Method for detecting engineered cells

Abstract

Provided herein are methods for detecting the expression of different bicistronic-expressed polypeptides, such as chimeric cytokine receptors, that have been engineered into a population of immune cells (e.g., a population of CAR T cells). In one aspect, the disclosure provides in vitro methods of detecting bicistronic expressed polypeptides in chimeric antigen receptor (CAR) T cells.

Description

Cross Reference to Related Applications

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

[0002] SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. The electronic file was created on June 14, 2024, is named “AT-061 / 02WO_ST26.xml” and has a size of 533,947 bytes. BACKGROUND

[0003] The adoptive transfer of genetically modified immune cells that recognize malignancy-associated antigens is showing promise as a 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 a chimeric antigen receptor (CAR), which is a fusion protein composed of an antigen recognition moiety and a T cell activating domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)). Immune cells containing a CAR (e.g., CAR-T cells (CAR-T)) are engineered to confer antigen specificity to them while maintaining or enhancing their ability to recognize and kill target cells.

[0004] T cell proliferation, cytotoxic potency, and persistence are driven by signaling pathways. Conventional CAR designs provide two signals: CD3 zeta activation (signal 1) and costimulation (signal 2, e.g., via 4-1BB, OX40, and / or CD28 expression). In some contexts, a third signal (signal 3) can be desirable, i.e., cytokine-induced cytokine receptor signaling (e.g., for immune-enhancing cytokine support). However, some approaches for providing signal 3 (e.g., systemic infusion of recombinant cytokines / cytokine mimetics) have encountered serious limitations, such as systemic toxicity in humans. Alternative approaches include engineering immune cells (such as CAR T cells) to express chimeric cytokine receptors (CCRs), as described in US2019-0292533A1, US2020-0291090A1, US2020-0276238A1, and US2021-0061881A1, each incorporated by reference herein in its entirety. Improvements to CAR-T cell therapy via the use of additional non-CAR polypeptides (such as CCRs) (e.g., to improve the efficacy and / or accuracy of treatment) would benefit the patient community.

[0005] There is a need for methods for detecting expression of different bi-cistronic expressed polypeptides (e.g., chimeric cytokine receptors) that have been engineered into a population of immune cells (e.g., a population of CAR T cells). Provided herein are compositions and methods that address this need. SUMMARY

[0006] In one aspect, the present disclosure provides an in vitro method of detecting a bi-cistronic expressed polypeptide in a chimeric antigen receptor (CAR) T cell. In one embodiment, the method comprises the step of contacting the CAR T cell with an exogenous agent. In another embodiment, the CAR T cell comprises a polynucleotide expressing the bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide. In some embodiments, the linker peptide is located at the C-terminus of the bi-cistronic expressed polypeptide. In another embodiment, the method further comprises the step of detecting the linker peptide after the contacting step. In yet another embodiment, detection of the linker peptide indicates the presence of the bi-cistronic expressed polypeptide in the CAR T cell.

[0007] In other embodiments, the bi-cistronic expressed polypeptide comprises a transmembrane domain, or is a membrane protein comprising a transmembrane domain. In one embodiment, the bi-cistronic expressed polypeptide further comprises an intracellular domain. In another embodiment, the linker peptide is located at the C-terminus of the bi-cistronic expressed polypeptide, or the linker peptide is located at the C-terminus of the intracellular domain. In other embodiments, the bi-cistronic expressed polypeptide further comprises an extracellular domain. In one embodiment, the linker peptide is heterologous to the bi-cistronic expressed polypeptide. In another embodiment, the linker peptide is adjacent to the intracellular domain of the bi-cistronic expressed polypeptide. In another embodiment, the intracellular domain is an intracellular signaling domain. In some embodiments, the additional polypeptide is a CAR.

[0008] In another embodiment, the exogenous agent used in the contacting step is selected from the group consisting of a small molecule agent and a target molecule, wherein the CAR specifically binds to the target molecule. In other embodiments, the small molecule agent stimulates cytokine production in the CAR T cell. In another embodiment, the small molecule agent 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 a target cell. In another embodiment, the contacting step comprises contacting the CAR T cell with the target cell. In other embodiments, the target cell is an inactivated target cell. In other embodiments, the contacting step comprises contacting the CAR T cell with the soluble target molecule. In one embodiment, the linker peptide is cleavable. In one embodiment, the linker peptide is cleavable by an enzyme. In other embodiments, the linker peptide is a self-cleaving peptide. In one embodiment, the self-cleaving peptide is a P2A or T2A peptide. In another embodiment, the linker peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 305-320. In another embodiment, the bi-cistronic expressed polypeptide is a chimeric cytokine receptor (CCR). In one embodiment, the CCR comprises a transmembrane domain and an intracellular domain. In another embodiment, the CCR further comprises an extracellular domain, or the CCR does not comprise 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 comprise an extracellular domain.

[0009] In another embodiment, the detecting step comprises detecting the linker peptide by flow cytometry and / or a Western blot assay.

[0010] In another aspect, the present disclosure provides an in vitro method for analyzing a population of CAR T cells, wherein the CAR T cells express a bi-cistronic expressed polypeptide, a linker peptide, and a CAR. In one embodiment, the method comprises the step of contacting a sample of the population of CAR T cells with an exogenous agent. In other embodiments, the method further comprises the step of detecting the bi-cistronic expressed polypeptide in the sample after the step of contacting. In one embodiment, the linker peptide is attached to the C-terminus of the bi-cistronic expressed polypeptide. In other embodiments, the step of detecting the bi-cistronic expressed polypeptide comprises detecting the linker peptide in the sample. In another embodiment, the method further comprises the step of analyzing whether a predetermined level or more than a predetermined level of the bi-cistronic expressed polypeptide is detected in the sample. In one embodiment, the predetermined level is the presence of the linker peptide 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 other embodiments, the population of CAR T cells constitutes a CAR T cell drug substance or a CAR T cell drug product. In one embodiment, the method further comprises the step of formulating the population of CAR T cells to form a drug product if a predetermined level or more than a predetermined level of the bi-cistronic expressed polypeptide is detected in the sample. In another embodiment, the method comprises the step of freezing the drug product. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 A- Figure 1 C shows the following schematics: engineered constitutively active chimeric cytokine receptor (CACCR) of the disclosure (A) and vectors for expression of CACCR (B-C).

[0012] Figures 2A-2B shows staining of cells with anti-idiotypic antibody against anti-CD19 antibody measured by flow cytometry at 24 hours (A) and 48 hours (B) with or without incubation with CD19-Fc (A) or irradiated cells (B).

[0013] Figures 3A-3B shows staining of cells with anti-idiotypic antibody against anti-CD19 antibody and anti-P2A antibody with or without incubation with CD19-Fc (A) or irradiated cells (B) measured by flow cytometry.

[0014] Figure 4 shows detection of P2A in engineered immune cells using western blot.

[0015] Figure 5 shows manufacturing workflow for engineered immune cells (e.g., CAR T cells).

[0016] Figures 6A-6B CCR detection in P+I stimulated samples and unstimulated samples. DETAILED DESCRIPTION

[0017] The present disclosure provides methods for the determination and / or detection of a bi-cistronic expressed polypeptide in an engineered immune cell (e.g., a chimeric antigen receptor (CAR) cell). In one embodiment, the method comprises the step of contacting the engineered immune cell with an exogenous agent. In another embodiment, the engineered immune cell comprises a polynucleotide sequence expressing a bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide. In another embodiment, the linker peptide is located at the C-terminus or the N-terminus of the bi-cistronic expressed polypeptide. In other embodiments, the method further comprises the step of detecting the linker peptide after the contacting step, thereby indicating the presence of the bi-cistronic expressed polypeptide in the CAR T cell.

[0018] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al. eds., 1994); Current Protocols in Immunology (J.E. Coligan et al. eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.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 J. D. Capra Eds., Harwood Academic Publishers, 1995). For example, gene editing technologies using TALENs, CRISPR / Cas9, and megaTAL nucleases are within the skill in the art and are well explained in the literature, such as T. Gaj et al., Genome-Editing Technologies: Principles and Applications,. Cold Spring Harb Perspect Biol 2016; 8: a023754 and citations therein.

[0019] Definitions 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."

[0020] As used herein "autologous" means that the cell, cell line, or cell population used to treat a subject is obtained from the subject.

[0021] As used herein "allogeneic" means that the cell or cell population used to treat a subject is not obtained from the subject, but from a donor.

[0022] As used herein, "bicistronic expressed polypeptide" refers to a polypeptide encoded by a first nucleic acid sequence, which is contained together with a second nucleic acid sequence encoding at least a second polypeptide in a polynucleotide or expression vector / cassette, wherein the first and second nucleic acid sequences are contained as consecutive nucleic acid sequences in the polynucleotide or vector. For bicistronic expression, the polynucleotide or expression vector / cassette includes a nucleic acid sequence encoding a ribosomal jumping sequence, such as, but not limited to, sequences encoding self-cleaving peptides (e.g., 2A peptides, including P2A, T2A, E2A, and F2A peptides). These peptides (identified in the aphthovirus subgroup of small RNA viruses) cause the ribosome to "jump" from one codon to the next, but no peptide bond is formed between the two amino acids encoded by the codon. A "codon" refers to three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single consecutive open reading frame within mRNA, where the polypeptides are separated by 2A oligopeptide sequences within the frame. This type of ribosome jumping mechanism is well known in the art and is known to be used by several vectors to express several proteins encoded by a single messenger RNA.

[0023] As used herein, the term "labeler" generally refers to an agent capable of interacting with cellular components, including but not limited to molecules on and / or within the cell membrane, and intracellular molecules. The interaction between the agent and the cellular component can be covalent or non-covalent, reversible or irreversible. The labeler can be specific to cellular components, including but not limited to cellular biomolecules (e.g., peptides, nucleic acids, lipids, etc.). In some embodiments, the labeler can be an agent specific to a biological target, such as an antibody or antibody fragment. In one embodiment, the labeler is an agent specific to cell surface molecules (e.g., cell surface proteins or cell membrane proteins). In some cases, the labeler may include one or more detectable markers. In some embodiments, the labeler comprises an antibody, which optionally conjugates to a detectable marker.

[0024] In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a molecule capable of undergoing a colorimetric reaction, a magnetic label, a radioactive label, an oligonucleotide label, and a hapten. In some embodiments, the fluorescent label is selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Renilla luciferin, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine 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, AmCyanl, mTFPl, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreenl, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellowl, KusabiraOrange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRedl, Katusha, P3, peridinin 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 can be directly or indirectly coupled or conjugated to a labeling agent.For the indirect form, one or more labels can be coupled or conjugated to a molecule capable of binding to a labeling agent. For example, a label can be conjugated to an oligonucleotide sequence that is complementary to another oligonucleotide sequence from an oligonucleotide conjugated to a labeling agent (e.g., an antibody conjugated to an oligonucleotide). In the case of a binding agent such as a secreted molecule binding agent, e.g., a secreted cytokine binding agent, a label can also be used with the methods and compositions of the disclosure.

[0025] As used herein, “immune cells” refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells (e.g., a / b T cells and g / d T cells, regulatory T (Treg) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0026] As used herein, the term “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0027] As used herein, “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors include all of those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating the recombinant polynucleotide.

[0028] As used herein, “expression cassette” refers to one or more expression units of one or more coding sequences operably linked to a promoter. In some embodiments, expression of multiple coding sequences can be driven by one promoter, and more than one coding sequence is linked by a sequence encoding a 2A peptide (e.g., P2A or T2A). In some embodiments, expression of multiple coding sequences is by ribosomal skipping. As an example, a bicistronic expression cassette allows for expression of two proteins from the same RNA transcript driven by one promoter. In some embodiments, expression of multiple coding sequences can be driven by multiple promoters, each operably linked to a coding sequence.

