Methods of detection for engineered cells

Abstract

Provided herein are approaches to detect the expression of different bicistronically expressed polypeptides, e.g., chimeric cytokine receptors, that have been engineered into immune cell populations, e.g., CAR T cell populations. In one aspect, the present disclosure provides in vitro methods of detecting a bicistronically expressed polypeptide in a chimeric antigen receptor (CAR) T cell.

Description

Attorney Docket No.: AT-061 / 02WO METHODS OF DETECTION FOR ENGINEERED CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No.63 / 509,070, filed on June 20, 2023, the contents of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING

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

[0003] Adoptive transfer of immune cells genetically modified to 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 chimeric antigen receptors (CARs), fusion proteins comprised of an antigen recognition moiety and T cell activation domains (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)). Immune cells that contain CARs, e.g., CAR-T cells (CAR-Ts), are engineered to endow them with antigen specificity while retaining or enhancing their ability to recognize and kill a target cell.

[0004] T-cell proliferation, cytotoxic potency and persistence is driven by signal transduction pathways. Conventional CAR designs provide two signals – CD3zeta activation (Signal 1) and co-stimulation (Signal 2, e.g. via 4-1BB, OX40, and / or CD28 expression). In some contexts, a third signal (Signal 3), cytokine-induced cytokine receptor signaling (e.g. cytokine support for immune potentiation), may be desirable. Some approaches to provide Signal 3, e.g., systemic infusions of recombinant cytokines / cytokine mimetics, have however been met with significant limitations, such as systemic toxicity in humans. Alternative approaches include the engineering of 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 of which isAttorney Docket No.: AT-061 / 02WO incorporated herein by reference in its entirety. Improvements in CAR-T cell therapy, e.g., to improve efficiency and / or accuracy of treatment, through the use of additional non-CAR polypeptides, such as CCRs, will benefit the patient community.

[0005] Needed are approaches to detect the expression of different bicistronically expressed polypeptides, e.g., chimeric cytokine receptors, that have been engineered into immune cell populations, e.g., CAR T cell populations. Provided herein and compositions and methods that address this need. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides in vitro methods of detecting a bicistronically 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 that expresses the bicistronically expressed polypeptide, a linking peptide, and an additional polypeptide. In some embodiments, the linking peptide is at the C-terminus of the bicistronically expressed polypeptide. In another embodiment, the method further comprises the step of detecting the linking peptide after the contacting step. In one other embodiment, the detection of the linking peptide indicates the presence of the bicistronically expressed polypeptide in the CAR T cell.

[0007] In other embodiments, the bicistronically expressed polypeptide comprises a transmembrane domain or is a membrane protein that comprises a transmembrane domain. In one embodiment, the bicistronically expressed polypeptide further comprises an intracellular domain. In another embodiment, the linking peptide is at the C-terminus of the bicistronically expressed polypeptide or the linking peptide is at the C-terminus of the intracellular domain. In other embodiments, the bicistronically expressed polypeptide further comprises an extracellular domain. In one embodiment, the linking peptide is heterologous to the bicistronically expressed polypeptide. In one other embodiment, the linking peptide is contiguous with the intracellular domain of the bicistronically expressed polypeptide. In another embodiment, the intracellular domain is an intracellular signaling domain. In some embodiments, the additional polypeptide is a CAR.Attorney Docket No.: AT-061 / 02WO

[0008] In one additional embodiment, the exogenous agent used in the contacting step is selected from a small molecule reagent and a target molecule, wherein the CAR specifically binds to the target molecule. In other embodiments, the small molecule reagent stimulates cytokine production in the CAR T cell. In a further embodiment, the small molecule reagent comprises phorbol myristate acetate (PMA) and / or ionomycin. In one embodiment, the target molecule is a soluble target molecule or the target molecule is expressed on the surface of 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 further embodiments, the contacting step comprises contacting the CAR T cell with the soluble target molecule. In one embodiment, the linking peptide is cleavable. In one embodiment, the linking peptide is cleavable by an enzyme. In other embodiments, the linking peptide is a self-cleaving peptide. In one embodiment, the self- cleaving peptide is a P2A or T2A peptide. In another embodiment, the linking peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 305-320. In a further embodiment, the bicistronically 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 a further embodiment, the detecting step comprises detecting the linking peptide by flow cytometry and / or a protein immunoblot assay.

[0010] In another aspect, the present disclosure provides in vitro methods for analyzing a population of CAR T cells wherein the CAR T cells express a bicistronically expressed polypeptide, a linking 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 bicistronically expressed polypeptide in the sample after the contacting step. In one embodiment, the linking peptide is attached to the C-terminus of the bicistronically expressed polypeptide. In other embodiments, the step of detecting the bicistronically expressed polypeptide comprises detecting the linking peptide in the sample. In another embodiment, the method further comprises the step of analyzing whether the bicistronically expressed polypeptide isAttorney Docket No.: AT-061 / 02WO detected at or exceeding a predetermined level in the sample. In one embodiment, the predetermined level is a presence of the linking 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 comprises 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 the bicistronically expressed polypeptide is detected in the sample at or exceeding a predetermined level. In another embodiment, the method comprises the step of freezing the drug product. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG.1A-C shows a schematic of an engineered constitutively active chimeric cytokine receptor (CACCR) of the disclosure (A) and a vector for expression of the CACCR (B-C).

[0012] FIG.2A-B shows staining of cells using an anti-idiotypic antibody for an anti- CD19 antibody at 24 hours (A) and 48 hours (B) by flow cytometry.

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

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

[0015] FIG.5 shows a manufacturing workflow for engineered immune cells, e.g., CAR T cells.

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

[0017] The present disclosure provides methods for the determination of and / or detection of a bicistronically expressed polypeptide in an engineered immune cell, e.g., a chimeric antigen receptor (CAR) cell. In one embodiment, the method includes the step ofAttorney Docket No.: AT-061 / 02WO contacting the engineered immune cell with an exogenous agent. In another embodiment, the engineered immune cell comprises a polynucleotide sequence that expresses the bicistronically expressed polypeptide, a linking peptide, and an additional polypeptide. In a further embodiment, the linking peptide is at the C-terminus or N-terminus of the bicistronically expressed polypeptide. In other embodiments, the method further comprises the step of detecting the linking peptide after the contacting step, thereby indicating the presence of the bicistronically expressed polypeptide in the CAR T cell.

[0018] The practice of the instant 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). Gene editing techniques using TALENs, CRISPR / Cas9, and megaTAL nucleases, for example, are within the skill of the art and explained fully 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.Attorney Docket No.: AT-061 / 02WO Definitions

[0019] 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 cells, a cell line, or population of cells used for treating subjects that are obtained from said subject.

[0021] As used herein “allogeneic” means that cells or population of cells used for treating subjects that are not obtained from said subject, but instead from a donor.

[0022] As used herein “bicistronically expressed polypeptide” means a polypeptide encoded by first nucleic acid sequence that is present in a polynucleotide or an expression vector / cassette along with at least a second nucleic acid sequence that encodes at least a second polypeptide, wherein the first and the second nucleic acid sequence are present in the polynucleotide or vector as a contiguous nucleic acid sequence. For bicistronic expression, the polynucleotide or expression vector / cassette includes a nucleic acid sequence encoding ribosomal skip sequences such as, for example without limitation, a sequence encoding a self-cleaving peptide, e.g., a 2A peptide, including P2A, T2A, E2A, and F2A peptides. This class of peptides, which was identified in the Aphthovirus subgroup of picornaviruses, cause a ribosomal "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons. By "codon" is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0023] As used herein, the term “labeling agent” generally refers to an agent capable of interacting with a component of a cell including, without limitation, the cell membrane, a molecule on and / or within the cell, an intracellular molecule of the cell, etc. The interaction between the agent and the cell component may be a covalent interaction or a non-covalent interaction, a reversible interaction, or an irreversible interaction. The labeling agent mayAttorney Docket No.: AT-061 / 02WO be specific to the cell component including, without limitation, a biological molecule of the cell (e.g., a polypeptide, a nucleic acid, a lipid, etc.). In some embodiments, the labeling agent may be an agent having specificity to a biological target, such as an antibody or an antibody fragment. In one embodiment, the labeling agent is an agent having specificity to a cell surface molecule, e.g., a cell surface or cell membrane protein. In some cases, the labelling agent can include one or more detectable labels. In some embodiments, the labeling agent comprises an antibody, optionally conjugated with a detectable label.

[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 a colorimetric reaction, a magnetic label, a radiolabel, an oligonucleotide label, and a hapten. In some embodiments, the fluorescent label is selected from the group consisting of an Atto dye, an Alexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin, Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T- Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima- Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In some embodiments, the one or more labeling agents are used for flow cytometry. The one or more labels may be directly or indirectly coupled to or conjugated to labelling agents. For an indirect format, the one or more labels may be coupled to or conjugated to a molecule that can bind to the labeling agent. For example, the label may be conjugated to an oligonucleotide sequence that is complementary to another oligonucleotide sequence from an oligonucleotide conjugated to the labeling agent (e.g., an antibody conjugated to an oligonucleotide). Labels may also be used withAttorney Docket No.: AT-061 / 02WO the methods and compositions of the present disclosure in the context of binding agents, such as secreted molecule binding agents, e.g., secreted cytokine binding agents.

[0025] As used herein, “immune cell” refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of innate and / or adaptative immune response. Examples of immune cells include T cells, e.g., alpha / beta T cells and gamma / delta 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 operatively linked to a nucleotide sequence to be expressed. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

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

[0029] As used herein, to “functionally express” a gene means that a gene is expressed and that expression yields a functioning gene end product. For example, if a gene encodes a protein, then a cell functionally expresses the gene if expression of the gene ultimately produces a properly functioning protein. Thus, if a gene is not transcribed, or expression of the gene ultimately produces an RNA that is not translated or translation yields only a non- functioning protein, e.g., the protein does not fold correctly or is not transported to its site ofAttorney Docket No.: AT-061 / 02WO action (e.g. membrane, for membrane-bound proteins), for example, then the gene is not functionally expressed. Functional expression can be measured directly (e.g. by assaying for the gene product itself) or indirectly (e.g. by assaying for the effects of the gene product).

[0030] The term “extracellular ligand-binding domain” as used herein refers to an oligo- 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 chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. The term “stalk domain” is used herein to refer to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand- binding domain. In particular, stalk domains are used to provide more flexibility and accessibility for the extracellular ligand-binding domain.

[0031] The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.

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

[0033] The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector signal 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. Antigen binding functions 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 Fab; Fab'; F(ab')2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (see,Attorney Docket No.: AT-061 / 02WO e.g., Ward et al., Nature 341 :544-546, 1989), and an isolated complementarity determining region (CDR).

[0035] An antibody, an antibody conjugate, or a polypeptide that “specifically binds” to a target is a term well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. It is also understood that under this definition, for example, an antibody (or moiety or epitope) that specifically binds to a first target may or may not specifically bind to a second target. “Specific binding” therefore does not necessarily require (although it can include) exclusive binding.

[0036] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are several techniques for determining CDRs, e.g., an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); an approach 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, a CDR can refer to CDRs defined by either approach or by a combination of both approaches.

[0037] Antibodies (and CARs) of the instant disclosure can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F.A., et al, J. Mol. Biol., 373(4) :924-40, 2007).Attorney Docket No.: AT-061 / 02WO

[0038] As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include 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. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. A 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, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'- azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR', CO or CH2 (“formacetal”), in which each R or R' is independently H or substituted orAttorney Docket No.: AT-061 / 02WO unsubstituted alkyl (1 -20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding 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 an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well-known in the art. A CAR is a fusion protein that comprises an antigen recognition moiety, a transmembrane domain and T cell activation domains (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 an antigen or capable of interacting with a cell surface molecule, such as a ligand or a surface antigen. For example, the extracellular ligand-binding or antigen-binding domain can be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, e.g., a tumor-specific antigen. In some embodiments, the antigen- binding domain comprises an antibody, or an antigen binding fragment or an antigen binding portion of an antibody. In some embodiments, the antigen binding domain comprises an Fv or scFv, an Fab or scFab, an F(ab’)2 or a scF(ab’)2, an Fd, a monobody, a 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 which transduces the effector signal function signal and directs the cell to perform a specialized function.

[0042] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% that value orAttorney Docket No.: AT-061 / 02WO parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.

[0043] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0044] An “antigen binding protein” comprises one or more antigen binding domains. An “antigen binding domain” as used herein means any polypeptide that binds 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 or to a viral or bacterial antigen.

[0045] Antigen binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term “immunologically functional fragment” (or “fragment”) of an antigen binding domain is a species of antigen binding domain comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain, but which is still capable of specifically binding to a target antigen. Such fragments are biologically active in that they bind to the target antigen 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, and the like), one or more complementarity determining regions (“CDRs”), a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprises 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 one of skill in the art, an antigen binding domain can include non-protein components.

[0047] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by the 3 hypervariable regions (CDRs). TheAttorney Docket No.: AT-061 / 02WO CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. By convention, 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, antigen binding domains comprise one or more complementarity binding regions (CDRs) present in the full-length light or heavy chain of an antibody, and in some embodiments comprise a single heavy chain 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 the complementarity determining regions (CDRs) thereof. 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] The assignment of amino acids to each of the framework, CDR, and variable domains is typically in accordance with numbering schemes of Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md.1991), Chothia numbering (see, e.g., Chothia & 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. Pat. 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 refers broadly to a non- naturally occurring surface receptor that comprises an extracellular antigen-binding domain or an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigenAttorney Docket No.: AT-061 / 02WO receptor (CAR). Chimeric antigen receptors (CARs) are well-known in the art. A CAR is a fusion protein that comprises an antigen recognition moiety, a transmembrane domain and T cell activation domains (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)).

[0052] Where aspects or embodiments of the instant disclosure are described in terms of a Markush group or other grouping of alternatives, the instant disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The instant disclosure also envisages the explicit exclusion of one or more of any of the group members in the disclosed and / or claimed embodiments.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. In case of conflict, the present specification, including definitions, will control. Throughout this 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, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the instant disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting. Analysis of Cell Populations

[0055] In one aspect, the present disclosure provides methods, assays, systems, compositions, and kits for the detection (and / or quantitation or counting) of one or more engineered immune cells that express a bicistronically 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 (or a vector comprising the same) encoding a bicistronically expressed polypeptide. In another embodiment, the polynucleotide sequence (or vector comprising the same) further encodes aAttorney Docket No.: AT-061 / 02WO linking peptide and an additional polypeptide. In general, bicistronically expressed polypeptides, linking peptides, and additional polypeptides (such as CAR polypeptides) are expressed in engineered immune cells and can be detected according to the methods described herein. In one embodiment, the methods described herein comprise the step of contacting the engineered immune cell, e.g., a CAR T cell, with an exogenous agent. In other embodiments, the method further comprises detecting the linking peptide after the contact step. In another embodiment, the detection of the linking peptide is indicative of the presence of the bicistronically expressed polypeptide in the engineered immune cell.

[0056] The engineered cells may be engineered immune cells, e.g., CAR-T cells. Detection of the bicistronically expressed polypeptide can provide important information about engineered immune cell populations prior to or after their formulation as a CAR T cell drug product and / or prior to their administration as part of a CAR T cell drug product to a subject. In addition, the detection of the bicistronically expressed polypeptide in the engineered immune cells can provide valuable information about the cells during and / or after the manufacturing process. For example, the disclosed methods allow for detection of the bicistronically expressed polypeptide during and / or after the manufacturing process.

[0057] In an additional aspect, the present disclosure relates to the analysis of different engineered immune cell populations. In one embodiment, the analysis includes a screening method for the detection and / or quantitation of the number of engineered immune cells from a population of engineered immune cells that express a bicistronically expressed polypeptide. As discussed herein, methods to understand the degree of expression of a bicistronically expressed polypeptide in an engineered immune cell population are valuable during the engineered cell manufacturing process. It may not only be desirable to clarify whether or not a bicistronically expressed polypeptide is present in one or more cells of a population of engineered immune cells but it may also be important to quantify with a high degree of precision and accuracy the frequency of expression of the bicistronically expressed polypeptide in the population. Screening methods of interest include, without limitation, screening of engineered immune cells during or after the manufacturing process. In one embodiment, the screening comprises the step of contacting the population of engineered immune cells with an exogenous agent, which is then followed by the step of detecting the bicistronically expressed polypeptide.Attorney Docket No.: AT-061 / 02WO

[0058] In one aspect, the screening method is a quantitative and / or a qualitative method. For screening methods described herein, the detection of the bicistronically expressed polypeptide in engineered immune cells from an engineered immune cell population may be performed using different methodologies including, without limitation, flow cytometry, fluorescence activated cell sorting, (FACS) flow cytometry, an Enzyme- Linked Immunosorbent Assay (ELISA), an Enzyme-linked immuno-filtration assay (ELIFA), an immunoblotting assay, an immunofluorescence assay, an immunochemistry (IHC) assay, western blot analysis, and immunoprecipitation, molecular binding assays.

[0059] Detection using Flow cytometry. Instruments for particle analysis, e.g., flow and scanning cytometers, allow individual particles or cells (e.g., single engineered immune cells from an engineered immune cell population) to be characterized using optical parameters such as light scatter and fluorescence. For example, a flow cytometer allows an aqueous suspension containing individual particles (e.g., beads comprising analytes of interest) or cells to be passed by a detection region that exposes the suspension to an excitatory light source, such as one or more lasers, thereby enabling a user to measure the light scattering and fluorescence properties of the particles or cells. The labeling of particles or cells with one or more fluorescent dyes can facilitate detection. A plurality of different particles or cells may be subjected to flow cytometry for simultaneous detection by using different dyes that are spectrally distinct to label different particles or cells. In other embodiments, multiple photodetectors can be deployed to measure different scatter parameters and / or different spectrally distinct dyes. For example, one or more detectors may be configured to measure one or more sets of scatter parameters and one or more additional detectors may be configured to measure one or more distinct dyes, which would allow the generation of data comprising signals for each light scatter parameter and each fluorescence emission.

