicistronally expressed polypeptide includes a transmembrane domain.

3. The method according to claim 1, wherein the bicistronally expressed polypeptide is a membrane protein containing a transmembrane domain.

4. The method according to claim 2 or 3, wherein the bicistronally expressed polypeptide further comprises an intracellular domain.

5. The method according to any one of claims 1 to 4, wherein the linked peptide is located at the C-terminus of the bicistronally expressed polypeptide.

6. The method according to any one of claims 3 to 5, wherein the linked peptide is located at the C-terminus of the intracellular domain.

7. The method according to any one of claims 1 to 6, wherein the bicistronally expressed polypeptide further comprises an extracellular domain.

8. The method according to any one of the prior claims, wherein the linked peptide is heterogeneous to the bicistronically expressed polypeptide.

9. The method according to any one of claims 4 to 8, wherein the linked peptide is continuous with the intracellular domain of the bicistronically expressed polypeptide.

10. The method according to any one of claims 4 to 9, wherein the intracellular domain is an intracellular signal transduction domain.

11. The method according to any one of claims 1 to 10, wherein the additional polypeptide is CAR.

12. The method according to any one of the prior claims, wherein the exogenous agent is selected from small molecule reagents and target molecules, and the CAR specifically binds to the target molecule.

13. The method according to claim 12, wherein the small molecule reagent stimulates cytokine production in the CAR T cells.

14. The method according to claim 12 or 13, wherein the small molecule reagent comprises phorbol myristate acetate (PMA) and / or ionomycin.

15. The method according to claim 12, wherein the target molecule is a soluble target molecule.

16. The method according to claim 12, wherein the target molecule is expressed on the surface of the target cell.

17. The method according to claim 16, wherein the contact includes bringing the CAR T cells into contact with the target cells.

18. The method according to claim 16 or 17, wherein the target cells are inactivated target cells.

19. The method according to claim 15, wherein the contact step includes bringing the CAR T cells into contact with the soluble target molecule.

20. The method according to any one of the prior claims, wherein the linked peptide is cleavable.

21. The method according to claim 20, wherein the linked peptide is cleavable by an enzyme.

22. The method according to claim 20, wherein the linked peptide is a self-cleaving peptide.

23. The method according to claim 22, wherein the self-cleaving peptide is a P2A or T2A peptide.

24. The method according to any one of the prior claims, wherein the linked peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 305 to 320.

25. The method according to any one of the prior claims, wherein the bicistronally expressed polypeptide is a chimeric cytokine receptor (CCR), and the CCR comprises a transmembrane domain and an intracellular domain.

26. The method according to claim 25, wherein the CCR further comprises an extracellular domain.

27. The method according to claim 25, wherein the CCR does not include an extracellular domain.

28. The method according to claim 25 or 26, wherein the CCR is an inducible CCR.

29. The method according to any one of claims 25 to 27, wherein the CCR is a constitutively active CCR.

30. The method according to any one of the prior claims, wherein the detection step comprises detecting the linked peptide by flow cytometry and / or protein immunoblotting assay.

31. The method according to claim 8, wherein the bicistronally expressed polypeptide further comprises an intracellular domain.

32. The method according to claim 31, wherein the linked peptide is located at the C-terminus of the intracellular domain.

33. An in vitro method for analyzing a population of CAR T cells, wherein the CAR T cells express bicistronically expressed polypeptides, linked peptides, and CAR, and the method is a) Contacting a sample of the CAR T cell population with an exogenous drug, b) The method comprising detecting the bisistronally expressed polypeptide in the sample after the contact step.

34. The method according to claim 33, wherein the linked peptide is bound to the C-terminus of the bicistronally expressed polypeptide, and the step of detecting the bicistronally expressed polypeptide includes detecting the linked peptide in the sample.

35. c) The method according to claim 33 or 34, further comprising analyzing whether the bicistronally expressed polypeptide is detected in the sample at or above a predetermined level.

36. The method according to claim 35, wherein the predetermined level means that the linked peptide is present in at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the CAR T cells in the sample.

37. The method according to any one of claims 33 to 36, wherein the population of CAR T cells comprises a CAR T cell active pharmaceutical ingredient or a CAR T cell preparation.

38. d) If the bicistronically expressed polypeptide is detected in the sample at or above a predetermined level, the method of claim 37 further comprises formulating the population of CAR T cells to form a formulation, and optionally e) freezing the formulation.

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