[0029] As used herein, "functionally expresses" a gene means that the gene is expressed and that the expression produces a functional gene end product. For example, if a gene encodes a protein, a cell functionally expresses the gene if expression of the gene ultimately results in a protein with the proper function. Thus, for example, a gene is not functionally expressed if the gene is not transcribed, or if expression of the gene ultimately results in an RNA that is not translated or that is translated only into a nonfunctional protein (e.g., a protein that is not properly folded or that is not transported to its site of action (e.g., a membrane, for a membrane-bound protein)). 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).

[0030] The term "extracellular ligand-binding domain" as used herein refers to an oligopeptide or polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell associated with a particular disease state. The term "stalk domain" is used herein to refer to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk domain serves to provide more flexibility and accessibility to the extracellular ligand-binding domain.

[0031] The term "intracellular signaling domain" refers to the portion of a protein that transduces a signal from a signal source to an effector function signal and directs the cell to perform a specialized function.

[0032] As used herein, the term "exogenous" refers to any material introduced or produced from outside of an organism, cell, tissue, or system. In some embodiments, an exogenous or recombinant sequence or an exogenous or recombinant protein is not a naturally occurring sequence or protein and is not endogenous or native to the cell, tissue, or organism.

[0033] The term "intracellular signaling domain" refers to the portion of a protein that transduces a signal from a signal source to an effector function signal and directs the cell to perform a specialized function.

[0034] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. It has been shown that the antigen- binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include a Fab; Fab'; F(ab')2; a Fd fragment consisting of a VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (see, e.g., Ward et al., Nature 341 :544-546, 1989), and an isolated complementarity determining region (CDR).

[0035] An antibody, antibody conjugate, or polypeptide that "specifically binds" to a target is a term of art well understood in the field, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if its reaction or association with a particular cell or substance is more frequent, faster, of longer duration, and / or of greater affinity than its reaction or association with alternative cells or substances. An antibody is said to "specifically bind" to a target if its binding to the target is of greater affinity, avidity, ease, and / or duration than its binding to other substances. It is also understood in accordance with this definition that, for example, an antibody (or portion or epitope) that specifically binds to a first target can or can not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but it can include) exclusive binding.

[0036] The "variable region" of an antibody refers to the variable region of the antibody light chain or variable region of the antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together and in relation to the CDRs from the other chain by the FRs to form an antigen binding site of an antibody. There are several techniques for determining CDRs, for example, techniques based on sequence variability across species (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), the Chothia system (i.e., Chothia and Lesk, J. Mol. Biol. (1987) 196(4):901-917). As used herein, CDRs can refer to CDRs defined by either method or by a combination of the two methods.

[0037] Antibodies (and CARs) of the present disclosure can be produced using techniques well known in the art, for example, recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques readily known in the art (see, e.g., Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F.A. et al., J. Mol. Biol., 373(4):924-40, 2007).

[0038] As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. The polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after the chain assembles. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino- phosphoramidates, amino -nucleoside phosphoramidates, etc.), with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), with intercalators (e.g., acridines, actinomycin, et al.), chelators, with similar nucleoside analogs, and the like. One or more of the nucleotides can be modified to confer either resistance to nuclease degradation, or to impart some other property, such as fluorescence or biotinylated. Non-nucleotide components can be present, such as, for example, a phosphate backbone of one or more of the internucleotide linkages. These and other modifications are well known to those skilled in the art and are described, for example, in the following references: Freier & Altmann, Nucleic Acids Res. 1997, 25: 4429-4443; Freier et al., Arch. Biochem. Biophys. 1987, 258: 30- 41; Uhlman & Peyman, Chemical Reviews 1990, 90: 543-584; Uhlman, Chimia 1993, 47: 139- 150; Sanghvi, Y.S., Antisense Research and Applications, CRC Press, 1993; and Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press, 1992.The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0039] In some embodiments, the antigen binding domain is a recombinant antigen receptor. The term "recombinant antigen receptor" as used herein refers broadly to a non-naturally occurring surface receptor that comprises 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. CARs are fusion proteins that comprise an antigen recognition moiety, a transmembrane domain, and a T cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)).

[0040] The term "extracellular ligand binding domain" or "extracellular antigen binding domain" as used herein refers to a polypeptide that is capable of binding a ligand or antigen or is capable of interacting with a cell surface molecule, such as a ligand or surface antigen. For example, the extracellular ligand binding or antigen binding domain can be selected to recognize a ligand that serves as a cell surface marker (e.g., a tumor-specific antigen) on a target cell associated with a particular disease state. In some embodiments, the antigen binding domain comprises an antibody or an antigen binding fragment or antigen binding portion of an antibody. In some embodiments, the antigen binding domain comprises an Fv or scFv, a Fab or scFab, a F(ab')2 or scF(ab')2, a Fd, a monobody, an affibody, a camelid antibody, a VHH antibody, a single domain antibody, or a darpin. In some embodiments, the ligand binding domain comprises a partner of a binding pair, such as a ligand that binds to a surface receptor, or an ectodomain of a surface receptor that binds to a ligand.

[0041] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signal functions signals and directs the cell to perform specialized functions.

[0042] Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X". Numerical ranges include the numbers defining the range.

[0043] It should be understood that wherever aspects are described herein with the language "comprising" either "a" or "an" or "the" feature, it is intended to leave open the possibility that "comprising" also includes aspects with the features of "consisting of" and / or "consisting essentially of".

[0044] An "antigen binding protein" comprises one or more antigen binding domains. An "antigen binding domain" as used herein means any polypeptide that binds to a specified target antigen. In some embodiments, an antigen binding domain binds to an antigen on a tumor cell. In some embodiments, an antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen.

[0045] Antigen binding domains include, but are not limited to, antibody combining regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen binding domain is an antigen binding domain comprising a portion of an antibody, regardless of how that portion is obtained or synthesized, that lacks at least some of the amino acids present in a full-length chain, but which still is capable of specifically binding to a target antigen. Such fragments are biologically active in that they bind to target antigens and can compete with other antigen binding domains, including intact antibodies, for binding to a given epitope.

[0046] 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 (a heavy chain variable domain paired with a light chain variable domain on the same polypeptide, connected by a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprising two antigen binding sites), multispecific antigen binding domains, and single chain antibodies. These fragments can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid, or rabbit. As will be appreciated by skilled artisans, an antigen binding domain can include non-protein components.

[0047] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions (CDRs). The CDRs from each chain of an antibody pair typically align to form a binding site that can bind to a specific epitope. From N- to C-terminus, both light and heavy chain variable regions typically comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are typically referred to as LC CDR1, CDR2, and CDR3.

[0048] In some embodiments, an antigen binding domain comprises one or more complementarity determining regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments comprises a single heavy and / or light chain or portion thereof. These fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding domains including intact antibodies.

[0049] In some embodiments, the antigen binding domain is an antibody or fragment thereof, including 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.

[0050] Assigning amino acids to each of the framework, CDRs, and variable domains generally follows the numbering scheme of: Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); Chothia numbering (see, e.g., Chothia and Lesk, (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, the AbM scheme (Antibody Modeling program, Oxford Molecular) or the AHo system (Honneger and Pluckthun, J Mol Biol (2001) 309(3):657-70).

[0051] In some embodiments, the antigen binding domain is a recombinant antigen receptor. The term “recombinant antigen receptor” as used herein broadly refers to a non-naturally occurring surface receptor that comprises 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. CARs are fusion proteins that comprise an antigen recognition moiety, a transmembrane domain, and a T cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)).

[0052] Where aspects or embodiments of the disclosure are described in terms of Markush groups or other alternatives, the disclosure encompasses not only the entire group listed as a whole, but also each individual member of the group and all possible subgroups of the main group, as well as the main group without one or more of the group members. The disclosure also contemplates explicitly excluding any one or more of the group members from the disclosed and / or claimed embodiments.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control. Throughout the present specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0054] Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0055] Analysis of cell populations In one aspect, the present disclosure provides methods, assays, systems, compositions, and kits for detecting (and / or quantifying or counting) one or more engineered immune cells expressing a bi-cistronic expressed polypeptide from a population of engineered immune cells, such as CAR T cells. In one embodiment, the one or more engineered immune cells comprise a polynucleotide sequence encoding a bi-cistronic expressed polypeptide (or a vector comprising the polynucleotide sequence). In another embodiment, the polynucleotide sequence (or a vector comprising the polynucleotide sequence) further encodes a linker peptide and an additional polypeptide. Typically, the bi-cistronic expressed polypeptide, the linker peptide, and the additional polypeptide (such as a CAR polypeptide) are expressed in the engineered immune cell and can be detected according to the methods described herein. In one embodiment, the methods described herein comprise the step of contacting the engineered immune cell (e.g., a CAR T cell) with an exogenous agent. In other embodiments, the methods further comprise detecting the linker peptide after the contacting step. In another embodiment, the detection of the linker peptide indicates the presence of the bi-cistronic expressed polypeptide in the engineered immune cell.

[0056] The engineered cells can be engineered immune cells (e.g., CAR-T cells). Detection of the bi-cistronic expressed polypeptide can provide important information about the engineered immune cell population prior to, after, and / or prior to administration of the engineered immune cell population to a subject as part of a CAR T cell drug product. Additionally, detection of the bi-cistronic expressed polypeptide in engineered immune cells during and / or after the manufacturing process can provide valuable information about the cells. For example, the disclosed methods allow for detection of the bi-cistronic expressed polypeptide during and / or after the manufacturing process.

[0057] In additional aspects, the disclosure relates to the analysis of different engineered immune cell populations. In one embodiment, the analysis includes a screening method for detecting engineered immune cells expressing the bi-cistronic expressed polypeptide from an engineered immune cell population and / or quantifying the number of the engineered immune cells. As discussed herein, methods for understanding the extent of expression of the bi-cistronic expressed polypeptide in engineered immune cell populations are valuable during the engineered cell manufacturing process. Not only are the methods desirable for clarifying whether the bi-cistronic expressed polypeptide is present in one or more cells of the engineered immune cell population, the methods are important for quantifying the frequency of expression of the bi-cistronic expressed polypeptide in the population with high precision and accuracy. The screening methods of interest include, but are not limited to, screening engineered immune cells during or after the manufacturing process. In one embodiment, the screening includes the step of contacting the engineered immune cell population with an exogenous agent, followed thereafter by the step of detecting the bi-cistronic expressed polypeptide.

[0058] In one aspect, the screening method is a quantitative method and / or a qualitative method. For the screening methods described herein, detection of the bi-cistronic expressed polypeptide in engineered immune cells from an engineered immune cell population can be performed using different methods including, but not limited to, flow cytometry, fluorescence-activated cell sorting (FACS) flow cytometry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), immunoblot assay, immunofluorescence assay, immunochemical (IHC) assay, Western blot analysis, and immunoprecipitation, molecular binding assay.

[0059] Detection using flow cytometry. Instruments for particle analysis (e.g., flow cytometers and scanning cytometers) allow for the use of optical parameters such as light scatter and fluorescence to characterize individual particles or cells (e.g., single engineered immune cells from a population of engineered immune cells). For example, a flow cytometer allows an aqueous suspension containing individual particles (e.g., beads comprising an analyte of interest) or cells to pass through a detection region that exposes the suspension to an excitation light source (such as one or more lasers), thereby enabling the user to measure the light scatter and fluorescence properties of the particles or cells. Labeling the particles or cells with one or more fluorescent dyes can facilitate detection. Multiple different particles or cells can be subjected to flow cytometry for simultaneous detection by labeling the different particles or cells with different dyes that are spectrally distinguishable. In other embodiments, multiple light detectors can be utilized to measure different scatter parameters and / or different spectrally distinguishable dyes. For example, one or more detectors can be configured to measure one or more sets of scatter parameters, and one or more additional detectors can be configured to measure one or more different dyes, which would allow for the generation of data comprising signals for each light scatter parameter and each fluorescence emission.

[0060] Generally measured flow cytometry parameters include, but are not limited to: (i) excitation light scattered by a particle or cell in a primarily forward direction, or forward scatter (FSC), (ii) excitation light scattered by a particle or cell in a primarily lateral direction, or side scatter (SSC), and (ii) light emission from a fluorescent molecule in one or more channels (frequency ranges) of the spectrum or from a fluorescent dye that is primarily detected in that channel. In one embodiment, different cell types from a population of engineered immune cells can be identified by FSC, SSC, and fluorescence emission obtained by labeling various cell surface proteins on the cells with dye-labeled antibodies.