[0060] Flow cytometry parameters that are commonly measured include, without limitation, (i) the excitation light that is scattered by the particle or cell along a mostly forward direction, or forward scatter (FSC), (ii) the excitation light that is scattered by the particle or cell in a mostly sideways direction, or side scatter (SSC), and (ii) the light emissions from fluorescent molecules in one or more channels (range of frequencies) of the spectrum or by the fluorescent dye that is primarily detected in that channel. In one embodiment, different cell types from an engineered immune cell population can beAttorney Docket No.: AT-061 / 02WO identified by the FSC, SSC, and fluorescence emissions resulting from labeling various cell- surface proteins on the cells with dye-labeled antibodies.

[0061] The data obtained from an analysis of particles or cells (e.g., single engineered immune cells from an engineered immune cell population) by multi-color flow cytometry are multidimensional, wherein each cell corresponds to a point in a multidimensional space defined by the parameters measured. Populations of cells or particles are identified as clusters of points in the data space. The identification of clusters and, thereby, populations can be carried out manually by drawing a gate around a population displayed 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 that define the limits of the populations, can be determined automatically. Flow cytometry is an important tool for the analysis and / or isolation of particle or cells (e.g., individual engineered immune cells from engineered immune cell populations) and cellular analytes or constituents thereof. Therefore, it can be used in the context of engineered immune cell analysis and / or isolation. In one embodiment, the present disclosure provides a method using a fluid stream to linearly space donor cells from an engineered immune cell population such that they pass individually through a detection apparatus. A single cell from the engineered immune cell population can be distinguished from other cells by their location in the fluid stream and the presence of detectable markers. As a result, a flow cytometer can be used to detect one or more bicistronically expressed polypeptides (as described herein) and / or generate a bicistronically expressed polypeptide expression profile for an engineered immune cell population. Such profiles may comprise a percentage of cells (as further described herein) expressing the bicistronically expressed polypeptide out of the total cell population that is being analyzed.

[0062] In one additional aspect, the instant disclosure provides a method of detecting engineered immune cells that express a bicistronically expressed polypeptide from an engineered immune cell population by detecting a level of the bicistronically expressed polypeptide. In one embodiment, the detected level indicates that some of the engineered immune cells express the bicistronically expressed polypeptide. In another aspect, the level of the bicistronically expressed polypeptide that is detected indicates the number of engineered immune cells expressing the bicistronically expressed polypeptide out of the engineered immune cell population being analyzed.Attorney Docket No.: AT-061 / 02WO

[0063] In some embodiments, the method provides a quantitative measurement of engineered immune cells from an engineered immune cell population that do not express bicistronically expressed polypeptide. Flow cytometry can be used to quantify cells expressing or not expressing the bicistronically expressed polypeptide, and / or quantifying cells of a specific cell type, within a population of cells.

[0064] As described herein, flow cytometry is a method for quantifying components or structural features of cells primarily by optical means using certain labeling agents (as further described herein). Since 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. A flow cytometry analysis involves two primary steps: 1) labeling selected cell types with one or more detectable labels or agents, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling cell types includes binding labeled antibodies to markers expressed by the specific cell type. The antibodies may be either directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescent-labeled second antibody which recognizes the first antibody.

[0065] In one other aspect, the present disclosure utilizes flow cytometry to provide methods for generating an expression profile for the bicistronically expressed polypeptide that is expressed in the engineered immune cell population based on the percentage of the cells in which the bicistronically expressed polypeptide is detected. In one embodiment, the method comprises providing an engineered immune cell population that is suspected of comprising the bicistronically expressed polypeptide. In another embodiment, the method further comprises contacting the engineered immune cell population with an exogenous agent. In a further embodiment, the method comprises detecting a level of the bicistronically expressed polypeptide in the engineered immune cell population after the contacting step, wherein the detected level of the bicistronically expressed polypeptide indicates that a percentage of the engineered immune cell population expresses the bicistronically expressed polypeptide. In other embodiments, the method further comprises detecting a level of the additional polypeptide in the engineered immune cell population after the contacting step, wherein the detected level of the additional polypeptide indicates that a percentage of the engineered immune cell population expresses the additional polypeptide. In some embodiments, the method comprises detecting levels of theAttorney Docket No.: AT-061 / 02WO bicistronically expressed polypeptide and the additional polypeptide, wherein the detected levels indicate a percentage expression in the engineered immune cell population of the bicistronically expressed polypeptide and the additional polypeptide.

[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 may be used at different stages of manufacturing of an engineered cell population. As shown in FIG.5, an immune cell population can go through manufacturing steps 10-15 before it is screened at 16 for expression of the bicistronically expressed polypeptide. The outcome of screening at 16 informs a decision on whether to proceed with using the engineered immune cell population to formulate a cell therapy product, e.g., a CAR T cell therapy product. If the bicistronically expressed polypeptide is detected 18, then the engineered immune cell population may be considered to have failed the screening assay. If the bicistronically expressed polypeptide detected 17, then the bicistronically expressed polypeptide may be considered to have passed the screening assay and can be moved forward in the cell therapy product manufacturing process.

[0067] In one embodiment, the cell therapy product lot that comprises the engineered immune cells that passed at 17 may have originated from a subject who will ultimately receive engineered immune cell population. For example, the engineered immune cells may comprise autologous immune cells obtained from a subject who 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 passed at 17 may have originated from a donor, who is a different individual than the subject who will receive the engineered cell population. For instance, the engineered immune cells may comprise allogeneic immune cells obtained from a healthy donor, who is a different individual than the subject who will receive engineered immune cells, e.g., CAR T cells, obtained from the allogeneic immune cells.

[0068] In one embodiment, screening 16 can be performed after 13 or after 14 according to the workflow in FIG.5, either in addition to screening after 15 or as an alternative to screening after 15. In addition, screening 16 can delayed until after 19, wherein the engineered immune cells complete the manufacturing process ending with 19 cryopreservation as a CAR T cell therapy product. The cryopreserved product can beAttorney Docket No.: AT-061 / 02WO thawed out and then screening 16 can be performed on a sample of the product to determine whether detection is a pass or a fail. Bicistronically expressed polypeptides

[0069] In one aspect, the disclosure concerns engineered immune cells and populations comprising the same, as well as methods of detecting polypeptides expressed by the cells and populations. In one embodiment, the polypeptides expressed by the cells are bicistronically expressed polypeptides. In one other embodiment, one or more bicistronically expressed polypeptides are expressed from a polynucleotide or a vector comprising the same, wherein the polynucleotide comprises several different elements. As described herein, a 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 sequence are separated by a third nucleic acid sequence encoding a linking 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 bicistronically expressed polypeptide during translation. In another embodiment, a second amino acid that is translated after translation of the first amino acid is the first N-terminal amino acid of the second polypeptide. In one other embodiment, the first amino acid and the second amino acid are characterized by the lack 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 concern the detection of the first polypeptide as the bicistronically expressed polypeptide. In another embodiment, the second polypeptide is a CAR. In certain embodiments, the first polypeptide, i.e., the bicistronically expressed polypeptide, that is detected is a different polypeptide than the second polypeptide, i.e., the CAR. The first polypeptide, i.e., the bicistronically expressed polypeptide, may be any polypeptide including without limitation, receptor polypeptides, membrane polypeptides, cytosolic polypeptides, and secreted polypeptides.

[0071] In one embodiment, the first polypeptide is a membrane polypeptide that comprises an intracellular domain. In one embodiment, the intracellular domain is anAttorney Docket No.: AT-061 / 02WO 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. In general, inducible CCRs are receptors that are responsive to a ligand, such as a small molecule (e.g., AP1903) or 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, which is incorporated herein by reference in its entirety). In one aspect, the invention 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 a Janus Kinase (JAK)-binding domain of, or derived from, a protein. In some of these embodiments, the tyrosine kinase activating domain further comprises a transmembrane domain. In some embodiments, the tyrosine kinase activating domain comprises a tyrosine kinase domain of, or derived from, 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 one other embodiment, the CCR is a constitutively active chimeric cytokine receptor (CACCR). The presence of a constitutively active, tunable chimeric cytokine receptor allows for the immune potentiation of Signal 3 to meet the need for immune potentiation (see US2020-0291090A1, which is incorporated herein by reference in its entirety). In general, the CACCRs of the disclosure are composed of two monomers, each monomer comprising: (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 disclosure does not comprise an extracellular ligand-binding domain. In some embodiments, the monomers are identical, giving rise to a constitutively active homodimer. In some embodiments, the monomers are not identical, giving rise a constitutively active heterodimer, which may be desirable under certain circumstances. The monomers of the CACCRs of the disclosure are capable of spontaneously dimerizing, and can activate signaling in the absence of any exogenous stimulation or ligand (ligand- independent dimerization).Attorney Docket No.: AT-061 / 02WO

[0074] Exemplary amino acid sequences useful in CACCRs are provided in Table 1A. Table 1A: Exemplary transmembrane domain amino acid sequences Transmembrane Amino acid sequence SEQ Domain ID TPOR / MPLR(478-582) SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 1 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478-582; SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 2 H499L,S505N) LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 3 582;S505N) LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLGLL 4 582;H499L,W515K) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 5 582;W515K) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 6 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 7 582;S505N,W515K) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLNLL 8 582;H499L,G509N) LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL

[0075] Exemplary transmembrane and JAK-binding sequences that are useful in CACCRs provided herein are shown in Table 1B.Attorney Docket No.: AT-061 / 02WO Table 1B: Exemplary Transmembrane + JAK2 Binding Domain Sequences Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: GCSFR(614-710) LTLMTLTPEGSELHIILGLFGLLLLLTCLCGTAWL 9 CCSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDA FQLPGLGTPPITKLTVLEEDEKKPVPWE GP130(609-700) TTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNK 10 RDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFN SKDQMYSDGNFTDVSVVEIEAND TPOR / MPLR(478-582) SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 11 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(N-1) SDPTRVETATETWISLVTALHLVLGLSAVLGLLL 12 LRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRD TAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLP L TPOR / MPLR(N-2) SDPTRVETATETISLVTALHLVLGLSAVLGLLLLR 13 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-2+1) SDPTRVETATETLISLVTALHLVLGLSAVLGLLLL 14 RWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-3) SDPTRVETATETSLVTALHLVLGLSAVLGLLLLR 15 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-4) SDPTRVETATETLVTALHLVLGLSAVLGLLLLRW 16 QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAA LSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-4+1) SDPTRVETATETILVTALHLVLGLSAVLGLLLLR 17 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-5) SDPTRVETATETVTALHLVLGLSAVLGLLLLRW 1824 QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAA LSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-6) SDPTRVETATETTALHLVLGLSAVLGLLLLRWQF 1925 PAHYRRLRHALWPSLPDLHRVLGQYLRDTAALS PPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-7) SDPTRVETATETALHLVLGLSAVLGLLLLRWQFP 20 AHYRRLRHALWPSLPDLHRVLGQYLRDTAALSP PKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-8) SDPTRVETATETLHLVLGLSAVLGLLLLRWQFPA 21 HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPP KATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-9) SDPTRVETATETHLVLGLSAVLGLLLLRWQFPAH 22 YRRLRHALWPSLPDLHRVLGQYLRDTAALSPPK ATVSDTCEEVEPSLLEILPKSSERTPLPLAttorney Docket No.: AT-061 / 02WO Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: TPOR / MPLR(N-10) SDPTRVETATETLVLGLSAVLGLLLLRWQFPAHY 23 RRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-11) SDPTRVETATETVLGLSAVLGLLLLRWQFPAHYR 24 RLRHALWPSLPDLHRVLGQYLRDTAALSPPKAT VSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-12) SDPTRVETATETLGLSAVLGLLLLRWQFPAHYRR 25 LRHALWPSLPDLHRVLGQYLRDTAALSPPKATV SDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-13) SDPTRVETATETGLSAVLGLLLLRWQFPAHYRRL 26 RHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-14) SDPTRVETATETLSAVLGLLLLRWQFPAHYRRLR 27 HALWPSLPDLHRVLGQYLRDTAALSPPKATVSD TCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-15) SDPTRVETATETSAVLGLLLLRWQFPAHYRRLRH 28 ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-16) SDPTRVETATETAVLGLLLLRWQFPAHYRRLRH 29 ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-17) SDPTRVETATETVLGLLLLRWQFPAHYRRLRHA 30 LWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCE EVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-18) SDPTRVETATETLGLLLLRWQFPAHYRRLRHAL 31 WPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPL TPOR / MPLR(N+1) SDPTRVETATETAWLISLVTALHLVLGLSAVLGL 32 LLLRWQFPAHYRRLRHALWPSLPDLHRVLGQYL RDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTP LPL TPOR / MPLR(N+2) SDPTRVETATETAWVLISLVTALHLVLGLSAVLG 33 LLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQY LRDTAALSPPKATVSDTCEEVEPSLLEILPKSSER TPLPL TPOR / MPLR(N+3) SDPTRVETATETAWLVLISLVTALHLVLGLSAVL 34 GLLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQ YLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPL TPOR / MPLR(N+4) SDPTRVETATETAWILVLISLVTALHLVLGLSAVL 35 GLLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQ YLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPL TPOR / MPLR(N+5) SDPTRVETATETAWLILVLISLVTALHLVLGLSAV 36 LGLLLLRWQFPAHYRRLRHALWPSLPDLHRVLG QYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSS ERTPLPLAttorney Docket No.: AT-061 / 02WO Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: TPOR / MPLR(N+6) SDPTRVETATETAWLLILVLISLVTALHLVLGLSA 37 VLGLLLLRWQFPAHYRRLRHALWPSLPDLHRVL GQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKS SERTPLPL TPOR / MPLR(N+7) SDPTRVETATETAWVLLILVLISLVTALHLVLGLS 38 AVLGLLLLRWQFPAHYRRLRHALWPSLPDLHRV LGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPK SSERTPLPL TPOR / MPLR(N+8) SDPTRVETATETAWLVLLILVLISLVTALHLVLGL 39 SAVLGLLLLRWQFPAHYRRLRHALWPSLPDLHR VLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILP KSSERTPLPL