[0061] Data obtained from analysis of particles or cells (e.g., individual engineered immune cells from an engineered immune cell population) by multicolor flow cytometry is multi-dimensional, with each cell corresponding to a point in a multi-dimensional space defined by the measured parameters. A population of cells or particles is identified as a cluster of points in the data space. Identification of clusters and, thus, populations can be performed manually by drawing gates around populations shown in one or more 2-dimensional plots (referred to as “scatter plots” or “dot plots”) of the data. Alternatively, clusters can be identified and gates defining population boundaries can be determined automatically. Flow cytometry is an important tool for analyzing and / or isolating particles or cells (e.g., individual engineered immune cells from an engineered immune cell population) and cellular analytes or components thereof. Thus, flow cytometry can be used in the context of engineered immune cell analysis and / or isolation. In one embodiment, the present disclosure provides a method of linearly segregating donor cells from an engineered immune cell population using a fluid stream such that they pass individually through a detection device. Individual cells from an engineered immune cell population can be distinguished from other cells by their position in the fluid stream and the presence of detectable markers. Thus, a flow cytometer can be used to detect one or more bi-cistronic expressed polypeptides (as described herein) and / or generate a bi-cistronic expressed polypeptide expression profile of an engineered immune cell population. Such a profile can comprise the percentage of cells expressing a bi-cistronic expressed polypeptide (as described further herein) in the total cell population being analyzed.

[0062] In a further aspect, the present disclosure provides a method of detecting engineered immune cells expressing a bi-cistronic expressed polypeptide from an engineered immune cell population by detecting the level of the bi-cistronic expressed polypeptide. In one embodiment, the detected level indicates that some engineered immune cells express the bi-cistronic expressed polypeptide. In another aspect, the detected level of the bi-cistronic expressed polypeptide is indicative of the number of engineered immune cells expressing the bi-cistronic expressed polypeptide in the engineered immune cell population being analyzed.

[0063] In some embodiments, the method provides a quantitative measure of engineered immune cells from an engineered immune cell population that do not express the bi-cistronic expressed polypeptide. Flow cytometry can be used to quantify cells within a cell population that express or do not express a bi-cistronic expressed polypeptide, and / or to quantify cells of a particular cell type.

[0064] Flow cytometry, as described herein, is a method of quantifying components or structural features of cells primarily through optical means using certain 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 involves two main steps: 1) labeling the selected cell types with one or more detectable labels or reagents, 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 a cell type includes allowing a labeled antibody to bind to a marker expressed by the particular cell type. The antibody can be directly labeled with a fluorescent compound, or indirectly labeled using, for example, a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0065] In another aspect, the present disclosure utilizes flow cytometry to provide methods for generating an expression profile of a bi-cistronic expressed polypeptide expressed in an engineered immune cell population based on detecting the percentage of cells expressing the bi-cistronic expressed polypeptide. In one embodiment, the method includes providing an engineered immune cell population suspected of comprising a bi-cistronic expressed polypeptide. In another embodiment, the method further includes contacting the engineered immune cell population with an exogenous agent. In another embodiment, the method includes detecting the level of the bi-cistronic expressed polypeptide in the engineered immune cell population after the contacting step, wherein the detected level of the bi-cistronic expressed polypeptide indicates that a certain percentage of the engineered immune cell population expresses the bi-cistronic expressed polypeptide. In other embodiments, the method further includes detecting the level of an additional polypeptide in the engineered immune cell population after the contacting 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 level of the bi-cistronic expressed polypeptide and the additional polypeptide, wherein the detected levels indicate the percentage expression of the bi-cistronic expressed polypeptide and the additional polypeptide in the engineered immune cell population.

[0066] In one embodiment, the quantitative screening method is a flow cytometry assay. In one aspect, the methods, assays, systems, compositions, and kits described herein can be used at various stages of manufacturing an engineered cell population. As Figure 5As shown in the middle, the population of immune cells can undergo manufacturing steps 10-15, after which the population is screened at 16 for expression of the bi-cistronic expressed polypeptide. The results of the screening performed at 16 inform the decision about whether to proceed with formulating a cell therapy product (e.g., a CAR T cell therapy product) using the engineered population of immune cells. If the bi-cistronic expressed polypeptide is detected at 18, then the engineered population of immune cells can be considered to have failed the screening assay. If the bi-cistronic expressed polypeptide is detected at 17, then the bi-cistronic expressed polypeptide can be considered to have passed the screening assay, and the cell therapy product manufacturing process can be moved forward.

[0067] In one embodiment, the cell therapy product lot comprising the engineered immune cells that pass at 17 can be derived from the subject that will ultimately receive the population of engineered immune cells. For example, the engineered immune cells can comprise autologous immune cells obtained from a subject that will ultimately receive engineered immune cells (e.g., CAR T cells) obtained from the autologous immune cells. In other embodiments, the engineered immune cells that pass at 17 can be derived from a donor that is a different individual than the subject that will receive the population of engineered cells. For example, the engineered immune cells can comprise allogeneic immune cells obtained from a healthy donor that is a different individual than the subject that will receive engineered immune cells (e.g., CAR T cells) obtained from the allogeneic immune cells.

[0068] In one embodiment, according to the workflow in Figure 5 In addition, screening 16 can be performed after 13 or after 14 in addition to or as an alternative to screening after 15, according to the workflow in

[0069] Polypeptides expressed in bicistron In one aspect, the present disclosure relates to engineered immune cells and populations comprising the engineered immune cells, and methods of detecting polypeptides expressed by the cells and populations. In one embodiment, the polypeptide expressed by the cell is a bi-cistronic expressed polypeptide. In another embodiment, one or more bi-cistronic expressed polypeptides are expressed from a polynucleotide or a vector comprising the polynucleotide, wherein the polynucleotide comprises several distinct 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, wherein the first and second nucleic acid sequences are separated by a third nucleic acid sequence encoding a linker peptide. In one embodiment, the third nucleic acid sequence encodes a series of amino acids, wherein a first amino acid is appended to the C-terminus of the bi-cistronic expressed polypeptide during translation. In another embodiment, a second amino acid translated after the first amino acid is the first N-terminal amino acid of the second polypeptide. In a further embodiment, the first amino acid and the second amino acid are characterized by the absence of a peptide bond between them. In one embodiment, the first polypeptide comprises the first amino acid at its C-terminus. In another embodiment, the second polypeptide comprises the second amino acid at its N-terminus. In one embodiment, the first amino acid is a glycine and / or the second amino acid is a proline.

[0070] In other embodiments, the methods described herein involve detecting a first polypeptide as a bi-cistronic expressed polypeptide. In another embodiment, the second polypeptide is a CAR. In certain embodiments, the first polypeptide detected (i.e., the bi-cistronic expressed polypeptide) is a different polypeptide than the second polypeptide (i.e., the CAR). The first polypeptide (i.e., the bi-cistronic expressed polypeptide) can be any polypeptide, including but not limited to, a receptor polypeptide, a membrane polypeptide, a cytoplasmic polypeptide, and a secreted polypeptide.

[0071] In one embodiment, the first polypeptide is a membrane polypeptide comprising an intracellular domain. In one embodiment, the intracellular domain is an intracellular signaling domain. In another embodiment, the intracellular signaling domain is a cytokine signaling domain.

[0072] 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 responds 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 engineered immune cells (e.g., CAR T cells) to improve cytokine-induced cytokine receptor signaling (see US2019-0292533A1, incorporated by reference in its entirety herein). In one aspect, the present disclosure provides an inducible chimeric cytokine receptor comprising: a dimerization domain; a tyrosine kinase activating domain; and a tyrosine effector domain. In some embodiments, the tyrosine kinase activating domain comprises or is derived from a Janus kinase (JAK) binding domain of 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 comprises or is derived from a tyrosine kinase domain of a receptor tyrosine kinase (RTK). In some of these embodiments, the tyrosine kinase activating domain further comprises a transmembrane domain.

[0073] In another embodiment, the CCR is a CCR lacking an extracellular domain. In yet another embodiment, the CCR is a constitutively active chimeric cytokine receptor (CACCR). The presence of a constitutively active tunable chimeric cytokine receptor allows for signal 3 immune enhancement to meet the need for immune enhancement (see US2020-0291090A1, incorporated by reference in its entirety herein). Generally, the CACCR of the present disclosure is composed of two monomers, each monomer comprising: (a) a transmembrane domain; (b) a JAK binding domain; and (c) a recruiting domain, wherein the monomers are constitutively dimerized. In some embodiments, the CACCR of the present disclosure does not comprise an extracellular ligand binding domain. In some embodiments, the monomers are identical, resulting in a constitutively active homodimer. In some embodiments, the monomers are different, resulting in a constitutively active heterodimer, which can be desirable in certain cases. The monomers of the CACCR of the present disclosure are capable of dimerizing spontaneously and can activate signaling in the absence of any exogenous stimulus or ligand (non-ligand dependent dimerization).

[0074] Exemplary amino acid sequences useful in CACCRs are provided in Table 1A.

[0075] Table 1A: Exemplary transmembrane domain amino acid sequences

[0076] Exemplary transmembrane and JAK binding sequences useful in the CACCRs provided herein are set forth in Table IB.

[0077] Table 1B: Exemplary transmembrane + JAK2 binding domain sequences

[0078] Table 1C provides exemplary amino acid sequences of recruitment domains of CACCRs of the disclosure. In some embodiments, a CACCR of the disclosure comprises a recruitment domain comprising an amino acid sequence selected from one or more of the receptor sequences in Table 1C. In some embodiments, a CACCR of the disclosure comprises 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.

[0079] Table 1C: Recruit domain (Cytotail) sequences

[0080] Table ID shows exemplary CACCR sequences (e.g., full-length CACCR sequences or components thereof) of the disclosure. CACCRs can be expressed with a signal sequence (e.g., CD8SS of sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127)).

[0081] Table 1D - Constitutively active chimeric cytokine receptor (CACCR) sequences

[0082] * underlined LE and SR are exemplary optional linkers that can be inserted between two domains.

[0083] In another embodiment, the CCR is an inducible PD-1 CCR that is active upon engagement with a PD-1 ligand or activation with an anti-PD-1 antibody (see US2020-0276238A1, incorporated by reference in its entirety herein). Also provided herein are constitutively active PD-1 chimeric cytokine receptors. Provided herein are engineered immune cells (e.g., CAR T cells) expressing the PD-1 CCRs of the disclosure. In one embodiment, the inducible PD-1 CCRs of the disclosure activate signaling upon, e.g., binding a PD-1 ligand (e.g., PD-L1, PD-L2) or a PD-1 antibody. These receptors activate signaling upon monomeric clustering and / or dimerization of the receptor. In some embodiments, the monomer of the PD-1 CCRs of the disclosure comprises: (a) a PD-1 ectodomain; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a STAT recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). In some embodiments, the monomer of the PD-1 CCRs of the disclosure comprises: (a) a PD-1 ectodomain; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). The recruiting domain can be a STAT recruiting domain, an AP1 recruiting domain, a Myc / Max recruiting domain; or a NFkB recruiting domain. The PD-1 chimeric cytokine receptors can bind to a PD-1 ligand, and / or be clustered and activated with an anti-PD-1 antibody. The PD-1 chimeric cytokine receptors activate signaling upon, e.g., binding a PD-L1 ligand, a PD-L2 ligand, and / or a PD-1 antibody.

[0084] Regardless of PD-1 ligand availability, the constitutively active PD-1 CCRs of the present disclosure are active, but can increase activity in the presence of a PD-ligand. In some embodiments, a monomer of the constitutively active PD-1 CCRs of the present disclosure comprises: (a) a PD-1 ectodomain; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a STAT recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). In some embodiments, a monomer of the constitutively active PD-1 CCRs of the present disclosure comprises: (a) a PD-1 ectodomain; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). The recruiting domain can be a STAT recruiting domain, an API recruiting domain, a Myc / Max recruiting domain; or a NFkB recruiting domain. In some embodiments, the constitutively active PD-1 CCRs can bind to a PD-1 ligand or an anti-PD-1 antibody, and / or undergo clustering, and the activity of the receptor can be further increased by engagement with a PD-1 ligand or an anti-PD-1 antibody.

[0085] Table 1E shows exemplary PD1 amino acid ectodomain sequences of the present disclosure. It is noted that expression and extracellular location of the exemplary PD1 amino acid sequences can be obtained by use of a signal sequence. In exemplary embodiments, a CD8 signal sequence (CD8SS) MALPVTALLLPLALLLHAARP (SEQ ID NO: 127) is utilized.

[0086] Table 1E: Exemplary ectodomain sequences

[0087] In some embodiments, the PD-1 ectodomain is dominant negative. Table 1F shows exemplary PD-1 dominant negative (DN) sequences of the present disclosure. The DN sequences of Table 1E can be expressed with the help of a signal sequence (e.g., the CD8SS signal sequence of SEQ ID NO: 127).

[0088] Table 1F: PD1 dominant negative (DN) sequences :

[0089] Table 1G shows exemplary transmembrane amino acid sequences coupled to an intracellular JAK2 binding domain sequence for use in a PD-1 CCR.

[0090] Table 1G: Exemplary transmembrane + JAK2 binding domain sequences

[0091] In some embodiments, the PD-1 chimeric cytokine receptor of the present 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 the amino acid sequence of any one of the sequences set forth in Table 1H.

[0092] Table 1H: Recruit domain sequences (Cytotail sequences)

[0093] Table 1I shows exemplary PD-1 CCR sequences of the present disclosure. The receptors can be expressed with a signal sequence, such as CD8SS of SEQ ID NO: 127.

[0094] Table 1I: Exemplary PD-1 CCR sequences (assembled PD1 chimeric cytokine receptors)

[0095] In another embodiment, the CCR is a CCR comprising a TGF-b binding domain. In one embodiment, the CCR is an inducible CCR that is active upon engagement with a TGF-b ligand (e.g., TGF-b1, TGF-b2, and / or TGF-b3) or activation with an anti-TGF-b receptor antibody (see US2021-0061881A1, incorporated by reference in its entirety herein). Provided herein are engineered immune cells (e.g., CAR T cells) expressing a TGF-b CCR of the disclosure. The CCRs of the disclosure activate signaling upon binding of a TGF-b ligand (e.g., TGF-b1, TGF-b2, and / or TGF-b3) or an anti-TGF-b receptor antibody. These receptors activate signaling upon monomeric clustering and / or dimerization of the receptor. The chimeric cytokine receptors of the disclosure are bifunctional chimeric cytokine receptors that can simultaneously neutralize the immunosuppressive effects of TGF-b ligands and mimic the transmission of immune-enhancing cytokine signals. In some embodiments, a monomer of a chimeric cytokine receptor of the disclosure comprises: (a) a binding domain capable of binding a TGF-b ligand or an anti-TGF-b receptor antibody; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a STAT recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). Each domain can be linked directly or via one or more peptide linkers. In some embodiments, a monomer of a chimeric cytokine receptor of the disclosure comprises: (a) a binding domain capable of binding a TGF-b ligand or an anti-TGF-b receptor antibody; (b) a transmembrane domain; (c) a Janus kinase (JAK) binding domain; and; (d) a recruiting domain (e.g., from a cytoplasmic domain of a receptor; e.g., from a cytokine receptor). The recruiting domain can be a STAT recruiting domain, an AP1 recruiting domain, a Myc / Max recruiting domain; or a NFkB recruiting domain. In some embodiments, the chimeric cytokine receptor clusters and activates upon its binding to a TGF-b ligand and / or is clustered and activated with an anti-TGF-b receptor antibody. The chimeric cytokine receptor activates signaling upon, e.g., binding to a TGF-b ligand and / or a TGF-b receptor antibody. In some embodiments, the TGF-b receptor antibody is, but is not limited to, PF-03446962 or LY3022859. In some embodiments, the chimeric cytokine receptor is constitutively clustered or dimerized.

[0096] Linker peptides In one aspect, the in vitro methods described herein utilize a linker peptide. In one embodiment, the linker peptide is part of a bi-cistronic expressed polypeptide. In other embodiments, the engineered immune cell (e.g., CAR T cell) comprises a polynucleotide sequence that encodes a bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide. Table 1J provides a list of exemplary linker peptides and their amino acid sequences.

[0097] Table 1J - Linker peptides

[0098] In one embodiment, the amino acid sequence of the linker peptide comprises D-(V or I)-E-x-N-P-G-P, where x is any amino acid. Upon translation, the amino acid sequence appended to the C-terminus of the first polypeptide (i.e., the bi-cistronic expressed polypeptide) comprises D-(V or I)-E-x-N-P-G, where x is any amino acid.

[0099] The present disclosure encompasses modifications to polypeptides, including bi-cistronic expressed polypeptides and CARs, and linker peptides comprising the sequences provided herein (e.g., Table 1J and Table 2), including functionally equivalent bi-cistronic expressed polypeptides, CARs, or linker peptides having modifications that do not significantly affect their properties, as well as variants with increased or decreased activity. For example, the amino acid sequence of a linker peptide can be altered (e.g., with deletions, insertions, and / or substitutions) to obtain a linker peptide with desirable properties. Modifications of peptides and polypeptides are routine practice in the art and thus need not be described in detail herein. Examples of modified polypeptides or peptides include polypeptides or peptides having conservative substitutions of amino acid residues, deletion or addition of one or more amino acids, which do not significantly alter the functional activity of the linker peptide, bi-cistronic expressed polypeptide comprising the linker peptide, and / or CAR, or which enhance their functionality. Deletions can include one or several terminal deletions in the linker peptide.

[0100] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one to several residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0101] Substitutional variants replace one or more amino acid residues with different residues. Conservative substitutions are shown in Table 2 under the heading of "Conservative Substitutions." If such substitutions result in a change in biological activity, more substantial changes, denominated "Exemplary Substitutions," or as further described below in reference to amino acid classes, can be introduced and the products screened.

[0102] Table 2: Amino Acid Substitutions

[0103] Immune cells Engineered immune cells are obtained from donor cells. Engineered cells derived from donor cells suitable for use with the methods and / or reagents described herein include immune cells.

[0104] Prior to manipulation or genetic modification in vitro (e.g., as described herein), donor cells (e.g., immune cells) for use in the methods described herein can be obtained from a subject. Donor cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, stem cell-derived or iPSC-derived immune cells, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to the skilled artisan can be used. In some embodiments, donor cells can be derived from a healthy donor, from a patient diagnosed with cancer, from a patient diagnosed with an autoimmune disorder, or from a patient diagnosed with an infection. In some embodiments, donor cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0105] In some embodiments, immune cells are autologous immune cells obtained from a subject who will ultimately receive the engineered immune cells. In some embodiments, immune cells are allogeneic immune cells obtained from a donor who is a different individual than the subject who will receive the engineered immune cells.

[0106] In some embodiments, immune cells include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, stem cell-derived or iPSC-derived T cells, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a volume of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation.

[0107] Donor cells can be obtained by apheresis from the circulating blood of an individual. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing.

[0108] Donor PBMCs can be used directly for genetic modification with immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, prior to or after genetic modification and / or expansion.

[0109] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting mononuclear cells (e.g., using centrifugation via a PERCOLL™ gradient). Particular 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 a T cell population by negative selection can be accomplished with a combination of antibodies against surface markers unique to the cells being negatively selected. One method used herein is cell sorting and / or selection via negative magnetic immunoadsorption or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate a population of cells of interest for use in the present disclosure.

[0110] In some embodiments, a population of donor cells (e.g., immune cells, such as T cells) is enriched for CD4+ cells.

[0111] In some embodiments, a population of donor cells (e.g., immune cells, such as T cells) is enriched for CD8+ cells.

[0112] In some embodiments, the CD8+ cells are further sorted into naive, central memory, and effector cells, by identifying cell surface antigens associated with each of these cell types. In some embodiments, the expression of phenotypic markers of naive T cells includes CD45RA+, CD95-, IL2Rβ-, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of stem cell memory T cells includes CD45RA+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of effector memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7-, and CD62L-. In some embodiments, the expression of phenotypic markers of T effector cells includes CD45RA+, CD95+, IL2Rβ+, CCR7-, and CD62L-. Thus, by identifying cell populations with cell surface antigens, CD4+ and / or CD8+ T helper cells can be sorted into naive, stem cell memory, central memory, effector memory, and T effector cells.

[0113] It will be appreciated that donor PBMCs can also include other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying coding sequences for chimeric receptors as disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Standard procedures are used for cryopreservation of T cells expressing CARs for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culturing, and / or expansion of T cells is performed in the absence of products of non-human animal origin, such as fetal calf serum and fetal bovine serum. In various embodiments, the cryopreservation medium can include, for example, CryoStor® CS2, CS5, or CS10, or another medium comprising DMSO or a medium comprising no DMSO.

[0114] Engineered immune cells Provided herein are engineered immune cells (e.g., CAR-T cells) expressing a polypeptide of the dual cistronic expression of the disclosure and an additional polypeptide (e.g., a CAR) obtained from donor cells as described herein.

[0115] In some embodiments, the engineered immune cell comprises a polynucleotide sequence encoding a bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide (e.g., a CAR). In one embodiment, the CAR comprises one or more extracellular antigen binding domains. In some embodiments, the engineered immune cell comprises a population of CARs, each CAR comprising a different extracellular antigen binding domain. In some embodiments, the immune cell comprises a population of CARs, each CAR comprising the same extracellular antigen binding domain.

[0116] The engineered immune cell can be allogeneic or autologous.

[0117] In some embodiments, the engineered immune cell is a T cell (e.g., inflammatory T lymphocyte, cytotoxic T lymphocyte, regulatory T lymphocyte, helper T lymphocyte, tumor infiltrating lymphocyte (TIL)), NK cell, NK-T cell, TCR-expressing cell, dendritic cell, killer dendritic cell, mast cell, or B cell.

[0118] In some embodiments, the engineered immune cell can be obtained or derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell. In some exemplary embodiments, the engineered immune cell is an a b T cell. In some exemplary embodiments, the engineered immune cell is a gammadelta T cell. In some exemplary embodiments, the engineered immune cell is a macrophage.

[0119] In some embodiments, the engineered immune cell can be derived from, for example, but not limited to, a stem cell. The stem cell can be an adult stem cell, a non-human embryonic stem cell (more particularly a non-human stem cell), a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell (iPSC), a totipotent stem cell, or a hematopoietic stem cell. The stem cell can be CD34+ or CD34-.

[0120] In some embodiments, the donor cells are obtained or prepared from peripheral blood. In some embodiments, the donor cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the donor cells are obtained or prepared from bone marrow. In some embodiments, the donor cells are obtained or prepared from umbilical cord blood. In some embodiments, the donor cells are human cells. In some embodiments, the donor cells will be transfected or transduced by a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), transfection, lipofection, polyplex, nanoparticle, viral transduction, or viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplex. In some embodiments, the donor cells are T cells that have been reprogrammed from non-T cells. In some embodiments, the donor cells are T cells that have been reprogrammed from T cells.

[0121] Detection agents (including antibodies and fragments thereof) In embodiments, the disclosed methods for detecting a bicistronic expressed polypeptide in an engineered immune cell include the use of an antibody or antigen binding agent (e.g., comprising an antigen binding domain or comprising an antibody or fragment thereof). As discussed below, in various embodiments, the engineered immune cells derived from donor cells of a donor cell population can also comprise a binding agent.