[0076] Table 1C provides exemplary amino acid sequences of recruiting domains for the CACCRs of the disclosure. In some embodiments, the CACCR of the disclosure comprises a recruiting domain comprising the amino acid sequence selected from one or more of the receptor sequences in Table 1C. In some embodiments, the CACCR of the disclosure comprises a recruiting domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences in Table 1C. Table 1C: Recruiting Domain (Cytotail) Sequences Cytotail sequences Amino acid sequence SEQ ID NO: IL7R(316-459) ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN 40 CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ IL2Rb(333-551) VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF 41 FHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAP TGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLL GGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDP QPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDA GPREGVSFPWSRPPGQGEFRALNARLPLNTDAYL SLQELQGQDPTHLV IFNAR1(508-557) ISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNE 42 DESESKTSEELQQDFV IFNAR2(310-515) KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 43 LTVRPLGQASATSTESQLIDPESEEEPDLPEVDVE LPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDY SSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEA PLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQAttorney Docket No.: AT-061 / 02WO Cytotail sequences Amino acid sequence SEQ ID NO: PTFPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYI MR IFNAR1 / 2(IFNAR1 ISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNE 44 residues 508-557-IFNAR2 DESESKTSEELQQDFVKKKVWDYNYDDESDSDT residues 310-515) EAAPRTSGGGYTMHGLTVRPLGQASATSTESQLI DPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPC ERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSV FLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPD NVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSD QSDTSESDVDLGDGYIMR IFNLR1(300-520) RGVRPTPRVRAPATQQTRWKKDLAEDEEEEDEE 45 DTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVD SGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWD RAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKD SGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPV SLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAE STQRTEDRGRTLGHYMAR IL2RG (335-369) IPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPE 46 T IL9R(356-521) TALLTCGPARPWKSVALEEEQEGPGTRLPGNLSS 47 EDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPA PPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSA LPGNTQSSGPIPALACGLSCDHQGLETQQGVAW VLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF IL21R(322-538) PRSPAKRLQLTELQEPAELVESDGVPKPSFWPTA 48 QNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCT WPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAG TTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADG EDWAGGLPWGGRSPGGVSESEAGSPLAGLDMD TFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQ WVVIPPPLSSPGPQAS GHR(353-638) PDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGR 49 TSCCEPDILETDFNANDIHEGTSEVAQPQRLKGE ADLLCLDQKNQNNSPYHDACPATQQPSVIQAEK NKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYA QVSDITPAGSVVLSPGQKNKAGMSQCDMHPEM VSLCQENFLMDNAYFCEADAKKCIPVAPHIKVES HIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSE MPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSS CGYVSTDQLNKIMP EpoR(339-508) WGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVL 50 DKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASS CSSALASKPSPEGASAASFEYTILDPSSQLLRPWT LCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQG AQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS murine IL2Rb(337-539) AVQLLLLQKDSAPLPSPSGHSQASCFTNQGYFFF 51 HLPNALEIESCQVYFTYDPCVEEEVEEDGSRLPEAttorney Docket No.: AT-061 / 02WO Cytotail sequences Amino acid sequence SEQ ID NO: GSPHPPLLPLAGEQDDYCAFPPRDDLLLFSPSLST PNTAYGGSRAPEERSPLSLHEGLPSLASRDLMGL QRPLERMPEGDGEGLSANSSGEQASVPEGNLHG QDQDRGQGPILTLNTDAYLSLQELQAQDSVHLI murine IL7Ra(316-459) ARDEVESFLPNDLPAQPEELETQGHRAAVHSAN 52 RSPETSVSPPETVRRESPLRCLARNLSTCNAPPLL SSRSPDYRDGDRNRPPVYQDLLPNSGNTNVPVPV PQPLPFQSGILIPVSQRQPISTSSVLNQEEAYVTMS SFYQNK EGFR(955-1186) VIQGDERMHLPSPTDSNFYRALMDEEDMDDVVD 53 ADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVA CIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSID DTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNP APSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTF DSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPN GIFKGSTAENAEYLRVAPQSSEFIGA EGFR(955- VIQGDERMHLPSPTDSNFFRALMDEEDMDDVVD 54 1186;Y974F,d1045-1057) ADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVA CIDRNGLQSCPIKEDSFLQRIDDTFLPVPEYINQSV PKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHS TAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSH QISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEY LRVAPQSSEFIGA EGFR(955-1009;Y974F) VIQGDERMHLPSPTDSNFFRALMDEEDMDDVVD 55 ADEYLIPQQGFFSSPSTSRTP EGFR(1019-1085) NNSTVACIDRNGLQSCPIKEDSFLQRIDDTFLPVP 56 EYINQSVPKRPAGSVQNPV EGFR(1037- KEDSFLQRIDDTFLPVPEFINQSVPKRPAGSVQNP 57 1103;Y1068 / 1101F,d1045 VYHNQPLNPAPSRDPHFQD -1057) EGFR(1066- VPEFINQSVPKRPAGSVQNPVFHNQPLNPAPSRD 58 1118;Y1068 / 1086F) PHYQDPHSTAVGNPEYLNTV EGFR(1122-1165) PEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDN 59 PDYQQDFFPKEAKPNGIFKG EGFR(1133- WAQKGSHQISLDNPDFQQDFFPKEAKPNGIFKGS 60 1186;Y1148F) TAENAEYLRVAPQSSEFIGA IL12Rb2(775-825) SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL 61 PSHEAPLADSLEELEPQ IL7Ra(376-416) ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 62 TTNSTLP IL7Ra(424-459) GILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ 63 NQ IL7Ra(376-416,424-459) ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 64 TTNSTLPQGQPILTSLGSNQEEAYVTMSSFYQNQ IL7Ra(424-459;Y456F) GILTLNPVAQGQPILTSLGSNQEEAYVTMSSFFQN 65 QAttorney Docket No.: AT-061 / 02WO Cytotail sequences Amino acid sequence SEQ ID NO: IL7R(376-416,424- ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 66 459,Y456F) TTNSTLPQGQPILTSLGSNQEEAYVTMSSFFQNQ IL2Rb`small(393-433) DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD 67 LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV IL2Rbsmall(518-551) GQGEFRALNARLPLNTDAYLSLQELQGQDPTHL 68 V IL2Rbsmall(339-379,393- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 69 433) LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPS IL2Rbsmall(339-379,518- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 70 551) LEIEACQ GQGEFRALNARLPLNTDAYLSLQELQGQDPTHL V IL2Rbsmall(393-433,518- DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD 71 551) LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV IL2Rbsmall(339-379,393- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 72 433,518-551) LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV IFNAR2small(310-352) KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 73 LTVRPLGQASA IFNAR2small(486-515) EGLWSEDAPSDQSDTSESDVDLGDGYIMR 74 IFNAR2small(310- KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 75 352,486-515) LTVRPLGQASA EGLWSEDAPSDQSDTSESDVDLGDGYIMR BLNK(53-208) ASESPADEEEQWSDDFDSDYENPDEHSDSEMYV 76 MPAEENADDSYEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR BLNK(53-208;Y72F) ASESPADEEEQWSDDFDSDFENPDEHSDSEMYV 77 MPAEENADDSYEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR BLNK(53- ASESPADEEEQWSDDFDSDFENPDEHSDSEMYV 78 208;Y72F,Y96F) MPAEENADDSFEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR EpoR(339-508) WGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVL 79 DKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASS CSSALASKPSPEGASAASFEYTILDPSSQLLRPWT LCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQG AQGGLSDGPYSNPYENSLIPAAEPLPPSYVACSAttorney Docket No.: AT-061 / 02WO Cytotail sequences Amino acid sequence SEQ ID NO: IL12Rb2(714-862) VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHS 80 ASSPPPPRALQAESRQLVDLYKVLESRGSDPKPE NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTL DQLKMRCDSLML IL12Rb1(622-662) WDKGERTEPLEKTELPEGAPELALDTELSLEDGD 81 RCKAKM IL10R1(304-578) VSPELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLP 82 DPHPQADRTLGNREPPVLGDSCSSGSSNSTDSGIC LQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQ NSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAA VAFQGYLRQTRCAEEKATKTGCLEEESPLTDGL GPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTL ASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWS FAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQ SSE IL2Rb(333-551, VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF 83 Y381S,Y384S,Y387S) FHLPDALEIEACQVSFTSDPSSEEDPDEGVAGAPT GSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLG GPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQ PLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAG PREGVSFPWSRPPGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV IL2Rb(333-551, VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGSFFF 84 Y364S,Y381S,Y384S,Y3 HLPDALEIEACQVSFTSDPSSEEDPDEGVAGAPTG 87S)  SSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGG PSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQP LGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGP REGVSFPWSRPPGQGEFRALNARLPLNTDAYLSL QELQGQDPTHLV

[0077] Table 1D shows exemplary CACCR sequences (e.g., full length CACCR sequences or components thereof) of the disclosure. The CACCRs may be expressed with a signal sequence, e.g., a CD8SS of sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127).Attorney Docket No.: AT-061 / 02WO Table 1D – Constitutively Active Chimeric Cytokine Receptor (CACCRs) sequences Receptor Amino acid sequence SEQ ID NO: TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 85 582).IL7Ra(316-459) LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478-582; SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 86 H499L,S505N).IL7Ra(31 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 6-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 87 582;S505N).IL7Ra(316- LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLGLL 88 582;H499L,W515K).IL7 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR Ra(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 89 582;W515K).IL7Ra(316- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 90 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL7Ra(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 91 582;S505N,W515K).IL7R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR a(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLNLL 92 582;H499L,G509N).IL7R LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR a(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ TPOR / MPLR(478-582; SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 93 H499L,S505N).IL7Ra(31 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 6-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ CD8SS- MALPVTALLLPLALLLHAARP 94 TPOR / MPLR(478- 582;S505N,W515K). SDPTRVETATETAWISLVTALHLVLGLNAVLGLL IL12Rb2(714-862) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHS ASSPPPPRALQAESRQLVDLYKVLESRGSDPKPE NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTL DQLKMRCDSLML CD8SS- MALPVTALLLPLALLLHAARP 95 TPOR / MPLR(478- 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL K).IL12Rb2(714-862) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHS ASSPPPPRALQAESRQLVDLYKVLESRGSDPKPE NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTL DQLKMRCDSLML CD8SS- MALPVTALLLPLALLLHAARP 96 TPOR / MPLR(478- 582;S505N,W515K).IL12 SDPTRVETATETAWISLVTALHLVLGLNAVLGLL Rb2(775-825) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ CD8SS- MALPVTALLLPLALLLHAARP 97 TPOR / MPLR(478- 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL K). IL12Rb2(775-825) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ CD8SS- MALPVTALLLPLALLLHAARP 98 TPOR / MPLR(478- 582;S505N,W515K). SDPTRVETATETAWISLVTALHLVLGLNAVLGLL IL2Rb(333-551) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF FHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAP TGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLL GGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDP QPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDA GPREGVSFPWSRPPGQGEFRALNARLPLNTDAYL SLQELQGQDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 99 TPOR / MPLR(478- 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL K). IL2Rb(333-551) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF FHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAP TGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLL GGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDP QPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDA GPREGVSFPWSRPPGQGEFRALNARLPLNTDAYL SLQELQGQDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 100 TPOR / MPLR(478- 582;S505N,W515K).IL2R SDPTRVETATETAWISLVTALHLVLGLNAVLGLL b(393-433,518-551) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 101 TPOR / MPLR(478- 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: K).IL2Rb(393-433,518- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 551) PL DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 102 TPOR / MPLR(478- 582;S505N,W515K). SDPTRVETATETAWISLVTALHLVLGLNAVLGLL IL2Rb(339-379,393- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 433,518-551) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 103 TPOR / MPLR(478- 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL K).IL2Rb(339-379,393- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 433,518-551) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV CD8SS- MALPVTALLLPLALLLHAARP 104 TPOR / MPLR(478- 582;S505N,W515K).IL7R SDPTRVETATETAWISLVTALHLVLGLNAVLGLL a(316-459).IL12Rb2(775- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 825) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ CD8SS- MALPVTALLLPLALLLHAARP 105 TPOR / MPLR(478-Attorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: 582;H499L,S505N,W515 SDPTRVETATETAWISLVTALLLVLGLNAVLGLL K).IL7Ra(316- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 459).IL12Rb2(775-825) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 106 582).IL7Ra(316-459) LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478-582; SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 107 H499L,S505N).IL7Ra(31 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 6-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 108 582;S505N).IL7Ra(316- LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLGLL 109 582;H499L,W515K).IL7 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR Ra(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 110 582;W515K).IL7Ra(316- LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR 459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 111 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL7Ra(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 112 582;S505N,W515K).IL7R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR a(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLSAVLNLL 113 582;H499L,G509N).IL7R LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR a(316-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478-582; SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 114 H499L,S505N).IL7Ra(31 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLR 6-459) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 115 582;S505N,W515K).IL2R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR b(333-551) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ GYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGV AGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFS PSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 116 582;S505N,W515K).IL12 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR Rb2(714-862) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEVTPVFRHPPCSNWPQREKGIQGHQASEKD MMHSASSPPPPRALQAESRQLVDLYKVLESRGS DPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLP SHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCG DKLTLDQLKMRCDSLML TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 117 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL2Rb(333-551) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ GYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGV AGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFS PSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRD WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 118 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL12Rb2(714-862) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEVTPVFRHPPCSNWPQREKGIQGHQASEKD MMHSASSPPPPRALQAESRQLVDLYKVLESRGS DPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLP SHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCG DKLTLDQLKMRCDSLML TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 119 582;S505N,W515K).IL12 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR Rb2(775-825) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLESDPKPENPACPWTVLPAGDLPTHDGYLPSN IDDLPSHEAPLADSLEELEPQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 120 582;H499L;S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL12Rb2(775-825) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLESDPKPENPACPWTVLPAGDLPTHDGYLPSN IDDLPSHEAPLADSLEELEPQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 121 582;S505N,W515K).IL7R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR a(316-459).IL12Rb2(775- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 825) PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: TMSSFYQNQSRSDPKPENPACPWTVLPAGDLPTH DGYLPSNIDDLPSHEAPLADSLEELEPQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 122 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL7Ra(316- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 459).IL12Rb2(775-825) PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAP ILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQSRSDPKPENPACPWTVLPAGDLPTH DGYLPSNIDDLPSHEAPLADSLEELEPQ TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 123 582;S505N,W515K).IL2R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR b(333-551; Y381S, DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL Y384S,Y387S) PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ GYFFFHLPDALEIEACQVSFTSDPSSEEDPDEGVA GAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSP SLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRD WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 124 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL2Rb(333- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 551;Y381S,Y384S,Y387S PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ ) GYFFFHLPDALEIEACQVSFTSDPSSEEDPDEGVA GAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSP SLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRD WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 125 582;S505N,W515K).IL2R LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR b(333- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 551;Y364S,Y381S,Y384S PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ ,Y387S) GSFFFHLPDALEIEACQVSFTSDPSSEEDPDEGVA GAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSP SLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRD WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 126 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K).IL2Rb(333- DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL 551;Y364S,Y381S,Y384S PLLEVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ ,Y387S) GSFFFHLPDALEIEACQVSFTSDPSSEEDPDEGVA GAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPAttorney Docket No.: AT-061 / 02WO Receptor Amino acid sequence SEQ ID NO: SLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRD WDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEV PDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV *The underlined LE and SR are exemplary optional linker that may be inserted between two domains.

[0078] In another embodiment, the CCR is an inducible PD-1 CCR, which is active when engaged with PD-1 ligands or activated with an anti-PD-1 antibody (see US2020- 0276238A1, which is incorporated herein by reference in its entirety). 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, for example, binding a PD-1 ligand (e.g. PD-L1, PD-L2), or a PD-1 antibody. These receptors activate signaling when monomers of the receptor cluster and / or dimerize. In some embodiments, a monomer of the PD-1 CCR 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 the cytoplasmic domain of a receptor; e.g. from a cytokine receptor). In some embodiments, a monomer of the PD-1 CCR 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 the 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 PD-1 ligands, and / or are clustered and activated with an anti-PD-1 antibody. The PD-1 chimeric cytokine receptors activate signaling upon for example binding a PD-L1 ligand, PD-L2 ligand, and / or a PD-1 antibody.

[0079] The constitutively active PD-1 CCRs of the disclosure are active regardless of PD- 1 ligand availability but may increase activity in the presence of a PD- ligand. In some embodiments, a monomer of the constitutively active PD-1 CCR 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 the cytoplasmic domain of a receptor; e.g. from a cytokine receptor). In some embodiments, a monomer of theAttorney Docket No.: AT-061 / 02WO constitutively active PD-1 CCR 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 the 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, constitutively active PD-1 CCRs can bind to PD-1 ligands or an anti-PD-1 antibody, and / or are clustered and the activity of the receptors can be further increased by engagement with PD-1 ligands or an anti-PD-1 antibody.

[0080] Table 1E shows exemplary PD1 amino acid ectodomain sequences of the disclosure. It is noted that the expression and extracellular location of the exemplary PD1 amino acid sequences can be achieved with the use of a signal sequence. In an exemplary embodiment, a CD8 signal sequence (CD8SS) MALPVTALLLPLALLLHAARP (SEQ ID NO: 127) is utilized. Table 1E: Exemplary Ectodomain Sequences Ectodomain Amino acid sequence SEQ ID NO: Wild type (WT) PD1 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 128 ectodomain (PD-1(21- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 207)) PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLV VGVVGGLLGSLVLLVWVLAVICSRAARGTIGAR RTGQ 2x WTPD1 ectodomain PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 129 (PD-1(21-207)-PD-1(21- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 207)) PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLA VICSRAARGTIGARRTGQPGWFLDSPDRPWNPPT FSPALLVVTEGDNATFTCSFSNTSESFVLNWYRM SPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG VVGGLLGSLVLLVWVLAVICSRAARGTIGARRT GQ 3x WTPD1 ectodomain PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 130 (PD-1(21-207)-PD-1(21- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 207)-PD-1(21-207)) PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLA VICSRAARGTIGARRTGQPGWFLDSPDRPWNPPTAttorney Docket No.: AT-061 / 02WO Ectodomain Amino acid sequence SEQ ID NO: FSPALLVVTEGDNATFTCSFSNTSESFVLNWYRM SPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG VVGGLLGSLVLLVWVLAVICSRAARGTIGARRT GQPGWFLDSPDRPWNPPTFSPALLVVTEGDNATF TCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDR SQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSG TYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA HPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWV LAVICSRAARGTIGARRTGQ High-affinity (HA) PD1 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 131 ectodomain SFSNTSESFHVIWHRESPSGQTDTLAAFPEDRSQP GQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY VCGVISLAPKIQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLA VICSRAARGTI GARRTGQ FKBP(F36V) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLED 132 GKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVA QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLV FDVELLKLE Wildtype (WT) PD1 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 133 ectodomain (PD-1(21- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 170)) PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLV High-affinity (HA) PD1 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 134 ectodomain (21-170) SFSNTSESFHVIWHRESPSGQTDTLAAFPEDRSQP GQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY VCGVISLAPKIQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLV 2x WTPD1 ectodomain PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 135 (PD-1(21-170)-G-PD- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 1(21-170)) PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVGPGWFLDSPDRPWNPPTFSP ALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDF HMSVVRARRNDSGTYLCGAISLAPKAQIKESLRA ELRVTERRAEVPTAHPSPSPRPAGQFQTLV 3x WTPD1 ectodomain PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 136 (PD-1(21-170)-G-PD- SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ 1(21-170)-G-PD-1(21- PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY 170)) LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVGPGWFLDSPDRPWNPPTFSP ALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFAttorney Docket No.: AT-061 / 02WO Ectodomain Amino acid sequence SEQ ID NO: HMSVVRARRNDSGTYLCGAISLAPKAQIKESLRA ELRVTERRAEVPTAHPSPSPRPAGQFQTLVGPGW FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQ DCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCG AISLAPKAQIKESLRAELRVTERRAEVPTAHPSPS PRPAGQFQTLV

[0081] In some embodiments, the PD-1 ectodomain is a dominant negative. Table 1F shows exemplary PD-1 dominant negative (DN) sequences of the disclosure. The DN sequences of Table 1E may be expressed with the aid of a signal sequence, e.g., a CD8SS signal sequence of SEQ ID NO: 127. Table 1F: PD1 dominant negative (DN) sequences: PD1 DN Amino acid sequence SEQ ID NO: Dominant PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESF 137 negative wild VLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR type PD1 DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTE (21-207) RRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWV LAVICSRAARGTI GARRTGQ Dominant PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESF 138 negative high HVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRD affinity PD1 FHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER RAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVL AVICSRAARGTI GARRTGQ