[0122] As used herein, the term "antibody" refers to a polypeptide comprising canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As known in the art, an intact antibody produced in nature is a tetrameric factor of approximately 150 kD, composed of two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD), associated with one another into a structure generally referred to as a "Y shape." Each heavy chain is composed of at least four domains (each about 110 amino acids long), namely an amino-terminal variable (VH) domain (at the top of the Y structure), followed by three constant domains: CHI, CH2, and carboxy-terminal CH3 (at the base of the Y-shaped stem). A short region called the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. In intact antibodies, two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another. Each light chain is composed of two domains, an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain. Those skilled in the art are very familiar with antibody structure and sequence elements, recognize "variable" and "constant" regions in the provided sequences, and understand that there can be some flexibility in the definition of the "boundaries" between such domains, such that different presentations of the same antibody chain sequence can for example indicate that this boundary is shifted by one or a few residues relative to different presentations of the same antibody chain sequence.

[0123] A complete antibody tetramer is composed of two heavy chain-light chain dimers, wherein the heavy chain and light chain are connected to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, such that the dimers are connected to one another and form the tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized as an "immunoglobulin fold," formed by two beta strands (e.g., 3-, 4-, or 5-stranded sheets) packed against one another in a compressed anti-parallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four "framework" regions (FR1, FR2, FR3, and FR4) that are somewhat invariant. Upon folding of a natural antibody, the FR regions form the beta strands that provide the structural framework of the domain, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space such that they create a single hypervariable antigen binding site at the top of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding properties of an Fc region for an Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present application include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0124] For purposes of the present disclosure, in certain embodiments, any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," whether such polypeptide is naturally produced (e.g., generated by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial system or method. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0125] Further, as used herein the term“antibody” can refer to any construct or form known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, in some embodiments, the antibodies utilized in the methods of the present disclosure are in a form selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments, and isolated CDRs or collections 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 to herein as nanobodies or VHH); shark antibodies, masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (SMIP™); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® microbodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibodies can lack covalent modifications (e.g., attachment of glycans) that would otherwise be present in naturally occurring instances. In some embodiments, the antibodies can contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendent groups (e.g., polyethylene glycol, etc.).

[0126] 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 that comprises immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent can comprise one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent can comprise one or more sequence elements that are humanized, primate- sourced, chimeric, etc., as known in the art. In many embodiments, the term“antibody agent” is used to refer to constructs or formats known or developed in the industry for exploiting the structural and functional features of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present application are in a format selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd fragments, and isolated CDRs or collections 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 diabodies (TandAb®); VHH; Anticalins®; Nanobodies® microbodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; and KALBITOR®.

[0127] The antibody or antibody agent used to perform the methods of the present disclosure can be single- or double-chained. In some embodiments, the antibody or antigen binding molecule is single-chained. 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.

[0128] Antibodies and antibody agents include antibody fragments. Antibody fragments comprise a portion of an intact antibody, such as the antigen binding 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, multi-specific antibodies formed from antibody fragment antibodies, and scFv fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single-chain, Fab, Fa, F(ab')2 fragments, and scFv. Antibodies can be intact antibodies, or immunoglobulins, or antibody fragments. Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells (e.g., E. coli, Chinese hamster ovary (CHO) cells, or phage), as known in the art.

[0129] In some embodiments, an antibody or antibody agent can be a chimeric antibody (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). A chimeric antibody can be an antibody that has a portion of the heavy and / or light chain derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In one example, a chimeric antibody can comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody can be a "class switched" antibody in which the class or subclass has been changed relative to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0130] In some embodiments, a chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, 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. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et 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 that contain both amino acid residues from non-human hypervariable regions and amino acid residues from human FRs. In certain embodiments, a humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the framework regions (FRs) correspond to those of a human antibody. A humanized antibody optionally can include at least a portion of an antibody constant region derived from a human antibody.

[0131] In some embodiments, an antibody or antibody agent provided herein is a human antibody. Human antibodies can be produced using a variety of techniques known in the art (see, e.g., van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol, 20:450-459 (2008)). A human antibody can be an antibody having an amino acid sequence corresponding to that produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of a human antibody explicitly includes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be prepared using methods well known in the art.

[0132] As used herein, the term "antibody agent" generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term covers any polypeptide or polypeptide complex comprising an immunoglobulin structural element sufficient 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 that are humanized, primate-derived, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, the antibody agents utilized according to the present invention are selected from, but not limited to, the following forms: 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 collections thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g., Probodies®); small modular antibodies. SMIP™ (a type of antiviral drug); TandAb® (single-chain or tandem biantibody); VHH; Anticalins®; Nanobodies® microantibodies; BiTE®; Ankylosing repeat protein or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®; Centyrins®; and KALBITOR®.

[0133] Chimeric antigen receptor As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). Upon binding to a target antigen, a CAR can activate immune cells to attack and destroy cells carrying that antigen (e.g., cancer cells). CARs can also be incorporated with costimulatory or signal transduction domains to increase their potency. See Krause et al., J. Exp. Med., Vol. 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.

[0134] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain that specifically binds to a target.

[0135] In some embodiments, the antigen-specific CAR further comprises a safety switch and / or one or more monoclonal antibody-specific epitopes.

[0136] 1. Antigen binding domain As discussed above, the CARs described herein comprise an antigen binding domain. By "antigen binding domain" as used herein is meant any polypeptide that binds to a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease.

[0137] In some embodiments, the antigen binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs 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.

[0138] An antigen binding domain is said to be "selective" when its binding to one target is tighter or of higher affinity than its binding to another target.

[0139] The antigen binding domain of the CAR selectively targets a cancer antigen. In some embodiments, the cancer antigen is selected from EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34. In some embodiments, the CAR comprises an antigen binding domain that targets EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34.

[0140] In some embodiments, the cancer antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4, folate binding protein (FBP), fetal acetylcholine receptor (AchR), folate receptor, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal 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 Wilms tumor protein (WT-1).

[0141] Variants of the antigen binding domain (e.g., variants of the CDRs, VH, and / or VL) are also within the scope of the present disclosure, e.g., variable light 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 to the amino acid sequence of the antigen binding domain sequence. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant form contains two variable light chains and two variable heavy chains (or subportions thereof). The skilled artisan will be able to determine suitable variants of the antigen binding domain as shown herein using well-known techniques. In certain embodiments, the skilled artisan can identify suitable regions of the molecule that can be varied without destroying activity by targeting regions deemed not to be important for activity.

[0142] In certain embodiments, the polypeptide structure of the antigen binding domain is based on an antibody, including but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, a human antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and fragments thereof, respectively. In some embodiments, the antigen binding domain comprises or consists of an avimer.

[0143] In some embodiments, the antigen binding domain is a scFv.

[0144] In some embodiments, the antigen-selective CAR comprises a leader sequence or signal peptide.

[0145] In other embodiments, the present disclosure relates to an isolated polynucleotide encoding any of the antigen binding domains described herein. In some embodiments, the present disclosure relates to an isolated polynucleotide encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making the same.

[0146] In other embodiments, the present disclosure relates to an isolated polynucleotide encoding any of the antigen binding domains described herein. In some embodiments, the present disclosure relates to an isolated polynucleotide encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making the same.

[0147] In some embodiments, the CAR-immune cells (e.g., CAR-T cells) that can be formed from components of the engineered immune cell population (derived from donor cells of a donor cell population as described herein) generated by practicing the methods of the present disclosure comprise a polynucleotide encoding a safety switch polypeptide (such as, for example, RQR8). See, e.g., WO2013153391A, which is hereby incorporated by reference in its entirety. In the CAR-immune cells (e.g., CAR-T cells) comprising the polynucleotide, the safety switch polypeptide can be expressed at the surface of the CAR-immune cells (e.g., CAR-T cells).

[0148] 2. Hinge domain The extracellular domain of a CAR of the present disclosure can comprise a "hinge" domain (or hinge region). The 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, the hinge domain can serve to provide more flexibility and accessibility to the extracellular antigen binding domain.

[0149] A hinge domain can comprise up to 300 amino acids, in some embodiments 10 to 100 amino acids or in some embodiments 25 to 50 amino acids. A hinge domain can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of an extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, a hinge region of IgG; it will be appreciated that a hinge region can contain some or all of an immunoglobulin family member, such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or a fragment thereof), or from all or a portion of an antibody heavy chain constant region. Alternatively, a hinge domain can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. In some embodiments, the hinge domain is a portion of a human CD8a chain (e.g., NP_001139345.1). In other embodiments, the hinge and transmembrane domains comprise a portion of a human CD8a chain. In some embodiments, a hinge domain of a CAR described herein comprises a subsequence of CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa, in particular a hinge region of any of CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa. In some embodiments, a hinge domain comprises a human CD8a hinge, a human IgGl hinge, a human IgG4, a human PD-1, or a human FcyRIIIa hinge. In some embodiments, a CAR disclosed herein comprises a scFv, a CD8a human hinge and transmembrane domain, a CD3 zeta signaling domain, and a 4-1BB signaling domain.

[0150] In some embodiments, a hinge domain in a CAR of the present disclosure is a CD8a transmembrane domain. In some embodiments, a hinge domain in a CAR of the present disclosure is a CD8a hinge domain comprising the amino acid sequence PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 332) or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 340). In some embodiments, a CD8a hinge domain comprises a nucleic acid sequence encoding the hinge amino acid sequence of SEQ ID NO: 332. In some embodiments, a hinge domain in a CAR of the present disclosure is a CD28 hinge domain.

[0151] 3. Transmembrane domain CARs of the present 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 can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In some embodiments, a short linker can form a link between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0152] Transmembrane domains suitable for the CARs disclosed herein have the ability to (a) be expressed at the surface of an immune cell, such as, for example, but not limited to, a lymphocyte, such as a T helper (T h ) cell, a cytotoxic T (T c ) cell, a T regulatory (T reg ) cell, or a natural killer (NK) cell, and / or (b) interact with the extracellular antigen-binding domain and the intracellular signaling domain for directing a cellular response of the immune cell against a target cell.

[0153] The transmembrane domain can be derived from a natural source or from a synthetic source. Where the source is a natural source, the domain can be derived from any membrane-bound or transmembrane protein.

[0154] The transmembrane region used particularly in the present disclosure can be derived from (comprising or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), Inducible T-cell Costimulator (ICOS), Lymphocyte Function-Associated Antigen-1 (LFA-1, CD1 -1a / CD18), CD3y, CD35, CD3s, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig a (CD79a), DAP-10, Fcy Receptors, MHC class 1 molecule, TNF Receptor proteins, Immunoglobulins, Cytokine Receptors, Integrins, Signaling Lymphocyte Activation Molecules (SLAM proteins), Activating 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, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, 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 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, a ligand that specifically binds with CD83, or any combination thereof.

[0155] As non-limiting examples, the transmembrane region can be derived from or be a portion of a T cell receptor, such as alpha, beta, gamma, or delta; a polypeptide that makes up the CD3 complex; an IL-2 receptor p55 (a chain), p75 (beta chain), or gamma chain; a subunit chain of an Fc receptor, particularly Fcy receptor III; or a CD protein. Alternatively, the transmembrane domain can be synthetic and can comprise primarily hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8a chain (e.g., NP_001139345.1).

[0156] In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8a transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8a transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT or IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 333). In some embodiments, the CD8a transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of IYIWAPLAGTCGVLLLSLVIT or SEQ ID NO: 333. In some embodiments, the hinge and transmembrane domain in the CAR of the present disclosure is a CD8a hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO: 323 or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT.

[0157] In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain.

[0158] 4. Intracellular domain The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide for activation of at least one of the normal effector functions of the immune cell comprising the CAR. For example, the effector function of a T cell can refer to cytolytic activity or helper activity, including secretion of cytokines.

[0159] In some embodiments, the activating intracellular signaling domain for use in the CAR can be, for example and without limitation, the cytoplasmic sequences of T cell receptors and accessory receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0160] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death 1 (PD-1), Inducible T-cell Costimulator (ICOS), Lymphocyte Function-Associated Antigen-1 (LFA-1, CD1 -1a / CD18), CD3y, CD35, CD3s, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig a (CD79a), DAP-10, Fcy receptors, MHC class 1 molecule, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, 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 protein), CEACAM1, CRT 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, a ligand that specifically binds with CD83, or any combination thereof.