[0082] Table 1G shows exemplary transmembrane amino acid sequences, coupled to intracellular JAK2 binding domain sequences for use in the PD-1 CCRs. Table 1G: Exemplary Transmembrane + JAK2 Binding Domain Sequences Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: GCSFR(614-710) LTLMTLTPEGSELHIILGLFGLLLLLTCLCGTAWL 139 CCSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDA FQLPGLGTPPITKLTVLEEDEKKPVPWE GP130(609-700) TTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNK 140 RDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFN SKDQMYSDGNFTDVSVVEIEAND TPOR / MPLR(478-582) SDPTRVETATETAWISLVTALHLVLGLSAVLGLL 141 LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRAttorney Docket No.: AT-061 / 02WO Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(N-1) SDPTRVETATETWISLVTALHLVLGLSAVLGLLL 142 LRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRD TAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLP L TPOR / MPLR(N-2) SDPTRVETATETISLVTALHLVLGLSAVLGLLLLR 143 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-2+1) SDPTRVETATETLISLVTALHLVLGLSAVLGLLLL 144 RWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-3) SDPTRVETATETSLVTALHLVLGLSAVLGLLLLR 145 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-4) SDPTRVETATETLVTALHLVLGLSAVLGLLLLRW 146 QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAA LSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-4+1) SDPTRVETATETILVTALHLVLGLSAVLGLLLLR 147 WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-5) SDPTRVETATETVTALHLVLGLSAVLGLLLLRW 148 QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAA LSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-6) SDPTRVETATETTALHLVLGLSAVLGLLLLRWQF 149 PAHYRRLRHALWPSLPDLHRVLGQYLRDTAALS PPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-7) SDPTRVETATETALHLVLGLSAVLGLLLLRWQFP 150 AHYRRLRHALWPSLPDLHRVLGQYLRDTAALSP PKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-8) SDPTRVETATETLHLVLGLSAVLGLLLLRWQFPA 151 HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPP KATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-9) SDPTRVETATETHLVLGLSAVLGLLLLRWQFPAH 152 YRRLRHALWPSLPDLHRVLGQYLRDTAALSPPK ATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-10) SDPTRVETATETLVLGLSAVLGLLLLRWQFPAHY 153 RRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-11) SDPTRVETATETVLGLSAVLGLLLLRWQFPAHYR 154 RLRHALWPSLPDLHRVLGQYLRDTAALSPPKAT VSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-12) SDPTRVETATETLGLSAVLGLLLLRWQFPAHYRR 155 LRHALWPSLPDLHRVLGQYLRDTAALSPPKATV SDTCEEVEPSLLEILPKSSERTPLPLAttorney Docket No.: AT-061 / 02WO Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: TPOR / MPLR(N-13) SDPTRVETATETGLSAVLGLLLLRWQFPAHYRRL 156 RHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-14) SDPTRVETATETLSAVLGLLLLRWQFPAHYRRLR 157 HALWPSLPDLHRVLGQYLRDTAALSPPKATVSD TCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-15) SDPTRVETATETSAVLGLLLLRWQFPAHYRRLRH 158 ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-16) SDPTRVETATETAVLGLLLLRWQFPAHYRRLRH 159 ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-17) SDPTRVETATETVLGLLLLRWQFPAHYRRLRHA 160 LWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCE EVEPSLLEILPKSSERTPLPL TPOR / MPLR(N-18) SDPTRVETATETLGLLLLRWQFPAHYRRLRHAL 161 WPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPL TPOR / MPLR(N+1) SDPTRVETATETAWLISLVTALHLVLGLSAVLGL 162 LLLRWQFPAHYRRLRHALWPSLPDLHRVLGQYL RDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTP LPL TPOR / MPLR(N+2) SDPTRVETATETAWVLISLVTALHLVLGLSAVLG 163 LLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQY LRDTAALSPPKATVSDTCEEVEPSLLEILPKSSER TPLPL TPOR / MPLR(N+3) SDPTRVETATETAWLVLISLVTALHLVLGLSAVL 164 GLLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQ YLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPL TPOR / MPLR(N+4) SDPTRVETATETAWILVLISLVTALHLVLGLSAVL 165 GLLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQ YLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPL TPOR / MPLR(N+5) SDPTRVETATETAWLILVLISLVTALHLVLGLSAV 166 LGLLLLRWQFPAHYRRLRHALWPSLPDLHRVLG QYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSS ERTPLPL TPOR / MPLR(N+6) SDPTRVETATETAWLLILVLISLVTALHLVLGLSA 167 VLGLLLLRWQFPAHYRRLRHALWPSLPDLHRVL GQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKS SERTPLPL TPOR / MPLR(N+7) SDPTRVETATETAWVLLILVLISLVTALHLVLGLS 168 AVLGLLLLRWQFPAHYRRLRHALWPSLPDLHRV LGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPK SSERTPLPLAttorney Docket No.: AT-061 / 02WO Transmembrane and Amino acid sequence SEQ JAK2 binding domain ID NO: TPOR / MPLR(N+8) SDPTRVETATETAWLVLLILVLISLVTALHLVLGL 169 SAVLGLLLLRWQFPAHYRRLRHALWPSLPDLHR VLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILP KSSERTPLPL TPOR / MPLR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLL 170 582;H499L,S505N,W515 LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR K) DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL TPOR / MPLR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLL 171 582;S505N,W515K) LLRKQFPAHYRRLRHALWPSLPDLHRVLGQYLR DTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PL

[0083] In some embodiments, the PD-1 chimeric cytokine receptor of the disclosure comprises an amino acid sequence of the STAT-recruiting 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 shown in Table 1H. Table 1H: Recruiting domain Sequences (Cytotail Sequences) Recruiting domain Amino acid sequence SEQ ID NO: IL7R(316-459) ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN 172 CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPP FSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMS SFYQNQ IL2Rb(333-551) VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF 173 FHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAP TGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLL GGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDP QPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDA GPREGVSFPWSRPPGQGEFRALNARLPLNTDAYL SLQELQGQDPTHLV IFNAR1(508-557) ISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNE 174 DESESKTSEELQQDFV IFNAR2(310-515) KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 175 LTVRPLGQASATSTESQLIDPESEEEPDLPEVDVE LPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDY SSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEA PLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQ PTFPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYI MRAttorney Docket No.: AT-061 / 02WO Recruiting domain Amino acid sequence SEQ ID NO: IFNAR1 / 2(IFNAR1 ISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNE 176 residues 508-557-IFNAR2 DESESKTSEELQQDFVKKKVWDYNYDDESDSDT residues 310-515) EAAPRTSGGGYTMHGLTVRPLGQASATSTESQLI DPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPC ERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSV FLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPD NVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSD QSDTSESDVDLGDGYIMR IFNLR1(300-520) RGVRPTPRVRAPATQQTRWKKDLAEDEEEEDEE 177 DTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVD SGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWD RAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKD SGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPV SLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAE STQRTEDRGRTLGHYMAR Common Gamma IPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPE 178 Chain(335-369) T IL9R(356-521) TALLTCGPARPWKSVALEEEQEGPGTRLPGNLSS 179 EDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPA PPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSA LPGNTQSSGPIPALACGLSCDHQGLETQQGVAW VLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF IL21R(322-538) PRSPAKRLQLTELQEPAELVESDGVPKPSFWPTA 180 QNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCT WPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAG TTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADG EDWAGGLPWGGRSPGGVSESEAGSPLAGLDMD TFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQ WVVIPPPLSSPGPQAS GHR(353-638) PDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGR 181 TSCCEPDILETDFNANDIHEGTSEVAQPQRLKGE ADLLCLDQKNQNNSPYHDACPATQQPSVIQAEK NKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYA QVSDITPAGSVVLSPGQKNKAGMSQCDMHPEM VSLCQENFLMDNAYFCEADAKKCIPVAPHIKVES HIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSE MPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSS CGYVSTDQLNKIMP EpoR(339-508) WGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVL 182 DKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASS CSSALASKPSPEGASAASFEYTILDPSSQLLRPWT LCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQG AQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS murine IL2Rb(337-539) AVQLLLLQKDSAPLPSPSGHSQASCFTNQGYFFF 183 HLPNALEIESCQVYFTYDPCVEEEVEEDGSRLPE GSPHPPLLPLAGEQDDYCAFPPRDDLLLFSPSLST PNTAYGGSRAPEERSPLSLHEGLPSLASRDLMGLAttorney Docket No.: AT-061 / 02WO Recruiting domain Amino acid sequence SEQ ID NO: QRPLERMPEGDGEGLSANSSGEQASVPEGNLHG QDQDRGQGPILTLNTDAYLSLQELQAQDSVHLI murine IL7Ra(316-459) ARDEVESFLPNDLPAQPEELETQGHRAAVHSAN 184 RSPETSVSPPETVRRESPLRCLARNLSTCNAPPLL SSRSPDYRDGDRNRPPVYQDLLPNSGNTNVPVPV PQPLPFQSGILIPVSQRQPISTSSVLNQEEAYVTMS SFYQNK EGFR(955-1186) VIQGDERMHLPSPTDSNFYRALMDEEDMDDVVD 185 ADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVA CIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSID DTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNP APSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTF DSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPN GIFKGSTAENAEYLRVAPQSSEFIGA EGFR(955- VIQGDERMHLPSPTDSNFFRALMDEEDMDDVVD 186 1186;Y974F,d1045-1057) ADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVA CIDRNGLQSCPIKEDSFLQRIDDTFLPVPEYINQSV PKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHS TAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSH QISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEY LRVAPQSSEFIGA EGFR(955-1009;Y974F) VIQGDERMHLPSPTDSNFFRALMDEEDMDDVVD 187 ADEYLIPQQGFFSSPSTSRTP EGFR(1019-1085) NNSTVACIDRNGLQSCPIKEDSFLQRIDDTFLPVP 188 EYINQSVPKRPAGSVQNPV EGFR(1037- KEDSFLQRIDDTFLPVPEFINQSVPKRPAGSVQNP 189 1103;Y1068 / 1101F,d1045 VYHNQPLNPAPSRDPHFQD -1057) EGFR(1066- VPEFINQSVPKRPAGSVQNPVFHNQPLNPAPSRD 190 1118;Y1068 / 1086F) PHYQDPHSTAVGNPEYLNTV EGFR(1122-1165) PEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDN 191 PDYQQDFFPKEAKPNGIFKG EGFR(1133- WAQKGSHQISLDNPDFQQDFFPKEAKPNGIFKGS 192 1186;Y1148F) TAENAEYLRVAPQSSEFIGA IL12Rb2(775-825) SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL 193 PSHEAPLADSLEELEPQ IL7R(376-416) ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 194 TTNSTLP IL7R(424-459) GILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ 195 NQ IL7R(376-416,424-459) ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 196 TTNSTLPQGQPILTSLGSNQEEAYVTMSSFYQNQ IL7R(424-459;Y456F) GILTLNPVAQGQPILTSLGSNQEEAYVTMSSFFQN 197 Q IL7R(376-416,424- ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG 198 459,Y456F) TTNSTLPQGQPILTSLGSNQEEAYVTMSSFFQNQAttorney Docket No.: AT-061 / 02WO Recruiting domain Amino acid sequence SEQ ID NO: IL2Rbsmall(393-433) DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD 199 LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV IL2Rbsmall(518-551) GQGEFRALNARLPLNTDAYLSLQELQGQDPTHL 200 V IL2Rbsmall(339-379,393- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 201 433) LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPS IL2Rbsmall(339-379,518- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 202 551) LEIEACQ GQGEFRALNARLPLNTDAYLSLQELQGQDPTHL V IL2Rbsmall(393-433,518- DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDD 203 551) LLLFSPSGQGEFRALNARLPLNTDAYLSLQELQG QDPTHLV IL2Rbsmall(339-379,393- QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA 204 433,518-551) LEIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV IFNAR2small(310-352) KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 205 LTVRPLGQASA IFNAR2small(486-515) EGLWSEDAPSDQSDTSESDVDLGDGYIMR 206 IFNAR2small(310- KKKVWDYNYDDESDSDTEAAPRTSGGGYTMHG 207 352,486-515) LTVRPLGQASA EGLWSEDAPSDQSDTSESDVDLGDGYIMR BLNK(53-208) ASESPADEEEQWSDDFDSDYENPDEHSDSEMYV 208 MPAEENADDSYEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR BLNK(53-208;Y72F) ASESPADEEEQWSDDFDSDFENPDEHSDSEMYV 209 MPAEENADDSYEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR BLNK(53- ASESPADEEEQWSDDFDSDFENPDEHSDSEMYV 210 208;Y72F,Y96F) MPAEENADDSFEPPPVEQETRPVHPALPFARGEY IDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLP ALTALQKPQVPPKPKGLLEDEADYVVPVEDNDE NYIHPTESSSPPPEKAPMVNR EpoR(339-508) WGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVL 211 DKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASS CSSALASKPSPEGASAASFEYTILDPSSQLLRPWT LCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQG AQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS IL12Rb2(714-862) VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHS 212 ASSPPPPRALQAESRQLVDLYKVLESRGSDPKPEAttorney Docket No.: AT-061 / 02WO Recruiting domain Amino acid sequence SEQ ID NO: NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTL DQLKMRCDSLML IL12Rb1(622-662) WDKGERTEPLEKTELPEGAPELALDTELSLEDGD 213 RCKAKM IL10R1(304-578) VSPELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLP 214 DPHPQADRTLGNREPPVLGDSCSSGSSNSTDSGIC LQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQ NSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAA VAFQGYLRQTRCAEEKATKTGCLEEESPLTDGL GPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTL ASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWS FAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQ SSE IL2Rb(333-551, VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFF 215 Y381S,Y384S,Y387S) FHLPDALEIEACQVSFTSDPSSEEDPDEGVAGAPT GSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLG GPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQ PLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAG PREGVSFPWSRPPGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLV IL2Rb(333-551, VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGSFFF 216 Y364S,Y381S,Y384S,Y3 HLPDALEIEACQVSFTSDPSSEEDPDEGVAGAPTG 87S) SSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGG PSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQP LGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGP REGVSFPWSRPPGQGEFRALNARLPLNTDAYLSL QELQGQDPTHLV