[0161] In addition to the activating domains described above, the intracellular domain of the CAR of the present disclosure can incorporate a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) to increase its potency. The costimulatory domain can provide a signal in addition to the primary signal provided by the stimulatory molecule as described herein.

[0162] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure can be derived from (or correspond to) CD28, OX40, 4-1BB / CD137, CD2, CD3 (a, b, d, e, g, z), 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, Ig a (CD79a), DAP-10, Fcy receptor, MHC class I molecule, TNFR, integrin, signaling lymphocytic activation 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, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, 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 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 ligand, or fragments or combinations thereof, for example. It will be appreciated that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0163] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a 4-1BB / CD137 domain alone or in combination with any other one or more desired intracellular domains useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI Reference Sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI Reference Sequence: NM_001561.5.

[0164] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a CD28 domain alone or in combination with any other one or more desired intracellular domains useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

[0165] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a CD3 zeta domain alone or in combination with any other one or more desired intracellular domains useful in the context of the CARs of the present disclosure.

[0166] For example, the intracellular domain of the CAR can include a portion of the CD3 zeta chain and a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CARs of the present disclosure can be linked to one another in random or specified order. In some embodiments, the intracellular domain is designed to include an activating domain of CD3 zeta and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to include an activating domain of CD3 zeta and a signaling domain of 4-1BB.

[0167] In some embodiments, the intracellular signaling domain of the CARs of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of the CARs of the present disclosure comprises a portion of a costimulatory molecule selected from the group consisting of a fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0168] Table 3 provides exemplary sequences of CAR components that can be used in the CARs disclosed herein as well as antibody and / or CAR sequences exemplified herein.

[0169] Table 3: Sequences related to CARs

[0170] Table 4 provides exemplary sequences of chimeric cytokine receptor (CCR) components that can be used in the CCRs disclosed herein. The CCRs can be expressed with a signal sequence (e.g., CD8SS of sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127)).

[0171] Table 4 - CCR sequences

[0172] Table 5 provides exemplary CACCR sequences with or without an anti-BCMA CAR showing a P2A linker sequence (GSGATNFSLLKQAGDVEENPG (SEQ ID NO: 307)).

[0173] Table 5 - CACCR sequences with P2A linker sequences

[0174] Engineered immune cells comprising a CAR Also provided herein are engineered immune cells and populations of engineered immune cells that i) comprise a polynucleotide encoding a bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide (e.g., a CAR), and / or ii) express a bi-cistronic expressed polypeptide, a linker peptide, and an additional polypeptide, such as a CAR (e.g., a CAR-T cell or a CAR+ cell).

[0175] In some embodiments, the engineered immune cells comprise CAR T cells, each CAR T cell comprising an extracellular antigen binding domain and having reduced or eliminated expression of an endogenous TCR. In some embodiments, the population of engineered immune cells comprises a population of CAR T cells, each CAR T cell comprising two or more different extracellular antigen binding domains and having reduced or eliminated expression of an endogenous TCR. In some embodiments, the immune cells comprise a population of CARs, each CAR T cell comprising the same extracellular antigen binding domain and having reduced or eliminated expression of one or more desired biomarkers (as described herein) and / or an endogenous TCR.

[0176] The engineered immune cells can be allogeneic or autologous.

[0177] In some embodiments, the engineered immune cell or population of engineered immune cells is a T cell (e.g., inflammatory T lymphocyte, cytotoxic T lymphocyte, regulatory T lymphocyte, helper T lymphocyte, tumor infiltrating lymphocyte (TIL)), NK cell, NK-T cell, TCR-expressing cell, dendritic cell, killer dendritic cell, mast cell, or B cell, and expresses a CAR. In some embodiments, the T cell can be derived from the group consisting of a CD4+ T lymphocyte, a CD8+ T lymphocyte, or a population comprising a combination of CD4+ and CD8+ T cells.

[0178] In some embodiments, the engineered immune cell or population of engineered immune cells generated using the disclosed methods can be derived from, for example, but not limited to, a stem cell. The stem cell can be an adult stem cell, a non-human embryonic stem cell (more particularly a non-human stem cell), a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell.

[0179] In some embodiments, the engineered immune cell or population of immune cells generated using the disclosed methods is obtained or prepared from peripheral blood. In some embodiments, the engineered immune cell is obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the engineered immune cell is obtained or prepared from bone marrow. In some embodiments, the engineered immune cell is obtained or prepared from cord blood. In some embodiments, the donor cell is a human cell. In some embodiments, the donor cell is transfected or transduced by a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipofection, polyplex, nanoparticle, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplex complex.

[0180] In some embodiments, the engineered immune cell expressing an antigen-specific CAR on the cell surface membrane comprises greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of a percentage of stem cell memory cells and central memory cells.

[0181] In some embodiments, the engineered immune cell expressing an antigen-specific CAR on the cell surface membrane comprises stem cell memory cells and central memory cells in 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%.

[0182] In some embodiments, the engineered immune cell expressing an antigen-specific CAR on the cell surface membrane is enriched for T CMcells and / or T SCM cells, such that the engineered immune cells comprise at least about 60%, 65%, 70%, 75%, or 80% combined T CM cells and T SCM cells. In some embodiments, the engineered immune cells expressing an antigen- specific CAR on the cell surface membrane are enriched for T CM cells and / or T SCM cells, such that the engineered immune cells comprise at least about 70% combined T CM cells and T SCM cells. In some embodiments, the engineered immune cells expressing an antigen- specific CAR on the cell surface membrane are enriched for T CM cells and / or T SCM cells, such that the engineered immune cells comprise at least about 75% combined T CM cells and / or T SCM cells.

[0183] In some embodiments, the engineered immune cells are inflammatory T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are cytotoxic T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are regulatory T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are helper T lymphocytes expressing a CAR.

[0184] Genetic modification of CAR T cells In some embodiments, engineered immune cells derived from donor cells having certain biomarker profiles according to the present disclosure can comprise one or more disrupted or inactivated genes. In some embodiments, a gene for a target antigen (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Mucin-18.2, Muc17, FAPa, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70) can be knocked out to introduce a CAR targeting the same antigen (e.g., a EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Mucin-18.2, Muc17, FAPa, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 CAR), thereby avoiding induced CAR activation. As described herein, in some embodiments, engineered immune cells according to the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of MHC1 (b2M), MHC2 (CIITA), EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Mucin-18.2, Muc17, FAPa, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70, TCRa and TCRp, and / or express a CAR or a multichain CAR. In some embodiments, the cells comprise a multichain CAR.In some embodiments, the isolated cell comprises two disrupted or inactivated genes selected from the group consisting of: CD52 and TCRa, CDR52 and TCRP, PD-1 and TCRa, PD-1 and TCRP, MHC-1 and TCRa, MHC-1 and TCRP, MHC2 and TCRa, MHC2 and TCRP, and / or expresses a CAR or a multi-chain CAR.

[0185] In some embodiments, the isolated cell comprises a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cell according to the present disclosure comprises two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRa, CDR52 and TCRP, DLL3 and CD52, DLL3 and TCRa, DLL3 and TCRP, GR and TCRa, GR and TCRP, TCRa and TCRP, PD-1 and TCRa, PD-1 and TCRP, CTLA-4 and TCRa, CTLA-4 and TCRP, LAG3 and TCRa, LAG3 and TCRP, TIM3 and TCRa, Tim3 and TCRP, BTLA and TCRa, BTLA and TCRP, BY55 and TCRa, BY55 and TCRP, TIGIT and TCRa, TIGIT and TCRP, B7H5 and TCRa, B7H5 and TCRP, LAIR1 and TCRa, LAIR1 and TCRP, SIGLEC10 and TCRa, SIGLEC10 and TCRP, 2B4 and TCRa, 2B4 and TCRP, and / or expresses a CAR (including a multi-chain CAR) and / or a pTa transgene. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the donor cell a

[0186] In some embodiments, the TCR is rendered non-functional in the cells according to the present disclosure by disruption or inactivation of the TCRa gene and / or one or more TCRb genes. In some embodiments, methods for obtaining modified cells derived from an individual are provided, wherein the cells can proliferate independent of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independent of the MHC signaling pathway that are readily obtained by this method are encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat a patient in need of treatment for host versus graft (HvG) rejection and graft versus host disease (GvHD); thus the scope of the present disclosure includes methods of treating a patient in need of treatment for host versus graft (HvG) rejection and graft versus host disease (GvHD), comprising treating the patient by administering to the patient an effective amount of modified cells comprising disrupted or inactivated TCRa and / or TCRb genes.

[0187] The present disclosure provides methods of 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 comprise less than 5.0%, less than 4.0%, less than 3.0% TCR+ cells, less than 2.0% TCR+ cells, less than 1.0% TCR+ cells, less than 0.9% TCR+ cells, less than 0.8% TCR+ cells, less than 0.7% TCR+ cells, less than 0.6% TCR+ cells, less than 0.5% TCR+ cells, less than 0.4% TCR+ cells, less than 0.3% TCR+ cells, less than 0.2% TCR+ cells, or less than 0.1% TCR+ cells. This population can be the product of the disclosed methods.

[0188] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic drugs. The chemotherapeutic drugs can be, for example, purine nucleotide analogs (PNAs), thereby adapting the immune cells for cancer treatment in combination with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to mono-, di-, and tri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production.

[0189] In some embodiments, the isolated cells or cell lines of the present disclosure can comprise pTa or a functional variant thereof. In some embodiments, the isolated cells or cell lines can be further genetically modified by disruption or inactivation of the TCRa gene.

[0190] The present disclosure also provides engineered immune cells comprising a polynucleotide encoding a bicistronic expressed polypeptide, a linker peptide, and any of the CAR polypeptides described herein. In some embodiments, the CAR can be introduced into an immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also contain, for example, a selection marker that provides for the identification and / or selection of cells that receive the vector.

[0191] The CAR polypeptide can be synthesized in situ in the cell after introducing the polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, the polynucleotide construct can be integrated into the cell genome using a stable transformation method (e.g., using a lentiviral vector). In other embodiments, the polynucleotide construct can be transiently expressed using a transient transformation method, as well as polynucleotide constructs that are not integrated into the cell genome. In other embodiments, a viral-mediated method can be used. The polynucleotide can be introduced into the cell by any suitable means, such as, for example, a recombinant viral vector (e.g., retrovirus, adenovirus), a liposome, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide can be included in a vector, such as, for example, a plasmid vector or a viral vector.

[0192] In some embodiments, an isolated nucleic acid is provided comprising a promoter operably linked to a first polynucleotide encoding an antigen binding domain, at least one costimulatory molecule, and an activating 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 a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0193] In some embodiments, the isolated nucleic acid construct is contained within a viral vector and is introduced into the genome of the engineered immune cell by random integration (e.g., lentiviral or retroviral-mediated random integration). In some embodiments, the isolated nucleic acid construct is contained within a viral vector or a non-viral vector and is introduced into the genome of the engineered immune cell by site-specific integration (e.g., adenoviral-mediated site-specific integration).

[0194] Manufacture of engineered immune cells, including CAR T cells Provided herein are methods of analyzing or determining various attributes of donor cells from a population of donor cells and / or engineered immune cells from a population of immune cells, including engineered immune cells such as CAR-expressing cells or CAR+ cells. As described herein, engineered immune cells such as CAR T cells can be modified to reduce or eliminate expression or activity of endogenous TCRs, and remaining TCR+ engineered immune cells can be depleted at the end of production according to the methods described herein. The present disclosure provides methods of characterizing or analyzing a population of engineered immune cells to characterize a drug product or as part of a manufacturing process. The present disclosure also provides methods of analyzing or determining other attributes of engineered immune cells, such as potency or polyfunctionality, to characterize a drug product 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).

[0195] A variety of known techniques can be utilized in the preparation of polynucleotides, polypeptides, vectors, antigen binding domains, immune cells, compositions, etc. according to the present disclosure.