[0084] Table 1I shows exemplary PD-1 CCR sequences of the disclosure. The receptors may be expressed with a signal sequence, e.g. a CD8SS of SEQ ID NO: 127. Table 1I: Exemplary PD-1 CCR sequences (assembled PD1 chimeric cytokine receptors) PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 217 207).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVG 5-825) GLLGSLVLLVWVLAVICSRAARGTIGARRTGQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 218 207).GCSFR(614 SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV -710).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVG 5-825) GLLGSLVLLVWVLAVICSRAARGTIGARRTGQ LTLMTLTPEGSELHIILGLFGLLLLLTCLCGTAWLCCSPN RKNPLWPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPI TKLTVLEEDEKKPVPWE ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 219 207).GP130(609- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 700).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVG 5-825) GLLGSLVLLVWVLAVICSRAARGTIGARRTGQ TTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIK KHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEAND ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 2xWTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 220 207).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVG 5-825) GLLGSLVLLVWVLAVICSRAARGTIGARRTGQPGWFLDS PDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLN WYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELR VTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV LLVWVLAVICSRAARGTIGARRTGQ SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ 3xWTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 221 207).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVG 5-825) GLLGSLVLLVWVLAVICSRAARGTIGARRTGQPGWFLDS PDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLN WYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELR VTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV LLVWVLAVICSRAARGTIGARRTGQPGWFLDSPDRPWN PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV RARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE VPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVL AVICSRAARGTIGARRTGQ SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(47 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 222 8- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG 5-825) LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 223 1).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETWISLVTALHLVLGLSAVLGLLLLRWQF PAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKAT VSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 224 2).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor SDPTRVETATETISLVTALHLVLGLSAVLGLLLLRWQFPA HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 225 2+1).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLISLVTALHLVLGLSAVLGLLLLRWQFP AHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATV SDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 226 3).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETSLVTALHLVLGLSAVLGLLLLRWQFPA HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 227 4).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLVTALHLVLGLSAVLGLLLLRWQFPAH YRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSD TCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 228 4+1).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETILVTALHLVLGLSAVLGLLLLRWQFPA HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 229 5).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETVTALHLVLGLSAVLGLLLLRWQFPAHY RRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 230 6).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETTALHLVLGLSAVLGLLLLRWQFPAHYR RLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCE EVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 231 7).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETALHLVLGLSAVLGLLLLRWQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 232 8).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825)Attorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLHLVLGLSAVLGLLLLRWQFPAHYRRL RHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 233 9).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETHLVLGLSAVLGLLLLRWQFPAHYRRLR HALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVE PSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 234 10).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLVLGLSAVLGLLLLRWQFPAHYRRLRH ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEP SLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 235 11).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETVLGLSAVLGLLLLRWQFPAHYRRLRHA LWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPS LLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 236 12).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLGLSAVLGLLLLRWQFPAHYRRLRHAL WPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSL LEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 237 13).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETGLSAVLGLLLLRWQFPAHYRRLRHAL WPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSL LEILPKSSERTPLPLAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 238 14).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLSAVLGLLLLRWQFPAHYRRLRHALW PSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLE ILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 239 15).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETSAVLGLLLLRWQFPAHYRRLRHALWPS LPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEIL PKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 240 16).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAVLGLLLLRWQFPAHYRRLRHALWPS LPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEIL PKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 241 17).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETVLGLLLLRWQFPAHYRRLRHALWPSLP DLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPK SSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 242 18).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETLGLLLLRWQFPAHYRRLRHALWPSLPD LHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKS SERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 243 +1).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWLISLVTALHLVLGLSAVLGLLLLRW QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPK ATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 244 +2).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWVLISLVTALHLVLGLSAVLGLLLLR WQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPP KATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 245 +3).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWLVLISLVTALHLVLGLSAVLGLLLL RWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSP PKATVSDTCEEVEPSLLEILPKSSERTPLPLAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 246 +4).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWILVLISLVTALHLVLGLSAVLGLLLL RWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSP PKATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 247 +5).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWLILVLISLVTALHLVLGLSAVLGLLL LRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALS PPKATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 248 +6).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825)Attorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWLLILVLISLVTALHLVLGLSAVLGLL LLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAAL SPPKATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 249 +7).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWVLLILVLISLVTALHLVLGLSAVLGL LLLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAA LSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1.TpoR(N PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 250 +8).IL7Ra(316- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 459).IL12Rb2(77 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 5-825) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ SDPTRVETATETAWLVLLILVLISLVTALHLVLGLSAVLG LLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTA ALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 251 170).TPOR / MPL SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT R(478- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 582;H499L,S505 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV N,W515K).IL7Ra (316-459) SDPTRVETATETAWISLVTALLLVLGLNAVLGLLLLRKQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 252 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582;S505N,W515 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES K).IL2small(393- LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV 433,518-551) SDPTRVETATETAWISLVTALHLVLGLNAVLGLLLLRKQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSP SGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV HAPD1DN PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 253 (HAPD1 SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT Dominant QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES Negative) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTI GARRTGQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 254 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGK NGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQ PILTSLGSNQEEAYVTMSSFYQNQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 255 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 582).IL2small(39 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 3-433,518-551) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPL DEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSP SGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 256 170).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRV 5-825) ETATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYR RLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCE EVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLE ESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 257 170).GCSFR(614 SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV -710).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVLTLMT 5-825) LTPEGSELHIILGLFGLLLLLTCLCGTAWLCCSPNRKNPL WPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPITKLTV LEEDEKKPVPWELEARDEVEGFLQDTFPQQLEESEKQRL GGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDA PILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFS LQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ GSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ WTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 258 170).GP130(609- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 700).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVTTPKFA 5-825) QGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPN VPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVS VVEIEANDLEARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGI LTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSR SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 2xWTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 259 170).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGPGWF 5-825) LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFV LNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPN GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVETATE TAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRH ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEP SLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEK QRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSA CDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLP PPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFY QNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNI DDLPSHEAPLADSLEELEPQ 3xWTPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 260 170).TpoR(478- SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV 582).IL7Ra(316- TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE 459).IL12Rb2(77 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGPGWF 5-825) LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFV LNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPN GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPSPRPAGQFQTLVGPGWFLDSPD RPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWY RMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDF HMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVT ERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVETATETAWI SLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWP SLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEI LPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLG GDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPI LSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSL QSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQG SGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPS HEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 261 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor FYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 262 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 1).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRRL RHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 263 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 2).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETISLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLR HALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVE PSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESE KQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVS ACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSF YQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 264 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 2+1).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLISLVTALHLVLGLSAVLGLLLLRWQFPAHYRRL RHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 265 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 3).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETSLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRH ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEP SLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEK QRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSAAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor CDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLP PPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFY QNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNI DDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 266 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 4).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRH ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEP SLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEK QRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSA CDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLP PPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFY QNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNI DDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 267 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 4+1).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETILVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRH ALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEP SLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEK QRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSA CDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLP PPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFY QNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNI DDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 268 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 5).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRHA LWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPS LLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQ RLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSAC DAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPP PFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ NQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNID DLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 269 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 6).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETTALHLVLGLSAVLGLLLLRWQFPAHYRRLRHAL WPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor LEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQR LGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACD APILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPF SLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ GSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 270 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 7).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETALHLVLGLSAVLGLLLLRWQFPAHYRRLRHALW PSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLE ILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRL GGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDA PILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFS LQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ GSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDL PSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 271 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 8).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWP SLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEI LPKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLG GDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPI LSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSL QSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQG SGSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPS HEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 272 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 9).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWPS LPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEIL PKSSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGG DVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPIL SSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQ SGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGS GSRSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPS HEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 273 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 10).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVEAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 459).IL12Rb2(77 TATETLVLGLSAVLGLLLLRWQFPAHYRRLRHALWPSLP 5-825) DLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPK SSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGI LTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSR SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 274 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 11).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETVLGLSAVLGLLLLRWQFPAHYRRLRHALWPSLP DLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPK SSERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGI LTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSR SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEA PLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 275 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 12).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLGLSAVLGLLLLRWQFPAHYRRLRHALWPSLPDL HRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSS ERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGIL TLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRS DPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 276 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 13).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETGLSAVLGLLLLRWQFPAHYRRLRHALWPSLPDL HRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSS ERTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQ SPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGIL TLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRS DPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAP LADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 277 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 14).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLSAVLGLLLLRWQFPAHYRRLRHALWPSLPDLH RVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQS PNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRS LDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILT LNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRSD PKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPL ADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 278 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 15).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETSAVLGLLLLRWQFPAHYRRLRHALWPSLPDLHR VLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSER TPLPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQSP NCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSL DCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTL NPVAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRSDP KPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLA DSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 279 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 16).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAVLGLLLLRWQFPAHYRRLRHALWPSLPDLHRV LGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTP LPLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNC PSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDC RESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNP VAQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRSDPKP ENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADS LEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 280 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 17).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETVLGLLLLRWQFPAHYRRLRHALWPSLPDLHRVL GQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPL PLLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCP SEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCR ESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPV AQGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRSDPKPE NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSL EELEPQAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 281 170).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 18).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETLGLLLLRWQFPAHYRRLRHALWPSLPDLHRVLG QYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLP LLEARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPS EDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRE SGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVA QGQPILTSLGSNQEEAYVTMSSFYQNQGSGSRSDPKPEN PACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLE ELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 282 170).TpoR(N+1). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWLISLVTALHLVLGLSAVLGLLLLRWQFPAHYR RLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCE EVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLE ESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDGYLPS NIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 283 170).TpoR(N+2). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWVLISLVTALHLVLGLSAVLGLLLLRWQFPAHY RRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDT CEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQ LEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLA GNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGT TNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVT MSSFYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHDG YLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 284 170).TpoR(N+3). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWLVLISLVTALHLVLGLSAVLGLLLLRWQFPAH YRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSD TCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQ QLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCL AGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLG TTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor TMSSFYQNQGSGSRSDPKPENPACPWTVLPAGDLPTHD GYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 285 170).TpoR(N+4). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWILVLISLVTALHLVLGLSAVLGLLLLRWQFPA HYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVS DTCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFP QQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTC LAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSL GTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAY VTMSSFYQNQGSGSRSDPKPENPACPWTVLPAGDLPTH DGYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 286 170).TpoR(N+5). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWLILVLISLVTALHLVLGLSAVLGLLLLRWQFP AHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATV SDTCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTF PQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLT CLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLS LGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEA YVTMSSFYQNQGSGSRSDPKPENPACPWTVLPAGDLPT HDGYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 287 170).TpoR(N+6). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWLLILVLISLVTALHLVLGLSAVLGLLLLRWQFP AHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATV SDTCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTF PQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLT CLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLS LGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEA YVTMSSFYQNQGSGSRSDPKPENPACPWTVLPAGDLPT HDGYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 288 170).TpoR(N+7). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWVLLILVLISLVTALHLVLGLSAVLGLLLLRWQ FPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKA TVSDTCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQD TFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor LTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLL LSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQE EAYVTMSSFYQNQGSGSRSDPKPENPACPWTVLPAGDL PTHDGYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 289 170).TpoR(N+8). SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459).IL12Rb2(77 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 5-825) TATETAWLVLLILVLISLVTALHLVLGLSAVLGLLLLRW QFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPK ATVSDTCEEVEPSLLEILPKSSERTPLPLLEARDEVEGFLQ DTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDS SLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDL LLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQ EEAYVTMSSFYQNQGSGSRSDPKPENPACPWTVLPAGD LPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 290 107).TPOR / MPL SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT R(478- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 582;H499L,S505 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE N,W515K).IL7Ra TATETAWISLVTALLLVLGLNAVLGLLLLRKQFPAHYRR (316-459) LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQ HAPD1.TpoR(47 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 291 8- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582;S505N,W515 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES K).IL2Rbsmall(3 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 93-433,518-551) TATETAWISLVTALHLVLGLNAVLGLLLLRKQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEDEGVAGAPTGSSPQPLQPL SGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 292 107).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582).IL7Ra(316- QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 459) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE TATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQ HAPD1.TpoR(47 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 293 8- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582).IL2Rbsmall( QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 393-433,518-551) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE TATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEDEGVAGAPTGSSPQPLQPL SGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV HAPD1 (21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 294 107)TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582;S505N,W515 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES K).IL2Rbsmall(3 LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 39-379,393- TATETAWISLVTALHLVLGLNAVLGLLLLRKQFPAHYRR 433,518-551) LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEQQDKVPEPASLSSNHSLTS CFTNQGYFFFHLPDALEIEACQDEGVAGAPTGSSPQPLQP LSGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNT DAYLSLQELQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 295 107).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582;H499L,S505 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES N,W515K).IL2R LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE bsmall(339- TATETAWISLVTALLLVLGLNAVLGLLLLRKQFPAHYRR 379,393-433,518- LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE 551) VEPSLLEILPKSSERTPLPLLEQQDKVPEPASLSSNHSLTS CFTNQGYFFFHLPDALEIEACQDEGVAGAPTGSSPQPLQP LSGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNT DAYLSLQELQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 296 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582). QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES IL2Rbsmall(339- LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 379,393-433,518- TATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRR 551) LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEQQDKVPEPASLSSNHSLTS CFTNQGYFFFHLPDALEIEACQDEGVAGAPTGSSPQPLQP LSGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNT DAYLSLQELQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 297 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 7).IL2Rbsmall(39 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 3-433,518-551) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE TATETALHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor PSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLE ILPKSSERTPLPLLEDEGVAGAPTGSSPQPLQPLSGEDDA YCTFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQE LQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 298 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 8).IL2Rbsmall(39 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 3-433,518-551) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE TATETLHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWP SLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEI LPKSSERTPLPLLEDEGVAGAPTGSSPQPLQPLSGEDDAY CTFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQEL QGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 299 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 9).IL2Rbsmall(39 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 3-433,518-551) LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE TATETHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWPS LPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEIL PKSSERTPLPLLEDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQELQ GQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 300 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 7).IL2Rbsmall(33 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 9-379,393- LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 433,518-551) TATETALHLVLGLSAVLGLLLLRWQFPAHYRRLRHALW PSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLE ILPKSSERTPLPLLEQQDKVPEPASLSSNHSLTSCFTNQGY FFFHLPDALEIEACQDEGVAGAPTGSSPQPLQPLSGEDDA YCTFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQE LQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 301 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 8).IL2Rbsmall(33 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES 9-379,393- LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE 433,518-551) TATETLHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWP SLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEI LPKSSERTPLPLLEQQDKVPEPASLSSNHSLTSCFTNQGY FFFHLPDALEIEACQDEGVAGAPTGSSPQPLQPLSGEDDA YCTFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQE LQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 302 107).TpoR(N- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 9).IL2Rbsmall(33 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVEAttorney Docket No.: AT-061 / 02WO PD1 chimeric Amino acid sequence SEQ cytokine ID NO: receptor 9-379,393- TATETHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWPS 433,518-551) LPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEIL PKSSERTPLPLLEQQDKVPEPASLSSNHSLTSCFTNQGYF FFHLPDALEIEACQDEGVAGAPTGSSPQPLQPLSGEDDA YCTFPSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQE LQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 303 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582;H499L,S505 QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES N,W515K).IL2R LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE bsmall(393- TATETAWISLVTALLLVLGLNAVLGLLLLRKQFPAHYRR 433,518-551) LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEDEGVAGAPTGSSPQPLQPL SGEDDAYCTFPSRDDLLLFSPSGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV HAPD1(21- PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT 304 170).TpoR(478- SESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVT 582; QLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKES S505N,W515K).I LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVSDPTRVE L7Ra(316-459) TATETAWISLVTALHLVLGLNAVLGLLLLRKQFPAHYRR LRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEE VEPSLLEILPKSSERTPLPLLEARDEVEGFLQDTFPQQLEE SEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGN VSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS TLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQ

[0085] In another embodiment, the CCR is a CCR comprising a TGF- ^ binding domain. In one embodiment, the CCR is an inducible CCR that is active when engaged with TGF-β ligands (e.g. TGF-β1, TGF-β2, and / or TGF-β3) or activation with an anti-TGF-β-receptor antibody (see US2021-0061881A1, which is incorporated herein by reference in its entirety). Provided herein are engineered immune cells, e.g., CAR T cells, expressing the TGF-β CCRs of the disclosure. The CCRs of the disclosure activate signaling upon binding of a TGF-β ligand (for example, TGF-β1, TGF-β2, and / or TGF-β3), or an anti-TGF-β- receptor antibody. These receptors activate signaling when monomers of the receptor cluster and / or dimerize. The chimeric cytokine receptors of the disclosure are dual-function chimeric cytokine receptors which can simultaneously neutralize the immune-suppressive effects of a TGF-β ligand, and mimic the transmission of an immune-potentiating cytokine signal. In some embodiments, a monomer of the chimeric cytokine receptor of theAttorney Docket No.: AT-061 / 02WO disclosure comprises: (a) a binding domain capable of binding a TGF-β ligand or an anti- TGF-β-receptor antibody; (b) a transmembrane domain; (c) a Janus Kinase (JAK)-binding domain; and; (d) a STAT-recruiting domain (e.g. from the cytoplasmic domain of a receptor; e.g. from a cytokine receptor). Each domain can be linked either directly or via one or more peptide linkers. In some embodiments, a monomer of the chimeric cytokine receptor of the disclosure comprises: (a) a binding domain capable of binding a TGF-β ligand or an anti-TGF-β-receptor antibody; (b) a transmembrane domain; (c) a Janus Kinase (JAK)-binding domain; and; (d) a recruiting domain (e.g. from the 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 receptors are clustered and activated when they bind to TGF-β ligands, and / or are clustered and activated with an anti-TGF-β- receptor antibody. The chimeric cytokine receptors activate signaling upon for example binding a TGF-β ligand, and / or a TGF-β-receptor antibody. In some embodiments, the TGF- ^ receptor antibody is, without limitation, PF-03446962 or LY3022859. In some embodiments, the chimeric cytokine receptors are constitutively clustered or dimerized. Linking Peptides

[0086] In one aspect, the in vitro methods described herein utilize linking peptides. In one embodiment, the linking peptides are part of a bicistronically expressed polypeptide. In other embodiments, the engineered immune cells, e.g., CAR T cells, comprise a polynucleotide sequence that encodes a bicistronically expressed polypeptide, a linking peptide, and an additional polypeptide. Table 1J provides a list of exemplary linking peptides and their amino acid sequences. Table 1J – Linking peptides Peptide origin Amino acid sequence SEQ ID NO P2A ATNFSLLKQAGDVEENPGP 305 P2A ATNFSLLKQAGDVEENPG 306 P2A GSGATNFSLLKQAGDVEENPGP 307 P2A GSGATNFSLLKQAGDVEENPG 308 T2A EGRGSLLTCGDVEENPGP 309 T2A EGRGSLLTCGDVEENPG 310 T2A GSGEGRGSLLTCGDVEENPGP 311 T2A GSGEGRGSLLTCGDVEENPG 312 E2A QCTNYALLKLAGDVESNPGP 313Attorney Docket No.: AT-061 / 02WO E2A QCTNYALLKLAGDVESNPG 314 E2A GSGQCTNYALLKLAGDVESNPGP 315 E2A GSGQCTNYALLKLAGDVESNPG 316 F2A VKQTLNFDLLKLAGDVESNPGP 317 F2A VKQTLNFDLLKLAGDVESNPG 318 F2A GSGVKQTLNFDLLKLAGDVESNPGP 319 F2A GSGVKQTLNFDLLKLAGDVESNPG 320

[0087] In one embodiment, the amino acid sequence of the linking peptide comprises D- (V or I)-E-x-N-P-G-P, wherein x is any amino acid. Following translation, the amino acid sequence that is appended to the C-terminus of the first polypeptide, i.e., the bicistronically expressed polypeptide, comprises D-(V or I)-E-x-N-P-G, wherein x is any amino acid.

[0088] The disclosure encompasses modifications to the polypeptides, including bicistronically expressed polypeptides and CARs, as well as linking peptides comprising the sequences provided herein, e.g., Tables 1J and 2, including functionally equivalent bicistronically expressed polypeptides, CARs or linking peptides having modifications which do not significantly affect their properties and variants which have enhanced or decreased activity. For example, the amino acid sequence of the linking peptide can be altered (e.g., with a deletion, insertion, and / or substitution) to obtain a linking peptide with the desired properties. Modification of peptides and polypeptides is routine practice in the art and thus need not be described in detail herein. Examples of modified polypeptides or peptides include polypeptides or peptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or which enhance the functionality of the linking peptide, the bicstronically expressed polypeptide hat comprises the linking peptide, and / or the CAR. Deletions may include one or several terminal deletions in the linking peptide.

[0089] 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.

[0090] Substitution variants have at least one amino acid residue in the polypeptides and peptides removed and a different residue inserted in its place. Conservative substitutions are shown in Table 2 under the heading of “conservative substitutions”. If suchAttorney Docket No.: AT-061 / 02WO substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 2, or as further described below in reference to amino acid classes, can be introduced and the products screened. Table 2: Amino Acid Substitutions Original Residue (naturally occurring amino Conservative acid) Substitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) LeuLeu; Val; Met; Ala; Phe;NorleucineLeu (L) IleNorleucine; Ile; Val; Met;Ala; PheLys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) LeuIle; Leu; Met; Phe; Ala;NorleucineImmune Cells

[0091] Engineered immune cells are obtained from donor cells. Engineered cells derived from the donor cells that are suitable for use with the methods and / or reagents described herein include immune cells.Attorney Docket No.: AT-061 / 02WO

[0092] Prior to the in vitro manipulation or genetic modification (e.g., as described herein), donor cells for use in methods described herein (e.g., immune cells) 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, cord blood, thymus tissue, stem cell- 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 those skilled in the art, can be used. In some embodiments, donor cells can be derived from a healthy donor, from a patient diagnosed with cancer, 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 which present different phenotypic characteristics.

[0093] 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 from the subject who will receive the engineered immune cells.

[0094] In some embodiments, immune cells comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, stem cell- or iPSC-derived T cells, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain some embodiments, T cells can be obtained from a volume of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation.

[0095] Donor cells can be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing.

[0096] Donor PBMCs can be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper TAttorney Docket No.: AT-061 / 02WO lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0097] In certain embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation 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 directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0098] In some embodiments, a population of donor cells, e.g., immune cells such as T cells, is enriched for CD4+ cells.

[0099] In some embodiments, a population of donor cells, e.g., immune cells such as T cells, is enriched for CD8+ cells.

[0100] In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of cells. In some embodiments the expression of phenotypic markers for naïve T cells include CD45RA+, CD95-, IL2R ^-, CCR7+, and CD62L+. In some embodiments the expression of phenotypic markers for stem cell memory T cells include CD45RA+, CD95+, IL2R ^ ^, CCR7+, and CD62L+. In some embodiments the expression of phenotypic markers for central memory T cells include CD45RO+, CD95+, IL2R ^ ^, CCR7+, and CD62L+. In some embodiments the expression of phenotypic markers for effector memory T cells include CD45RO+, CD95+, IL2R ^ ^, CCR7-, and CD62L-. In some embodiments the expression of phenotypic markers for T effector cells include CD45RA+, CD95+, IL2R ^ ^, CCR7-, and CD62L-. Thus, CD4+ and / or CD8+ T helper cells can be sorted intoAttorney Docket No.: AT-061 / 02WO naive, stem cell memory, central memory, effector memory and T effector cells by identifying cell populations that have cell surface antigens.

[0101] It will be appreciated that donor PBMCs can further include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor 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 the CAR for storage and / or preparation for use in a human subject. In one embodiment, the in vitro transduction, culture and / or expansion of T cells are performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum. In vaious embodiments a crypreservative media can comprise, for example, CryoStor® CS2, CS5, or CS10 or other medium comprising DMSO, or a medium that does not comprise DMSO. Engineered Immune Cells

[0102] Provided herein are engineered immune cells expressing the bicistronically expressed polypeptides and additional polypeptides, e.g., CARs, of the disclosure (e.g., CAR-T cells) that have been obtained from donor cells as described herein.

[0103] In some embodiments, an engineered immune cell comprises a polynucleotide sequence encoding a bicistronically expressed polypeptide, a linking 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, an engineered immune cell comprises a population of CARs, each CAR comprising different extracellular antigen- binding domains. In some embodiments, an immune cell comprises a population of CARs, each CAR comprising the same extracellular antigen-binding domains.

[0104] The engineered immune cells can be allogeneic or autologous.

[0105] 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, a mast cell, or a B-cell.Attorney Docket No.: AT-061 / 02WO

[0106] In some embodiments, the engineered immune cell can be obtained from 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 alpha beta T cell. In some exemplary embodiments, the engineered immune cell is a gamma delta T cell. In some exemplary embodiments, the engineered immune cell is a macrophage.

[0107] In some embodiments, the engineered immune cell can be derived from, for example without limitation, a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSC), totipotent stem cells or hematopoietic stem cells. Stem cells can be CD34+ or CD34-.

[0108] 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 transfected or transduced by the nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), transfection, lipid transfection, polymer transfection, nanoparticles, viral transduction or viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplexes. In some embodiments the donor cell is a T cell that has been re-programmed from a non-T cell. In some embodiments the donor cell is a T cell that has been re-programmed from a T cell. Detection Agents (including antibodies and fragments thereof)

[0109] In embodiments, the disclosed methods for the detection of a bicistronically expressed polypeptide in an engineered immune cell comprises 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 engineered immune cells derived from donor cells of a donor cell population can also comprise a binding agent.

[0110] As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particularAttorney Docket No.: AT-061 / 02WO target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y's stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The "hinge" connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains – an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain. Those skilled in the art are well familiar with antibody structure and sequence elements, recognize “variable” and “constant” regions in provided sequences, and understand that there may be some flexibility in definition of a “boundary” between such domains such that different presentations of the same antibody chain sequence may, for example, indicate such a boundary at a location that is shifted one or a few residues relative to a different presentation of the same antibody chain sequence.

[0111] Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5- stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including forAttorney Docket No.: AT-061 / 02WO example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0112] For purposes of the instant disclosure, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody,” whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.

[0113] Moreover, the term “antibody” as used herein, can refer to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody utilized in the methods of the instant disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments, and isolated CDRs or sets thereof; single chain variable fragments (scFVs); 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 VHHs); shark antibodies, masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans- bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g.,Attorney Docket No.: AT-061 / 02WO a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.).

[0114] 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 includes 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 may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc. as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art- known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans- bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s.

[0115] An antibody or antibody agent used in performing the methods of the instant disclosure can be single chained 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 an scFv, a Fab, a Fab’, a Fv, a F(ab’)2, a dAb, and any combination thereof.

[0116] Antibodies and antibody agents include antibody fragments. An antibody fragment comprises a portion of an intact antibody, such as the antigen binding or variable region ofAttorney Docket No.: AT-061 / 02WO the intact antibody. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, diabody, linear antibodies, multispecific formed from antibody fragments antibodies and scFv fragments, and other fragments. Antibodies also include, but are not limited to, polyclonal monoclonal, chimeric dAb (domain antibody), single chain, Fab, Fa, F(ab’)2fragments, and scFvs. An antibody can be a whole antibody, or immunoglobulin, or an antibody fragment. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli, Chinese Hamster Ovary (CHO) cells, or phage), as known in the art.

[0117] In some embodiments, an antibody or antibody agent can be a chimeric antibody (see, e.g., U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody can be an antibody in which a portion of the heavy and / or light chain is 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 a further example, a chimeric antibody can be a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0118] 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)). A humanized antibody is a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise 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 antibodyAttorney Docket No.: AT-061 / 02WO optionally can comprise at least a portion of an antibody constant region derived from a human antibody.

[0119] In some embodiments, an antibody or antibody agent provided herein is a human antibody. Human antibodies can be produced using various 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 one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen- binding residues. Human antibodies may be prepared using methods well known in the art.

[0120] 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 includes 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 may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc. as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art- known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-Attorney Docket No.: AT-061 / 02WO bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. Chimeric Antigen Receptors

[0121] As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). When bound to the target antigen, the CAR can activate the immune cell to attack and destroy the cell bearing that antigen (e.g., the cancer cell). CARs can also incorporate costimulatory or signaling domains to increase their potency. See Krause et al., J. Exp. Med., Volume 188, No.4, 1998 (619–626); Finney et al., Journal of Immunology, 1998, 161: 2791–2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med.3:95 (2011); Porter et al., N. Engl. J. Med.365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol.56:59–83 (2016); U.S. Patent Nos.7,741,465, and 6,319,494.

[0122] 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 the target.

[0123] In some embodiments, antigen-specific CARs further comprise a safety switch and / or one or more monoclonal antibody specific-epitope. 1. Antigen Binding Domains

[0124] As discussed above, CARs described herein comprise an antigen binding domain. An “antigen binding domain” as used herein means any polypeptide that binds 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.

[0125] In some embodiments, the antigen binding domain comprises a variable heavy chain, 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.Attorney Docket No.: AT-061 / 02WO

[0126] An antigen binding domain is said to be “selective” when it binds to one target more tightly or with higher affinity than it binds to a second target.

[0127] 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, Liv1, 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, Liv1, 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.

[0128] 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 receptors, 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), κ-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).

[0129] Variants of the antigen binding domains (e.g., variants of the CDRs, VH and / or VL) are also within the scope of the disclosure, e.g., variable light and / or variable heavy chains that each have at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to the amino acid sequences of antigen binding domain sequences. InAttorney Docket No.: AT-061 / 02WO some instances, such molecules include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two variable light chains and two variable heavy chains (or subparts thereof). A skilled artisan will be able to determine suitable variants of the antigen binding domains as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity.

[0130] In certain some embodiments, the polypeptide structure of the antigen binding domains is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the antigen binding domain comprises or consists of avimers.

[0131] In some embodiments, an antigen binding domain is a scFv.

[0132] In some embodiments, an antigen-selective CAR comprises a leader or signal peptide.

[0133] In other embodiments, the disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the disclosure relates to isolated polynucleotides encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making same.

[0134] In other embodiments, the disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the disclosure relates to isolated polynucleotides encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making same.

[0135] In some embodiments, a CAR-immune cell (e.g., CAR-T cell) which can form a component of an engineered immune cell population (derived from donor cells of a donor cell population as described herein) generated by practicing the methods of the instant disclosure comprises a polynucleotide encoding a safety switch polypeptide, such as for example RQR8. See, e.g., WO2013153391A, which is hereby incorporated by reference inAttorney Docket No.: AT-061 / 02WO its entirety. In a CAR-immune cell (e.g., a CAR-T cell) comprising the polynucleotide, the safety switch polypeptide can be expressed at the surface of a CAR-immune cell (e.g., CAR-T cell). 2. Hinge Domain

[0136] The extracellular domain of the CARs of the disclosure can comprise a “hinge” domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain in a CAR to the extracellular antigen binding domain in a CAR. In particular, hinge domains can be used to provide more flexibility and accessibility for the extracellular antigen binding domain.

[0137] 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. The hinge domain can be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, the hinge region of an IgG; it will be appreciated that the hinge region can contain some or all of a member of the immunoglobulin family such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragment thereof), or from all or part of an antibody heavy-chain constant region. Alternatively, the hinge domain can be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or can be an entirely synthetic hinge sequence. In some embodiments said hinge domain is a part of human CD8α chain (e.g., NP_001139345.1). In other embodiments, said hinge and transmembrane domains comprise a part of human CD8α chain. In some embodiments, the hinge domain of CARs described herein comprises a subsequence of CD8α, CD28, an IgG1, IgG4, PD-1 or an FcγRIIIα, in particular the hinge region of any of an CD8α, a CD28, an IgG1, IgG4, PD-1 or an FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1 or a human FcγRIIIα hinge. In some embodiments the CARs disclosed herein comprise a scFv, CD8α human hinge and transmembrane domains, the CD3ζ signaling domain, and 4-1BB signaling domain.

[0138] In some embodiments, the hinge domain in the CAR of the disclosure is a CD8α transmembrane domain. In some embodiments, the hinge domain in the CAR of the disclosure is a CD8α hinge domain comprising the amino acid sequence PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:Attorney Docket No.: AT-061 / 02WO 332) or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 340). In some embodiments, the CD8α hinge domain comprises the nucleic acid sequence that encodes the hinge amino acid sequence of SEQ ID NO: 332. In some embodiments, the hinge domain in the CAR of the disclosure is a CD28 hinge domain. 3. Transmembrane Domain

[0139] The CARs of the disclosure are designed with a transmembrane domain that is fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In some embodiments, short linkers can form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0140] Suitable transmembrane domains for a CAR disclosed herein have the ability to (a) be expressed at the surface an immune cell such as, for example without limitation, a lymphocyte cell, such as a T helper (Th) cell, cytotoxic T (Tc) cell, T regulatory (Treg) cell, or Natural killer (NK) cells, and / or (b) interact with the extracellular antigen binding domain and intracellular signaling domain for directing the cellular response of an immune cell against a target cell.

[0141] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0142] Transmembrane regions of particular use in this disclosure can be derived from (comprise, or correspond 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), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80Attorney Docket No.: AT-061 / 02WO (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, 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.

[0143] As non-limiting examples, the transmembrane region can be derived from, or be a portion of a T cell receptor such as α, β, γ or δ, polypeptide constituting CD3 complex, IL-2 receptor p55 ( ^ chain), p75 (β chain) or γ chain, subunit chain of Fc receptors, in particular Fcγ receptor III or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments said transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0144] In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT or IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 333). In some embodiments, the CD8α transmembrane domain comprises the nucleic acid sequence that encodes 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 disclosure is a CD8α hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO: 323 or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVIT.

[0145] In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD28 transmembrane domain. 4. Intracellular DomainAttorney Docket No.: AT-061 / 02WO

[0146] The intracellular (cytoplasmic) domain of the CARs of the disclosure can provide activation of at least one of the normal effector functions of the immune cell comprising the CAR. Effector function of a T cell, for example, can refer to cytolytic activity or helper activity, including the secretion of cytokines.

[0147] In some embodiments, an activating intracellular signaling domain for use in a CAR can be the cytoplasmic sequences of, for example without limitation, the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0148] 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), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, 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.

[0149] The intracellular domains of the CARs of the disclosure can incorporate, in addition to the activating domains described above, co-stimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to increase theirAttorney Docket No.: AT-061 / 02WO potency. Co-stimulatory domains can provide a signal in addition to the primary signal provided by an activating molecule as described herein.

[0150] It will be appreciated that suitable co-stimulatory domains within the scope of the disclosure can be derived from (or correspond to) for example, CD28, OX40, 4- 1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, 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 alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocytic activation molecule, 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, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, 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. It will be appreciated that additional costimulatory molecules, or fragments thereof, not listed above are within the scope of the disclosure.

[0151] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the 4-1BB / CD137 domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the 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.

[0152] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the CD28 domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the disclosure. The completeAttorney Docket No.: AT-061 / 02WO 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.

[0153] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the disclosure.

[0154] For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of the disclosure can be linked to each other in a random or specified order. In some embodiments, the intracellular domain is designed to comprise the activating domain of CD3 zeta and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to comprise the activating domain of CD3 zeta and a signaling domain of 4-1BB.

[0155] In some embodiments the intracellular signaling domain of the CAR of the disclosure comprises a domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR of the disclosure comprises a part of co-stimulatory molecule selected from the group consisting of fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0156] Table 3 provides exemplary sequences of CAR components that can be used in the CARs disclosed herein and the antibody and / or CAR sequences exemplified herein. Table 3: Sequences relating to CARs Domain Amino acid sequence SEQ ID NO V5 epitope tag IPNPLLGLDST 321 2173 scFv EIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHW 322 VRQMPGKGLEWMGRIDPENDETKYGPIFQGHVTIS ADTSINTVYLQWSSLKASDTAMYYCAFRGGVYW GQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVV MTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYL NWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSG TDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTK VEIK CD8 hinge and TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH 323 transmembrane TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCAttorney Docket No.: AT-061 / 02WO Domain Amino acid sequence SEQ ID NO 4-1BB intracellular KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE 324 signaling EEGGCEL CD3z intracellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV 325 signaling LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR BFP MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGK 326 PYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTF INHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTL GWEAFTETLYPADGGLEGRNDMALKLVGGSHLIA NIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEAN NETYVEQHEVAVARYCDLPSKLGHKLN P2A GSGATNFSLLKQAGDVEENPGP 307 2173 anti-EGFRvIII MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKP 327 scFv GESLRISCKGSGFNIEDYYIHWVRQMPGKGLEWM GRIDPENDETKYGPIFQGHVTISADTSINTVYLQWS SLKASDTAMYYCAFRGGVYWGQGTTVTVSSGGG GSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGE RATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRL ISLVSKLDSGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCWQGTHFPGTFGGGTKVEIKTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYKQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR Mouse kappa light METDTLLLWVLLLWVPGSTG 328 chain leader sequence Anti-human CD19 EVQLQQSGPELIKPGASVKMSCKASGYTFTSYVM 329 (4G7) heavy chain HWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGK ATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYY YGSRVFDYWGQGTTLTVSS Glycine-serine GGGGSGGGGSGGGGS 330 linker Anti-human CD19 DIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNT 331 (4G7) light chain YLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSG SGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAG TKLELKRSD CD8 hinge PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV 332 HTRGLDFACDAttorney Docket No.: AT-061 / 02WO Domain Amino acid sequence SEQ ID NO CD8 transmembrane IYIWAPLAGTCGVLLLSLVITLYC 333 domain 41-BB domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE 324 EEGGCEL CD3 zeta domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV 325 LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR Anti-CD194G7 METDTLLLWVLLLWVPGSTGEVQLQQSGPELIK 334 chimeric antigen PGASVKMSCKASGYTFTSYVMHWVKQKPGQGLE receptor (with signal WIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAY sequence shown in MELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQ bold text) GTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSIP VTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQS PQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVE AEDVGVYYCMQHLEYPFTFGAGTKLELKRSDPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR Anti-CD194G7 EVQLQQSGPELIKPGASVKMSCKASGYTFTSYVM 335 chimeric antigen HWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGK receptor (without ATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYY signal sequence) YGSRVFDYWGQGTTLTVSSGGGGSGGGGSGGGG SDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNT YLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSG SGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAG TKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR CD8 signal sequence MALPVTALLLPLALLLHAARP 127 Anti-human BCMA EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMN 336 (P5A2) heavy chain WVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPM DIWGQGTLVTVSS Glycine-serine GGGGSGGGGSGGGGS 330 linker Anti-human BCMA EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAW 337 (P5A2) light chain YQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDAttorney Docket No.: AT-061 / 02WO Domain Amino acid sequence SEQ ID NO FTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEI K P5A2 anti-BCMA EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMN 338 scFv WVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPM DIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQS PGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG QAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSWPLTFGQGTKVEIK Safety switch GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGG 339 GS CD8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH 340 TRGLDFACD CD8 transmembrane IYIWAPLAGTCGVLLLSLVITLYC 333 domain 41-BB domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE 324 EEGGCEL CD3 zeta domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV 325 LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR P5A2 anti-BCMA MALPVTALLLPLALLLHAARP 341 CAR (with CD8 signal sequence) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAM NWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPM DIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQS PGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG QAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSWPLTFGQGTKVEIKGSGG GGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR Anti-BCMA (P5A2) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMN 342 chimeric antigen WVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFT receptor with a ISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPM safety switch DIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQS (without signal PGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG sequence) QAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSWPLTFGQGTKVEIKGSGG GGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGSTAttorney Docket No.: AT-061 / 02WO Domain Amino acid sequence SEQ ID NO TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