[0196] Prior to in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. CAR-expressing cells can be derived from allogeneic or autologous sources, and can be depleted of endogenous TCRs as described herein.

[0197] 1. Source Material In some embodiments, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a volume of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation.

[0198] Cells can be obtained by apheresis from the circulating blood of an individual. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis can be washed to remove the plasma fraction, and then placed in an appropriate buffer or medium for subsequent processing.

[0199] In certain embodiments, T cells are isolated from PBMC by lysing red blood cells and depleting mononuclear cells, for example, using centrifugation via a PERCOLL™ gradient. Particular subpopulations of T cells (e.g., CD28+, CD4+, CD45RA-, and CD45RO+ T cells or CD28+, CD4+, CDS+, CD45RA-, CD45RO+, and CD62L+ T cells) can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies against surface markers unique to the cells being negatively selected. One method used herein is cell sorting and / or selection by negative magnetic immunoadsorption or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the cell populations of interest for use in the present disclosure.

[0200] PBMCs can be used directly for genetic modification with immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes can be further isolated, both cytotoxic and helper T lymphocytes, and sorted into naive, memory, and effector T cell subpopulations, prior to or after genetic modification and / or expansion. In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells, by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of 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. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

[0201] 2. Stem cell-derived immune cells In some embodiments, the immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSCs, mesenchymal, iPS cells, and other types of stem cells can be cultivated immortal cell lines or isolated directly from a patient. Various methods for isolating, developing, and / or cultivating stem cells are known in the art and can be used in practicing the present disclosure.

[0202] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the source material can be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The source material can be embryonic stem cells. The source material can be B cells, or any other cell from a peripheral blood mononuclear cell isolate, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0203] 3. Genetic modification of isolated cells The donor immune cells (e.g., T cells) of the donor immune cell population can be genetically modified using known methods after isolation, or the donor immune cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In some embodiments, the isolated donor immune cells are genetically modified to reduce or eliminate expression or activity of endogenous TCRa and / or CD52. In some embodiments, the cells are genetically modified using gene editing technology (e.g., CRISPR / Cas9, CRISPR / Casl2a, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, base editing, or prime editing) to reduce or eliminate expression or activity of an endogenous protein (e.g., TCRa and / or CD52). In another embodiment, the immune cells (e.g., T cells) are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro.

[0204] Certain methods for making the constructs and engineered immune cells of the present disclosure are described in PCT Application PCT / US 15 / 14520, the contents of which are hereby incorporated by reference in their entirety.

[0205] It will be appreciated that PBMCs can also include other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying coding sequences for chimeric receptors as disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3 positive 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, and then further propagated to increase the number of these CAR-expressing T cells. 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 expansion of T cells is performed in the absence of products of non-human animal origin, such as fetal calf serum and fetal bovine serum.

[0206] For cloning of polynucleotides, a vector can be introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby amplifying the copy number of the polynucleotide contained in the vector. Cloning vectors can contain sequence components, typically including but not limited to an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be selected as appropriate by one of skill in the art. For example, an origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0207] In some embodiments, the present disclosure provides an isolated host cell containing a vector provided herein. Host cells containing vectors can be used for expression or cloning of the polynucleotides contained in the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells, particularly human cells.

[0208] Vectors can be introduced into host cells 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 bombardment, receptor-mediated gene delivery, delivery mediated by poly-lysine, histones, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of vectors of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used when genetically modifying a donor population of immune effector cells, with each vector encoding a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a portion of the engineered cells expressing more than one different CAR.

[0209] In one embodiment, the disclosure provides a method of sorting genetically engineered cells expressing a CAR or TCR. The method involves cryopreserving immune cells such that the cells remain viable upon thawing. A portion of the CAR-expressing immune cells can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignancies. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded to obtain more of such cells.

[0210] In some embodiments, the cells are formulated by first harvesting the cells from the culture medium of the cells, then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic media formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or Ringer's lactate can also be utilized. The infusion media can be supplemented with human serum albumin.

[0211] 4. Allogeneic CAR T cells The process for manufacturing allogeneic CAR T therapy involves harvesting healthy, selected, screened, and tested donor immune cells (including T cells) from a healthy donor. Next, the T cells of the donor immune cells are engineered to express a CAR that recognizes certain cell surface proteins expressed in hematological tumors or solid tumors. The allogeneic T cells are genetically edited to reduce the risk of graft versus host disease (GvHD) and to prevent allogeneic rejection. The T cell receptor genes (e.g., TCRa, TCRP) are knocked out to avoid GvHD. The CD52 gene can be knocked out to render the CAR T product resistant to anti-CD52 antibody therapy. Thus, anti-CD52 antibody therapy can be used to suppress the host immune system and allow the CAR T to engraft for full therapeutic effect. The engineered T cells then undergo further processing, which can optionally include a depletion step to remove unwanted T cells (e.g., unwanted T cells expressing TCR genes) that express the biomarkers described herein, as well as a purification step, and the T cells are finally cryopreserved in vials for delivery to a patient.

[0212] 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 and kills the cancer cells. The engineered cells are then cryopreserved for subsequent administration to the patient.

[0213] In vitro sorting methods In some embodiments, methods for in vitro sorting of a population of immune cells are provided, wherein a subset of the population of immune cells comprises engineered immune cells expressing an antigen-specific CAR comprising an epitope specific for a monoclonal antibody (e.g., an exemplary mimotope sequence). The methods comprise contacting the population of immune cells with a monoclonal antibody specific for the epitope, and selecting immune cells bound to the monoclonal antibody to obtain a population of cells enriched for engineered immune cells expressing the antigen-specific CAR.

[0214] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells bound to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0215] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a magnetic particle. In this embodiment, the step of selecting cells bound to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0216] In some embodiments, the mAb used in the method for sorting immune cells expressing CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10. In other embodiments, the mAb binds to TCRa or TCRP.

[0217] In some embodiments, the population of immune cells expressing CAR obtained when using the above-described method for sorting immune cells expressing CAR in vitro comprises at least 70%, 75%, 80%, 85%, 90%, 95% of immune cells expressing CAR. In some embodiments, the population of immune cells expressing CAR obtained when using the method for sorting immune cells expressing CAR in vitro comprises at least 85% of immune cells expressing CAR.

[0218] In some embodiments, the population of CAR-expressing immune cells obtained upon use of the above-described methods for sorting CAR-expressing immune cells in vitro show increased cytotoxic activity in vitro compared to the initial (unsorted) population of cells. In some embodiments, the cytotoxic activity in vitro is increased by 10%, 20%, 30%, 40%, or 50%. In some embodiments, the immune cells are T cells.

[0219] In some embodiments, the mAb is pre-bound to a support or surface. Non-limiting examples of solid supports can include beads, agarose beads, magnetic beads, plastic multiwell plates, glass multiwell plates, ceramic multiwell plates, columns, or cell culture bags.

[0220] CAR-expressing immune cells to be administered to a recipient can be enriched in vitro from a source population. Methods of expanding a source population can include selecting cells expressing an antigen (such as a CD34 antigen) using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0221] Flow cytometry can be used to quantify specific cell types within a population of cells. In general, flow cytometry is a method for quantifying components or structural features of cells, primarily by optical means. 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.

[0222] In some embodiments, the method for sorting CAR-expressing T cells is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method for separating cell populations by using superparamagnetic nanoparticles and columns according to surface antigens (e.g., CD molecules) of various cell populations. MACS can be used to obtain pure cell populations. Cells in a single cell suspension can be magnetically labeled with microbeads. The sample is applied to a column composed of ferromagnetic spheres, which are covered with a cell-friendly coating, allowing rapid and gentle isolation of cells. Unlabeled cells pass through, while magnetically labeled cells remain 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.

[0223] 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).

[0224] Pharmaceutical compositions and therapies In some embodiments, the engineered immune cells described herein are formulated by first harvesting the cells from the culture medium of the cells, then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration ("pharmaceutically acceptable" carriers). Suitable infusion media can be any isotonic media formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's can also be utilized. The infusion media can be supplemented with human serum albumin.

[0225] In embodiments, the desired therapeutic amount of cells in the composition is typically at least 2 cells (e.g., at least 1 CD8+ central or stem cell memory T cell and at least 1 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 greater than 10 2 cells, and up to and including 10 6 cells, up to and including 10 7 cells, 10 8 cells, or 10 9 cells, and can be more than 10 10 cells. The number of cells will depend on the desired use for which the composition is intended, as well as the type of cells included in the composition. The density of the desired cells is typically greater than 10 6 cells / ml and typically greater than 10 7 cells / ml, typically 10 8 cells / ml or greater. A clinically relevant number of immune cells can be allocated into multiple infusions, cumulatively equaling or exceeding 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12cells. In some aspects of the disclosure, particularly because all of the infused cells will be redirected to a particular target antigen, lower numbers of cells can be administered, in the range of about 10 5 / kilogram or about 10 6 / kilogram (10 6 - 10 11 / patient). CAR therapy can be administered multiple times at doses within these ranges. For patients undergoing therapy, the cells can be autologous, allogeneic, or heterologous.

[0226] The CAR-expressing cell populations of the disclosure can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components, such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the disclosure can comprise a population of CAR- or TCR-expressing cells (such as T cells) as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates, such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the disclosure are preferably formulated for intravenous administration.

[0227] Pharmaceutical compositions (solutions, suspensions, etc.) can include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycols, glycerine, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0228] Methods for treating diseases or disorders, including cancer and autoimmune diseases, are provided. In some embodiments, the present disclosure relates to generating a T cell-mediated immune response in a subject, including administering to the subject an effective amount of an engineered immune cell of the present application. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). The CAR-containing immune cells of the present disclosure can be used to treat CD19-related diseases or disorders and / or BCMA-related diseases or disorders. In some embodiments, the disease or disorder can be an autoimmune disease, including but not limited to lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, and myositis.

[0229] All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, are hereby incorporated by reference in their entirety, to the same extent as if they were all fully set forth herein.

[0230] The following examples are provided for illustrative purposes only. In fact, various modifications of the application, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the applications.

[0231] Example Example 1 - Immune cells engineered to express chimeric antigen receptors (CARs) and chimeric cytokine receptors (CCRs) Figure 1 In the context of adoptive immune cell transfer, the beneficial activity (e.g., activation, proliferation, persistence, and / or potency) of CAR T cells can be enhanced via cytokine-induced cytokine receptor signaling (also known as signal 3). Current approaches for signal 3 include combining CAR T cell therapy with systemic infusion of recombinant cytokines / cytokine mimetics, and engineering CAR T cells to extracellularly secrete / express cytokines, but these approaches 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 US20200291090A1, which is incorporated by reference herein in its entirety). An exemplary CACCR was designed using sequences from the thrombopoietin receptor (TpoR). TpoR is capable of activating the JAK-Stat signaling pathway and signals as a homodimeric receptor. In this example, immune cells were engineered to express a CD19-specific CAR and a constitutively active chimeric cytokine receptor (CACCR).

[0232] CAR-T cells comprising an exemplary CD19 CAR with a 4G7 scFv against CD19 coupled to 4-1BB and CD3z signaling domains were selected for further engineering of CACCR (see, e.g., U.S. Patent 10,874,693, incorporated by reference in its entirety herein). CD19-specific CAR-T cells co-expressing CACCR comprised of a truncated IL2Rb tail were generated. CAR-T cells comprising an exemplary BCMA CAR with a P5A2 scFv against BCMA coupled to 4-1BB and CD3z signaling domains and the same CACCR were also selected for testing (see, e.g., published U.S. Application No. US20210260118A1, incorporated by reference in its entirety herein).

[0233] Figure 1 A schematic showing an engineered constitutively active chimeric cytokine receptor. Figure 1 B shows a schematic of a lentiviral vector for co-expression of a constitutively active chimeric cytokine receptor and an anti-CD19 CAR. A construct encoding an anti-CD19 CAR comprising SEQ ID NO: 335 (see Table 3) and a CACCR comprising SEQ ID NO: 343 (see Table 4) was used.