[0157] Table 4 provides exemplary sequences of chimeric cytokine receptor (CCR) components that can be used in the CCRs disclosed herein. The CCRs may be expressed with a signal sequence, e.g., a CD8SS of sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 127). Table 4 – CCR sequences Domain Amino acid sequence SEQ ID NO CD8 signal sequence MALPVTALLLPLALLLHAARP 127 Transmembrane and SDPTRVETATETAWISLVTALLLVLGLNAVLGLLL 6 JAK-binding LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT domain AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR (478- 582;H499L,S505N, W515K) Transmembrane and SDPTRVETATETAWISLVTALHLVLGLNAVLGLLL 7 JAK-binding LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT domain AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL TPOR / MPLR (478- 582;S505N,W515K) Intracellular QQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALE 72 signaling domain IEACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPS RDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQEL IL2Rbsmall (339- QGQDPTHLV 379,393-433,518- 551) P2A GSGATNFSLLKQAGDVEENPGP 307 CACCR SDPTRVETATETAWISLVTALLLVLGLNAVLGLLL 343 LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT CD8SS- AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLQ TPOR / MPLR(478- QDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEI 582;H499L;S505N, EACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPS W515K).Attorney Docket No.: AT-061 / 02WO IL2Rb(339-379,393- RDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQEL 433,518-551) QGQDPTHLV CACCR SDPTRVETATETAWISLVTALHLVLGLNAVLGLLL 344 LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT CD8SS- AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLQ TPOR / MPLR(478- QDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEI 582;S505N,W515K) EACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPS . IL2Rb(339- RDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQEL 379,393-433,518- QGQDPTHLV 551)

[0158] Table 5 provides exemplary CACCR sequences showing the P2A linkage sequence – GSGATNFSLLKQAGDVEENPG (SEQ ID NO: 307) with or without an anti- BCMA CAR. Table 5 – CACCR sequences with P2A linker sequence Domain Amino acid sequence SEQ ID NO TpoR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLLL 345 582;H499L,S505N, LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT W515K).IL2Rb(339 AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLL -379,393-433,518- EQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDAL 551).P2A EIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTF PSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQ ELQGQDPTHLVGSGATNFSLLKQAGDVEENPG TpoR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLLL 346 582;S505N,W515K) LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT .IL2Rb(339- AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLL 379,393-433,518- EQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDAL 551).P2A EIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTF PSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQ ELQGQDPTHLVGSGATNFSLLKQAGDVEENPG TpoR(478- SDPTRVETATETAWISLVTALLLVLGLNAVLGLLL 347 582;H499L,S505N, LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT W515K).IL2Rb(339 AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLL -379,393-433,518- EQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDAL 551) EIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTF PSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQ P2A (bolded) ELQGQDPTHLVGSGATNFSLLKQAGDVEENPGM ALPVTALLLPLALLLHAARPEVQLLESGGGLVQPG P5A2 anti-BCMA GSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVS CAR (with CD8 AISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMN signal sequence) SLRAEDTAVYYCARYWPMDIWGQGTLVTVSSGG GGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLAWYQQKPGQAPRLLMYDASIRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGAttorney Docket No.: AT-061 / 02WO SWPLTFGQGTKVEIKGSGGGGSCPYSNPSLCSGGG GSCPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCEL TpoR(478- SDPTRVETATETAWISLVTALHLVLGLNAVLGLLL 348 582;S505N,W515K) LRKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDT .IL2Rb(339- AALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLL 379,393-433,518- EQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDAL 551).P2A EIEACQDEGVAGAPTGSSPQPLQPLSGEDDAYCTF PSRDDLLLFSPSGQGEFRALNARLPLNTDAYLSLQ P2A (bolded) ELQGQDPTHLVGSGATNFSLLKQAGDVEENPG P5A2 anti-BCMA MALPVTALLLPLALLLHAARPEVQLLESGGGLVQP CAR (with CD8 GGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEW signal sequence) VSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS GGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERAT LSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASI RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSWPLTFGQGTKVEIKGSGGGGSCPYSNPSLCS GGGGSCPYSNPSLCSGGGGSTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCEL Engineered Immune Cells Comprising CARs

[0159] Also provided herein are engineered immune cells and populations of engineered immune cells i) comprising a polynucleotide encoding a bicistronically expressed polypeptide, a linking peptide and an additional polypeptide (e.g., a CAR) and / or ii) expressing a bicistronically expressed polypeptide, a linking peptide and an additional polypeptide, such as a CAR (e.g., CAR-T cells or CAR+ cells).

[0160] In some embodiments, an engineered immune cell comprises a CAR T cell, each CAR T cell comprising an extracellular antigen-binding domain and has reduced or eliminated expression of endogenous TCR. In some embodiments, a population of engineered immune cells comprises a population of CAR T cells, each CAR T cell comprising two or more different extracellular antigen-binding domain and has reduced or eliminated expression of endogenous TCR. In some embodiments, an immune cell comprises a population of CARs, each CAR T cell comprising the same extracellularAttorney Docket No.: AT-061 / 02WO antigen-binding domains and has reduced or eliminated expression of one or more wanted biomarkers (as described herein) and / or endogenous TCR.

[0161] The engineered immune cells can be allogeneic or autologous.

[0162] In some embodiments, an 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, a mast cell, or a B- cell, and expresses a CAR. In some embodiments, the T cell can be derived from the group consisting of CD4+ T lymphocytes, CD8+ T lymphocytes or population comprising a combination of CD4+ and CD8+ T cells.

[0163] In some embodiments, an engineered immune cell or population of engineered immune cells that are generated using the disclosed methods can be derived from, for example without limitation, a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.

[0164] In some embodiments, an engineered immune cell or a population of immune cells that are generated using the disclosed methods is obtained or prepared from peripheral blood. In some embodiments, an engineered immune cell is obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, an engineered immune cell is obtained or prepared from bone marrow. In some embodiments, an engineered immune cell is obtained or prepared from umbilical cord blood. In some embodiments, the donor cell is a human cell. In some embodiments, the donor cell is transfected or transduced by the nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplexes.

[0165] In some embodiments, the engineered immune cells expressing at their cell surface membrane an antigen-specific CAR comprise a percentage of stem cell memory and central memory cells greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Attorney Docket No.: AT-061 / 02WO

[0166] In some embodiments, engineered immune cells expressing at their cell surface membrane an antigen-specific CAR comprise a percentage of stem cell memory and central memory cells 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%.

[0167] In some embodiments, engineered immune cells expressing at their cell surface membrane an antigen-specific CAR enriched in TCM and / or TSCM cells such that the engineered immune cells comprise at least about 60%, 65%, 70%, 75%, or 80% combinedTCM and TSCM cells. In some embodiments, engineered cells expressing at their cell surface membrane an antigen-specific CAR are enriched in TCMand / or TSCMcells such that the engineered immune cells comprise at least about 70% combined and TSCM cells. Insome embodiments, engineered immune cells expressing at their anantigen-specific CAR e enriched in TCMand / or TSCMcells such that the engineered immune cells comprise at least about 75% TSCM cells.

[0168] In some embodiments, anis an inflammatory T- lymphocyte that expresses a CAR. In some embodiments, an engineered immune cell is a cytotoxic T-lymphocyte that expresses a CAR. In some embodiments, an engineered immune cell is a regulatory T-lymphocyte that expresses a CAR. In some embodiments, an engineered immune cell is a helper T-lymphocyte that expresses a CAR. Genetic modification of CAR T cells

[0169] In some embodiments, an engineered immune cell 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, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAP alpha, 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, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 CAR) to avoid induced CARAttorney Docket No.: AT-061 / 02WO activation. As described herein, in some embodiments, an engineered immune cell according to the present disclosure comprises one disrupted or inactivated gene selected from the group consisting of MHC1 (β2M), MHC2 (CIITA), EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70, TCRα and TCRβ and / or expresses a CAR or a multi-chain CAR. In some embodiments, a cell comprises a multi-chain CAR. In some embodiments, the isolated cell comprises two disrupted or inactivated genes selected from the group consisting of: CD52 and TCRα, CDR52 and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, MHC-1 and TCRα, MHC-1 and TCRβ, MHC2 and TCRα, MHC2 and TCRβ and / or expresses a CAR or a multi-chain CAR.

[0170] In some embodiments, an isolated cell comprises polynucleotides encoding polypeptides 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 TCRα, CDR52 and TCRβ, DLL3 and CD52, DLL3 and TCRα, DLL3 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ and / or expresses a CAR, including a multi-chain CAR, and / or a pTα transgene. In some embodiments the method comprises disrupting or inactivating one or more genes by introducing into the donor cells an endonuclease capable of selectively inactivating a gene by selective DNA cleavage. In some embodiments the endonuclease can be, for example, a zinc finger nuclease (ZFN), megaTAL nuclease, meganuclease, transcription activator-like effector nuclease (TALE- nuclease, or TALEN®), or CRISPR (e.g., Cas9 or Cas12) endonuclease.

[0171] In some embodiments, TCR is rendered not functional in the cells according to the disclosure by disrupting or inactivating TCRα gene and / or TCRβ gene(s). In some embodiments, a method to obtain modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complexAttorney Docket No.: AT-061 / 02WO (MHC) signaling pathway. Modified cells, which can proliferate independently of the MHC signaling pathway, susceptible to be obtained by this method are encompassed in the scope of the present disclosure. Modified cells disclosed herein can be used in for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present disclosure is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising disrupted or inactivated TCRα and / or TCRβ genes.

[0172] The present disclosure provides methods of determining the purity of a population of engineered immune cells lacking or having 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. Such a population can be a product of the disclosed methods.

[0173] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapy drugs. The chemotherapy drug can be, for example, a purine nucleotide analogue (PNA), thus making the immune cell suitable for cancer treatment combining 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) into 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), which is involved in trinucleotide production.

[0174] In some embodiments, isolated cells or cell lines of the disclosure can comprise a pTα or a functional variant thereof. In some embodiments, an isolated cell or cell line can be further genetically modified by disrupting or inactivating the TCRα gene.

[0175] The disclosure also provides engineered immune cells that comprise polynucleotides encoding a bicistronically expressed polypeptide, linking peptide, and anyAttorney Docket No.: AT-061 / 02WO of the CAR polypeptides described herein. In some embodiments, a 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 which provides for identification and / or selection of cells which received the vector.

[0176] CAR polypeptides can be synthesized in situ in the cell after introduction of polynucleotides encoding the CAR polypeptides into the cell. Alternatively, CAR polypeptides can be produced outside of cells, and then introduced into cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, stable transformation methods (e.g., using a lentiviral vector) can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. The polynucleotides can be introduced into a cell by any suitable means such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example without limitation, microinjection, electroporation or particle bombardment. Polynucleotides can be included in vectors, such as for example plasmid vectors or viral vectors.

[0177] In some embodiments, isolated nucleic acids are 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 retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, Herpes virus vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained within a plasmid.

[0178] In some embodiments, the isolated nucleic construct is contained within a viral vector and is introduced into the genome of an engineered immune cell by random integration, e.g., lentiviral- or retroviral-mediated random integration. In some embodiments, the isolated nucleic acid construct is contained in a viral vector or a non-viralAttorney Docket No.: AT-061 / 02WO vector and is introduced into the genome of an engineered immune cell by site-specific integration, e.g., adenovirus-mediated site-specific integration. Manufacture of Engineered Immune Cells (including CAR T cells)

[0179] Provided herein are methods of analyzing or determining various attributes of donor cells from a donor cell population and / or engineered immune cells from a population of immune cells (including engineered immune cells such as CAR expressing 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 TCR, and the remaining TCR+ engineered immune cells can be depleted according to the methods described herein, at the end of production. The instant disclosure provides methods of characterizing or analyzing a population of engineered immune cells to characterize the drug product or as part of the manufacturing process. The instant disclosure also provides methods of analyzing or determining other attributes, such as the potency or polyfunctionality of the engineered immune cells to characterize the drug product or as part of the manufacturing process. In some embodiments, the engineered immune cells, such as CAR T cells, are manufactured according to Good Manufacturing Practice (GMP).

[0180] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, antigen binding domains, immune cells, compositions, and the like according to the disclosure.

[0181] Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. Cells expressing a CAR can be derived from an allogeneic or autologous source and can be depleted of endogenous TCR as described herein. 1. Source Material

[0182] In some embodiments, the immune cells comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain some embodiments, T cells can be obtained from a volume of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation.Attorney Docket No.: AT-061 / 02WO

[0183] Cells can be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction, and then placed in an appropriate buffer or media for subsequent processing.

[0184] In certain some embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation 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 directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0185] PBMCs can be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the 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 either before 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 that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells include CD27, CD45RA, CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and are 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,Attorney Docket No.: AT-061 / 02WO CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain some 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 that have cell surface antigens. 2. Stem cell derived immune cells

[0186] In some embodiments, the immune cells can be derived from embryonic stem (ES) 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 to practice the present disclosure.

[0187] In some embodiments, the immune cell is an induced pluripotent stem cell (iPSC) derived from a reprogrammed T-cell. In some embodiments, the source material can be an induced pluripotent stem cell (iPSC) derived from a T cell or a non-T cell. The source material can be an embryonic stem cell. The source material can be a B cell, or any other cell from peripheral blood mononuclear cell isolates, hematopoietic progenitor, hematopoietic stem cell, mesenchymal stem cell, adipose stem cell, or any other somatic cell type. 3. Genetic Modification of isolated cells

[0188] The donor immune cells, e.g., T cells, of a donor immune cell population can be genetically modified following isolation using known methods, or the donor immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In some embodiments, the isolated donor immune cells are genetically modified to reduce or eliminate expression or activity of endogenous TCRα and / or CD52. In some embodiments, the cells are genetically modified using gene editing technology (e.g., CRISPR / Cas9, CRISPR / Cas12a, a zinc finger nuclease (ZFN), a TALEN, a MegaTAL, a meganuclease, base editing or prime editing) to reduce or eliminate expression or activity of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, the immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and / or expanded in vitro.Attorney Docket No.: AT-061 / 02WO

[0189] Certain methods for making the constructs and engineered immune cells of the disclosure are described in PCT application PCT / US15 / 14520, the contents of which are hereby incorporated by reference in their entirety.

[0190] It will be appreciated that PBMCs can further include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of these CAR expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing the CAR for storage and / or preparation for use in a human subject. In one embodiment, the in vitro transduction, culture and / or expansion of T cells are performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum.

[0191] For cloning of polynucleotides, the vector can be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors can contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0192] In some embodiments, the present disclosure provides isolated host cells containing the vector provided herein. The host cells containing the vector can be useful in expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells, particularly human cells.

[0193] The vector can be introduced to the host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection,Attorney Docket No.: AT-061 / 02WO electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing more than one different CARs.

[0194] In one embodiment, the disclosure provides a method of storing genetically engineered cells expressing CARs or TCRs. This involves cryopreserving the immune cells such that the cells remain viable upon thawing. A fraction of the immune cells expressing the CARs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.

[0195] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment- effective amount. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. 4. Allogeneic CAR T cells

[0196] The process for manufacturing allogeneic CAR T therapy involves harvesting healthy, selected, screened and tested donor immune cells, including T cells, from healthy donors. Next, the T cells of the donor immune cells are engineered to express CARs, which recognize certain cell surface proteins that are expressed in hematologic or solid tumors. Allogeneic T cells are gene editing to reduce the risk of graft versus host disease (GvHD) and to prevent allogeneic rejection. A T cell receptor gene (e.g., TCRα, TCRβ) is knocked out to avoid GvHD. The CD52 gene can be knocked out to render the CAR T product resistant to anti-CD52 antibody treatment. Anti-CD52 antibody treatment can therefore beAttorney Docket No.: AT-061 / 02WO used to suppress the host immune system and allow the CAR T to stay engrafted to achieve full therapeutic impact. The engineered T cells then undergo further processing, which may optionally include a depletion step to remove unwanted T cells that express a biomarker described herein (e.g., unwanted T cells expressing a TCR gene), as well as a purification step and are ultimately cryopreserved in vials for delivery to patients. 5. Autologous CAR T cells

[0197] Autologous chimeric antigen receptor (CAR) T cell therapy, involves collecting a patient’s own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express CARs that recognize target expressed on the cell surface of one or more specific cancer cells and kill cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient. Methods of in vitro Sorting

[0198] In some embodiments, provided are methods for in vitro sorting of a population of immune cells, wherein a subset of the population of immune cells comprises engineered immune cells expressing an antigen-specific CARs comprising epitopes specific for monoclonal antibodies (e.g., exemplary mimotope sequences). The method comprises contacting the population of immune cells with a monoclonal antibody specific for the epitopes and selecting the immune cells that bind to the monoclonal antibody to obtain a population of cells enriched in engineered immune cells expressing an antigen-specific CAR.

[0199] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting the cells that bind to the monoclonal antibody can be done by Fluorescence Activated Cell Sorting (FACS).

[0200] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a magnetic particle. In this embodiment, the step of selecting the cells that bind to the monoclonal antibody can be done by Magnetic Activated Cell Sorting (MACS).Attorney Docket No.: AT-061 / 02WO

[0201] In some embodiments, the mAb used in the method for sorting immune cells expressing the CAR is chosen 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, said mAb is rituximab. In another embodiment, said mAb is QBEND- 10. In other embodiments the mAb binds to TCR ^ or TCR ^.

[0202] In some embodiments, the population CAR-expressing immune cells obtained when using the method for in vitro sorting CAR-expressing immune cells described above, comprises at least 70%, 75%, 80%, 85%, 90%, 95% of CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained when using the method for in vitro sorting CAR-expressing immune cells, comprises at least 85% CAR- expressing immune cells.

[0203] In some embodiments, the population of CAR-expressing immune cells obtained when using the method for in vitro sorting CAR-expressing immune cells described above shows increased cytotoxic activity in vitro compared with the initial (non-sorted) cell population. In some embodiments, said cytotoxic activity in vitro is increased by 10%, 20%, 30%, 40% or 50%. In some embodiments, the immune cells are T-cells.

[0204] In some embodiments, the mAbs are previously bound onto a support or surface. Non-limiting examples of solid support can include a bead, agarose bead, a magnetic bead, a plastic welled plate, a glass welled plate, a ceramic welled plate, a column, or a cell culture bag.

[0205] The CAR-expressing immune cells to be administered to the recipient can be enriched in vitro from the source population. Methods of expanding source populations can include selecting cells that express an antigen such as CD34 antigen, using combinations of density centrifugation, immuno-magnetic bead purification, affinity chromatography, and fluorescent activated cell sorting.Attorney Docket No.: AT-061 / 02WO

[0206] 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. Since 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.

[0207] In some embodiments, the method used for sorting T cells expressing CAR is the Magnetic- Activated Cell Sorting (MACS). Magnetic-activated cell sorting (MACS) is a method for separation of various cell populations depending on their surface antigens (e.g., CD molecules) by using superparamagnetic nanoparticles and columns. MACS can be used to obtain a pure cell population. 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 fast and gentle separation of cells. The unlabeled cells pass through while the magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0208] Detailed protocol for the purification of specific cell population such as T-cell 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). Pharmaceutical Compositions and Therapy

[0209] In some embodiments, the engineered immune cells described herein are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin.

[0210] In embodiments, desired treatment amounts of cells in the composition are generally at least 2 cells (for example, 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 memoryAttorney Docket No.: AT-061 / 02WO T cell; or two or more CD4+ helper T cell subset) or is more typically greater than 102cells, and up to and including 106, up to and including 107, 108or 109cells and can be more than 1010cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 106cells / ml and generally is greater than 107cells / ml, generally 108cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012cells. In some aspects of the present disclosure, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of about 105 / kilogram or about 106 / kilogram (106-1011per patient) can be administered. CAR treatments can be administered multiple times at dosages within these ranges. The cells can be autologous, allogeneic, or heterologous to the patient undergoing therapy.

[0211] The CAR expressing cell populations of the present disclosure can be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present disclosure can comprise a CAR or TCR expressing cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise 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. Compositions of the present disclosure are preferably formulated for intravenous administration.

[0212] The pharmaceutical compositions (solutions, suspensions or the like), 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 which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; 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.Attorney Docket No.: AT-061 / 02WO The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0213] Methods are provided for treating diseases or disorders, including cancer and autoimmune diseases. In some embodiments, the disclosure relates to creating a T cell- mediated immune response in a subject, comprising administering an effective amount of the engineered immune cells of the present application to the subject. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). The CAR containing immune cells of the disclosure can be used to treat a CD19-associated disease or disorder and / or a BCMA-associated disease or disorder. In some embodiments, the disease or disorder can be an autoimmune disease, including, without limitation, lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, and myositis.

[0214] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety.

[0215] The following examples are offered for illustrative purposes only. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. EXAMPLES Example 1 – Immune cells engineered to express a chimeric antigen receptor (CAR) and a chimeric cytokine receptor (CCR)

[0100] In the context of adoptive immune cell transfer, the beneficial activities of CAR T cells, e.g., activation, proliferation, persistence, and / or potency, can be enhanced through cytokine-induced cytokine receptor signaling, also known as Signal 3. Current approaches for signal 3 include combining CAR T cell therapy with systemic infusions of recombinant cytokines / cytokine mimetic and engineering CAR T cells to secrete / express cytokines extracellularly but these have drawbacks, such as the risk of systemic toxicity. In an alternative approach, Signal 3 can be enhanced using constitutively active chimeric cytokineAttorney Docket No.: AT-061 / 02WO receptor (CACCR) (see US20200291090A1, which is incorporated herein by reference 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 homodimeric receptor. In this example, immune cells were engineered to express a CD19-specific CAR and a constitutively active chimeric cytokine receptor (CACCR).

[0101] CAR-T cells comprising an exemplary CD19 CAR bearing the 4G7 scFv directed towards CD19 coupled to 4-1BB and CD3z signaling domains (see e.g., US Pat. 10,874,693, incorporated herein by reference in its entirety) were selected for further engineering of a CACCR. CD19-specific CAR-T cells co-expressing a CACCR composed of a truncated IL2Rb tail were generated. CAR-T cells comprising an exemplary BCMA CAR bearing the P5A2 scFv directed towards BCMA coupled to 4-1BB and CD3z signaling domains and the same CACCR (see e.g., Published US Application No. US20210260118A1, incorporated herein by reference in its entirety) were also selected for testing.

[0102] FIG.1A shows a schematic of the engineered constitutively active chimeric cytokine receptor. FIG.1B shows a schematic of the lentiviral vector used to co-express the constitutively active chimeric cytokine receptor and the anti-CD19 CAR. Constructs encoding an anti-CD19 CAR comprising SEQ ID NO: 335 (see Table 3) and a CACCR comprising SEQ ID NO: 343 (see Table 4) were used.

[0103] FIG.1C shows a schematic of the lentiviral vector used to co-express the constitutively active chimeric cytokine receptor and the anti-BCMA CAR. Constructs encoding an anti-BCMA CAR comprising SEQ ID NO: 342 (Table 3) and / or a CACCR comprising SEQ ID NO: 343 (Table 4) were used.

[0104] In an exemplary protocol for making lentivirus encoding a CACCR and CD19 CAR, HEK293T cells were plated at 0.45 million cells per mL in 2mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, the lentivirus was prepared by mixing together lentiviral packaging vectors 1.5 ug psPAX2, 0.5 ug pMD2G, and 0.5 ug of the appropriate transfer CAR vector in 250 uL Opti-MEM (Gibco) per well of the 6-well plate (“DNA mix”).10 uL Lipofectamine 2000 (Invitrogen) in 250 uL Opti-MEM was incubated at roomAttorney Docket No.: AT-061 / 02WO temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and the total volume of 500 uL was slowly added to the sides of the wells containing HEK293T. One day post-transfection, the media from each well of HEK293T cells in the 6-well plate was replaced with 2mL per well of T cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. Two days post- transfection, the lentiviral supernatants from HEK293T cells were harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and crude lentiviral supernatants were used directly for T cell transduction. On Day 0, purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat# 130- 111-160, 1:100 dilution) in a Grex-24 plate (Wilson Wolf, cat# 80192M). On Day 2, T cells were resuspended at 0.5 million cells per mL in T cell transduction media, transduced with an equal volume of crude lentiviral supernatant along with 100 IU / mL human IL-2 in a Grex-24 plate. On Day 5, CACCR-expressing CAR-T cells were fed by replacing the spent media with T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), along with 100 IU / mL human IL-2. At this time, control CAR-T cells lacking CACCR were expanded in either 100 U / mL human IL-2 only, or 100 U / mL human IL-2 and 10 ng / mL human IL-15 (Miltenyi Biotec). Cells were expanded into larger G-Rex vessels (Wilson Wolf) as needed using T cell expansion media and the respective concentrations of recombinant cytokines. On Day 13, cells were stained with the Zombie NIR Fixable Viability Kit (Biolegend), labelled with a BUV395-conjugated CD3 antibody (Biolegend) and an anti-idiotype antibody specific for the P5A2 scFv, then FACS-sorted to enrich for CAR+ T cells. Sorted CAR+ T cells were then cultured in Grex-24 plates for a further 2 days in T cell expansion media, with CACCR CD19 CAR+ T cells left in the absence of exogenous cytokines, and with sorted control CAR+ T cells either left in the absence of exogenous cytokines, treated with 100 U / mL human IL-2, or treated with 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 cell pellets were snap-frozen for subsequent RNA extraction and NanoString gene expression analysis (Human CAR-T Panel; NanoString Technologies). Example 2 – Detection of CACCR in CAR T cells after stimulationAttorney Docket No.: AT-061 / 02WO

[0105] Co-culture setup for CAR T cell stimulation. Two different CAR T cell drug product lots were used as effector cells in this experiment – a) PBMCs used as a starting material to provide cells engineered to express a CD19-specific CAR and a CACCR (Lot 02) and b) CD4 / CD8+ T cells used as a starting material to provide cells engineered to express a CD19-specific CAR and a CACCR (Lot 04). Both CAR T cell drug product lots were engineered from the same donor. Irradiated-DGL (Daudi cell were purchased from ATCC(CCL-213™), engineered with GFP-luciferase and irradiated for assay) or CD19-Fc (Acro bio, USA) at 2.5ug / ml were used to stimulate the two CAR T drug product lot cells. Both effector cells were thawed and cultured in 1E6 cells / ml in X-vivo media with 10% FBS. Irradiated-DGL was added to the culture in 2:1 ratio of effector cells to target cells. After the setup, co-cultures were sampled at 20 hours, 24 hours, and 48 hours for western blot and flow cytometry.

[0106] Intracellular staining of P2A using flow cytometry. Harvest cells were stained with a CD19-specific CAR anti-idiotypic antibody PE (Acro Biosystems) and FVS780 (BD Bioscience) in flow stain buffer (554656, BD). Cells were fixed and permeabilized using FOXP3 Transcription factor staining buffer set (00-5523-00, eBioscience) according to the protocol. Generally, cells were fixed using Fixation / Permeabilization buffer for 20 min at room temperature (RT) and stained with P2A-APC (NBP2-59627AF647, Novus Bio) in permeabilization buffer for 30 min at RT. Cells were resuspended in stain buffer and analyzed using LSRFortessa™ cell analyzer (BD Bioscience 0.Data were analyzed FlowJo 9.2 software (Tree Star, Inc).

[0107] As described above, the CAR T cell drug product cells engineered to express a CD19-specific CAR and a CACCR were incubated with or without IR-DGL. As shown in FIG.2, cells were harvested and stained with a CD19-specific CAR anti-idiotypic antibody PE (Acro Biosystems) and FVS780 (Live / Dead Viability Stain) at 24 hours (FIG.2A) or 48 hours (FIG.2B), then fixed for intracellular staining of P2A using Transcription factor staining buffer set. The P2A and CAR+ double positive cell population were gated from singlet viable cells in different culture conditions.

[0108] FIG.3 shows the results of an additional experiment where cells were incubated with (FIG.3A) or without (FIG.3B) CD19Fc or IR-DGL. Cells were harvested at 24 hours and stained with a CD19-specific CAR anti-idiotypic antibody PE (see US20200331998A1,Attorney Docket No.: AT-061 / 02WO which is incorporated herein by reference in its entirety) (Customized from Biolegend) and FVS780 (Live / Dead Viability Stain), then fixed for intracellular staining of P2A using Transcription factor staining buffer set. The P2A+ and CAR+ double positive cell population were gating from singlet viable cells from different culture conditions.

[0109] P2A Detection using western blot. HEK293T cells with BCMA CAR + CACCR transfection were applied as positive control for P2A protein detection.0.2E6cells / ul cell lysates of all the samples were prepared using mix of M-PER buffer, protease inhibitor and reducing reagent.0.26E6 cells for positive control and 2.6E6 cells for other samples were loaded into 10% Bis-tris gel. Mix of 2A Peptide(3H4) antibody (Novsubio, Cat#NBP2- 59627) in 1:200 and beta-Tubulin (Cell Signaling, Cat#2128L) in 1:1000 was used as primary antibody. Mix of IRD-800CW Goat anti-Rabbit IgG (LI-COR, Cat#926-32211) and IRD-680CW Goat anti-Mouse IgG (LI-COR, Cat#926-68070) in 1:5000 was used as secondary antibody. The blot was visualized using Odyssey CLx. Table 6 lists the samples by lane for the western blot. Table 6 Lane Sample 1 Stable CD19 CAR expressing HEK293T 10E6 2 02 Lot – stimulation for 20 hours 3 02 Lot – stimulation for 24 hours 4 02 Lot – stimulation for 48 hours 5 02 Lot – No stimulation for 48 hours 6 04 Lot – stimulation for 20 hours 7 04 Lot – stimulation for 24 hours 8 04 Lot – stimulation for 48 hours 9 04 Lot – No stimulation for 48 hours

[0110] As shown in FIG.4, tubulin was shown in bands at 50kDa (see upper arrow) and served as a technical control. P2A protein was shown in bands at 24.3kDa (see lower arrow). P2A protein was detected in positive control and stimulated samples, shown in lanes 1, 2, 3, 4, 6, 7, 8. No P2A protein was detected in unstimulated samples, shown in lanes 5 and 9.Attorney Docket No.: AT-061 / 02WO

[0111] The CAR T cells comprising the anti-BCMA CAR bearing the P5A2 scFv and expressing the CACCR (see Example 1 and Table 5 above) were treated with PMA + ionomycin (P+I) to observe the level of detection of the CACCR. CAR T cells comprising the anti-BCMA CAR bearing the P5A2 scFv but not expressing the CACCR (see Example 1 and Table 5 above) were included as a negative control.

[0112] The following samples listed in Table 7 were tested. Table 7 CAR T cells Whole Blood Stimulation – 24h 1 Anti-BCMA (no CACCR) - - 2 Anti-BCMA (no CACCR) - P+I 3 Anti-BCMA with CACCR - - 4 Anti-BCMA with CACCR - P+I 5 Anti-BCMA (no CACCR) + - 6 Anti-BCMA (no CACCR) + P+I 7 Anti-BCMA with CACCR + - 8 Anti-BCMA with CACCR + P+I

[0113] Frozen whole blood with EDTA as anticoagulant (Stem Cell Technologies) was used. Frozen CAR T cell lots (Anti-BCMA: 14e6 CAR+ cells / ml in 1ml; Anti-BCMA + CACCR: 40e6 CAR+ cells / 2ml – see Table 8) were used. Freeze-thawed CAR T cells were spiked into freeze-thawed whole blood (WB). 10% CAR+ cells among viable whole blood cells. Table 8 Cell type # per sample Thawed whole blood 600,000 CAR+ cells 60,000Attorney Docket No.: AT-061 / 02WO

[0114] The anti-BCMA CAR bearing the P5A2 scFv was detected using an anti-idiotypic antibody for the CAR (see Published US Patent Application No.20240101710A1, incorporated herein by reference in its entirety).

[0115] The anti-BCMA CAR was detected in all samples (data not shown). The CACCR was detected via P2A in WB / CAR mixtures that are a) unstimulated and stained after mixing the cells; or b) simulated 24h with PMA+ionomycin before staining. Cells were stained for viability and surface markers before fix / perm for intracellular P2A staining. As shown in FIG.6A-B, CCR was detected in P+I stimulated samples (right panels) using the anti-P2A antibody P2A-APC (NBP2-59627AF647, Novus Bio) but was not detected in the unstimulated samples (left panels). As expected, the CACCR was not detected in any of the samples (data not shown).

Claims

Attorney Docket No.: AT-061 / 02WO WHAT IS CLAIMED IS:

1. An in vitro method of detecting a bicistronically expressed polypeptide in a chimeric antigen receptor (CAR) T cell, comprising: a)  contacting the CAR T cell with an exogenous agent, wherein the CAR T cell comprises a polynucleotide that expresses the bicistronically expressed polypeptide, a linking peptide, and an additional polypeptide, wherein the linking peptide is at the C- terminus of the bicistronically expressed polypeptide; and b)  detecting the linking peptide after step a), thereby indicating the presence of the bicistronically expressed polypeptide in the CAR T cell.

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

3. The method of claim 1, wherein the bicistronically expressed polypeptide is a membrane protein that comprises a transmembrane domain.

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

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

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

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

8. The method of any one of the preceding claims, wherein the linking peptide is heterologous to the bicistronically expressed polypeptide.

9. The method of any one of claims 4-8, wherein the linking peptide is contiguous with the intracellular domain of the bicistronically expressed polypeptide.

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

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

12. The method of any one of the preceding claims, wherein the exogenous agent is selected from a small molecule reagent and a target molecule, wherein the CAR specifically binds to the target molecule.Attorney Docket No.: AT-061 / 02WO 13.  The method of claim 12, wherein the small molecule reagent stimulates cytokine production in the CAR T cell.

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 a target cell.

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

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 cell with the soluble target molecule.

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

21. The method of claim 20, wherein the linking peptide is cleavable 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 one of the preceding claims, wherein the linking peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 305-320.

25. The method of any one of the preceding claims, wherein the bicistronically 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 comprise an extracellular domain.

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

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

30. The method of any one of the preceding claims, wherein the detecting step comprises detecting the linking peptide by flow cytometry and / or a protein immunoblot assay.Attorney Docket No.: AT-061 / 02WO 31.  The method of claim 8, wherein the bicistronically expressed polypeptide further comprises an intracellular domain.

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

33. An in vitro method for analyzing a population of CAR T cells wherein the CAR T cells express a bicistronically expressed polypeptide, a linking peptide, and a CAR, the method comprising: a) contacting a sample of the population of CAR T cells with an exogenous agent; and b) detecting the bicistronically expressed polypeptide in the sample after the contacting step.

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

35. The method of claim 33 or 34, further comprising: c) analyzing whether the bicistronically expressed polypeptide is detected at or exceeding a predetermined level in the sample.

36. The method of claim 35, wherein the predetermined level is a presence of the linking 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 population of CAR T cells comprises a CAR T cell drug substance or a CAR T cell drug product.

38. The method of claim 37, further comprising: d) if the bicistronically expressed polypeptide is detected in the sample at or exceeding a predetermined level, formulating the population of CAR T cells to form a drug product; and optionally e) freezing the drug product.

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