[0234] Example 2 - Detection of CACCR in CAR T cells upon stimulation C shows a schematic of a lentiviral vector for co-expression of a constitutively active chimeric cytokine receptor and an anti-BCMA CAR. A construct encoding an anti-BCMA CAR comprising SEQ ID NO: 342 (Table 3) and / or a CACCR comprising SEQ ID NO: 343 (Table 4) was used.

[0235] In an exemplary protocol for preparing lentivirus encoding CACCR and CD19 CAR, HEK293T cells were plated at 450,000 cells / 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, lentivirus was prepared by mixing 1.5 ug of lentivirus packaging vector, 0.5 ug pMD2G, and 0.5 ug of the appropriate transfer CAR vector together in 250 uL of Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 uL of Lipofectamine 2000 (Invitrogen) in 250 uL of Opti-MEM was incubated at room temperature for 5 minutes before adding it to the DNA mix. The mix was incubated at room temperature for 20 minutes and 500 uL total volume was slowly added to the side of the well containing the HEK293T. The day after transfection, the media of the HEK293T cells in each well of the 6-well plate was changed to 2 mL / well of T cell transduction media (i.e., X-Vivo-15 supplemented with 10% FBS). Two days after transfection, lentivirus supernatant was harvested from the HEK293T cells and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and the crude lentivirus supernatant was used directly for T cell transduction. On day 0, purified T cells were activated in Grex-24 plates (Wilson Wolf, catalog number 80192M) in X-Vivo-15 media (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1: 100 dilution). On day 2, T cells were resuspended at 5 million cells / mL in T cell transduction media and transduced in Grex-24 plates with an equal volume of crude lentivirus supernatant, and 100 IU / mL human IL-2. On day 5, CACCR-expressing CAR-T cells were fed by changing the spent media to T cell expansion media (i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio)) and 100 IU / mL human IL-2. At this time, control CAR-T cells lacking CACCR were expanded in 100 U / mL human IL-2 alone or 100 U / mL human IL-2 and 10 ng / mL human IL-15 (Miltenyi Biotec). Cells were expanded as needed into larger G-Rex vessels (Wilson Wolf) using T cell expansion media and the corresponding concentrations of recombinant cytokines.On day 13, cells were stained with the ZombieNIR fixation viability kit (Biolegend), labeled with a BUV395-conjugated CD3 antibody (Biolegend) and an anti-idiotype antibody specific to P5A2scFv, and then FACS sorted to enrich CAR+ T cells. The sorted CAR+ T cells were then cultured in Grex-24 plates in T cell expansion medium for another 2 days, with CACCR CD19 CAR+ T cells kept in the absence of exogenous cytokines, and sorted control CAR+ T cells kept in the absence of exogenous cytokines and treated with 100 U / mL human IL-2 or 10 ng / mL human IL-15. On day 15, live CAR+ T cells were enriched using the Easy Sep dead cell removal kit (StemCell Technologies), and the cell pellet was flash-frozen for subsequent RNA extraction and NanoString gene expression analysis (Human CAR-T Panel; NanoString Technologies).

[0236] Figure 2A Co-culture setup for CAR T cell stimulation. Two different CAR T cell drug product batches were used as effector cells in this experiment: a) PBMCs were used as starting material to provide cells engineered to express CD19-specific CAR and CACCR (batch 02), and b) CD4 / CD8+ T cells were used as starting material to provide cells engineered to express CD19-specific CAR and CACCR (batch 04). Both CAR T cell drug product batches were engineered from the same donor. Cells from both CAR T cell drug product batches were stimulated at 2.5 μg / ml with irradiated DGL (Daudi cells purchased from ATCC (CCL-213™), engineered with GFP-luciferase and irradiated for assays) or CD19-Fc (Acro bio, USA). 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 at an effector cell to target cell ratio of 2:1. After setup, co-culture samples were taken at 20, 24, and 48 hours for Western blotting and flow cytometry.

[0237] Intracellular staining of P2A cells using flow cytometry. Harvested cells were stained with CD19-specific CAR anti-idiotype antibody PE (Acro Biosystems) and FVS780 (BD Bioscience) in flow cytometry staining buffer (554656, BD). Cells were fixed and permeabilized using FOXP3 transcription factor staining buffer (00-5523-00, eBioscience) according to protocol. Typically, cells were fixed at room temperature (RT) for 20 min using fixation / permeabilization buffer and stained with P2A-APC (NBP2-59627AF647, Novus Bio) in permeabilization buffer at RT for 30 min. Cells were resuspended in staining buffer and analyzed using an LSL Ortessa™ cell analyzer (BD Bioscience). Data were analyzed using FlowJo 9.2 software (Tree Star, Inc.).

[0238] As described above, CAR T cell drug products engineered to express CD19-specific CAR and CACCR were incubated with or without IR-DGL. As shown in Figure 2, after 24 hours (… Figure 2B ) or 48 hours Figure 3A Cells were harvested and stained with CD19-specific CAR anti-idiotype antibody PE (Acro Biosystems) and FVS780 (Live / Dead activity staining agent), and then fixed with transcription factor staining buffer for intracellular staining against P2A. Gating was performed on P2A and CAR+ double-positive cell populations from single-peak live cells under different culture conditions.

[0239] Figure 3 shows the results of another experiment in which cells were compared with ( Figure 3B ) or not with ( Table 6 Incubate with CD19Fc or IR-DGL. Harvest cells at 24 hours and stain with CD19-specific CAR anti-idiotype antibody PE (see US20200331998A1, which is incorporated herein by reference in its entirety) (custom-made from Biolegend) and FVS780 (Live / Dead activity staining agent), then fix with intracellular staining against P2A using transcription factor staining buffer. Gating of P2A+ and CAR+ double-positive cell populations from single-peak live cells from different culture conditions.

[0240] 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 0.2E6 cells / µL were prepared for all samples using a mixture of M-PER buffer, protease inhibitor, and reducing agent. 0.26E6 cells from the positive control and 2.6E6 cells from other samples were loaded into 10% Bis-tris gels. A mixture of 1:200 dilution of 2A peptide (3H4) antibody (Novsubio, catalog number NBP2-59627) and 1:1000 dilution of β-tubulin (Cell Signaling, catalog number 2128L) was used as the primary antibody. A mixture of 1:5000 IRD-800CW goat anti-rabbit IgG (LI-COR, catalog number 926-32211) and IRD-680CW goat anti-mouse IgG (LI-COR, catalog number 926-68070) was used as the secondary antibody. The blot was visualized using an Odyssey CLx. Table 6 lists the samples by lane of the protein blot.

[0241] Figure 4

[0242] like Table 7 As shown, tubulin is displayed in the 50 kDa band (see arrow above) and used as a technical control. P2A protein is displayed in the 24.3 kDa band (see arrow below). P2A protein was detected in the positive control and stimulated samples, shown in lanes 1, 2, 3, 4, 6, 7, and 8. P2A protein was not detected in the unstimulated samples, shown in lanes 5 and 9.

[0243] CAR T cells containing anti-BCMA CARs with P5A2 scFv and expressing CACCR (see Example 1 and Table 5 above) were treated with PMA + iomycin (P+I) to observe the detection level of CACCR. CAR T cells containing anti-BCMA CARs with P5A2 scFv but not expressing CACCR (see Example 1 and Table 5 above) were included as negative controls.

[0244] The following samples are listed in Test Table 7.

[0245] Table 8

[0246] Frozen whole blood with EDTA as an anticoagulant was used (Stem Cell Technologies). Frozen CAR T cell batches were used (anti-BCMA: 14e6 CAR+ cells / ml, in 1 ml; anti-BCMA + CACCR: 40e6 CAR+ cells / 2 ml – see Table 8). Frozen-thawed CAR T cells were adulterated into frozen-thawed whole blood (WB). 10% CAR+ cells were present in the live whole blood cells.

[0247] Figures 6A-6B

[0248] Detection of anti-BCMA CARs with P5A2 scFv using anti-idiotype antibodies against CARs (see published U.S. Patent Application No. 20240101710A1, which is incorporated herein by reference in its entirety).

[0249] Anti-BCMA CAR was detected in all samples (data not shown). CACCR was detected via P2A in a WB / CAR mixture a) unstimulated and stained after mixing cells; or b) stimulated with PMA + iomycin for 24 h prior to staining. Cells were stained for viability and surface markers, followed by fixation / permeabilization for intracellular P2A staining. ​ As shown, CCR was detected in P+I stimulated samples using the anti-P2A antibody P2A-APC (NBP2-59627AF647, Novus Bio) (right inset), but not in unstimulated samples (left inset). As expected, CACCR was not detected in any sample (data not shown).

Claims

1. An in vitro method for detecting bicistronic expressed polypeptides in chimeric antigen receptor (CAR) T cells, the method comprising: a) Contact the CAR T cells with an exogenous drug, wherein the CAR T cells contain a polynucleotide expressing the bicistronic polypeptide, a linker peptide, and another polypeptide, wherein the linker peptide is located at the C-terminus of the bicistronic polypeptide; as well as b) After step a), the linker peptide is detected, thereby indicating the presence of the bicistron-expressed polypeptide in the CAR T cells.

2. The method of claim 1, wherein the bicistronic expressed polypeptide comprises a transmembrane domain.

3. The method of claim 1, wherein the bicistronic expressed polypeptide is a membrane protein containing a transmembrane domain.

4. The method of claim 2 or 3, wherein the bicistronic expressed polypeptide further comprises an intracellular domain.

5. The method of any one of claims 1-4, wherein the linker peptide is located at the C-terminus of the bicistron-expressed polypeptide.

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

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

8. The method of any of the preceding claims, wherein the linker peptide is heterologous to the bicistronic expressed polypeptide.

9. The method of any one of claims 4-8, wherein the linker peptide is adjacent to the intracellular domain of the bicistron-expressed polypeptide.

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

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

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

13. The method of claim 12, wherein the small molecule reagent stimulates the production of cytokines in the CAR T cells.

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

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

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

17. The method of claim 16, wherein the contact comprises contacting the CAR T cells with the target cells.

18. The method of claim 16 or 17, wherein the target cell is an inactivated target cell.

19. The method of claim 15, wherein the contacting step comprises contacting the CAR T cells with the soluble target molecule.

20. The method of any of the preceding claims, wherein the linker peptide is cleavable.

21. The method of claim 20, wherein the linker peptide is cleaved by an enzyme.

22. The method of claim 20, wherein the linking peptide is a self-cleaving peptide.

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

24. The method of any of the preceding claims, wherein the linker peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 305-320.

25. The method of any of the preceding claims, wherein the bicistronic expressed polypeptide is a chimeric cytokine receptor (CCR), wherein the CCR comprises a transmembrane domain and an intracellular domain.

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

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

28. The method of claim 25 or 26, wherein the CCR is an induced CCR.

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

30. The method of any of the preceding claims, wherein the detection step comprises detecting the linker peptide by flow cytometry and / or Western blotting.

31. The method of claim 8, wherein the bicistronic expressed polypeptide further comprises an intracellular domain.

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

33. An in vitro method for analyzing a CAR T cell population, wherein the CAR T cells express a bicistronic expressed polypeptide, a linker peptide, and CAR, the method comprising: a) Contact the sample of the CAR T cell population with the exogenous drug; as well as b) Detect the bicistronic expressed polypeptide in the sample after the contact step.

34. The method of claim 33, wherein the linker peptide is attached to the C-terminus of the bicistronic expressed polypeptide, and the step of detecting the bicistronic expressed polypeptide includes detecting the linker peptide in the sample.

35. The method of claim 33 or 34, further comprising: c) Analyze whether a predetermined level or more of the bicistronic expressed polypeptide is detected in the sample.

36. The method of claim 35, wherein the predetermined level is the presence of the linker peptide 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 of any one of claims 33-36, wherein the CAR T cell population constitutes a CAR T cell drug substance or a CAR T cell drug product.

38. The method of claim 37, further comprising: d) If a predetermined level or more of the bicistronic expressed polypeptide is detected in the sample, the CAR T cell population is formulated to form a drug product; and optionally e) the drug product is frozen.

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