Compositions and methods for stimulating t cells used in cell therapy with a bispecific bridging molecule

Abstract

The disclosure relates to methods and compositions for stimulating immune cells used in cell therapy with a bispecific bridging molecule that can be used to treat a disease or disorder, for example, cancer.

Description

COMPOSITIONS AND METHODS FOR STIMULATING T CELLS USED IN CELL THERAPY WITH A BISPECIFIC BRIDGING MOLECULECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application 63 / 671,692, filed on July 15, 2024, the contents of which are incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTINGThe present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 061250-564001WO, created on July 14, 2025 and is 504 kilobytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0002] Adoptive cell transfer (ACT) therapy is an immunotherapy that uses modified immune cells of the adaptive immune system. ACT is a powerful tool for the treatment of various diseases, particularly cancers and has shown efficacy in blood cancers. Solid tumors pose unique challenges for ACT therapy compared to blood cancers. One of the major obstacles that limits effectiveness of ACT is access to the solid tumor tissue and to the specific antigens targeted by the therapeutic immune cells. Because antigen, otherwise known as signal 1, is the ignition and the fuel of the adaptive immune response, it is advantageous that immune cells such as T cells receive activation signals through the signal 1 pathway.

[0003] T cells are regulated in complex ways, but the three most important groups of stimuli that generate and maintain an antigen-specific response in T cells are: (i) antigen, otherwise called signal 1; (ii) costimulation, otherwise called signal 2; and (iii) cytokine, otherwise called signal 3. These three major stimuli interact with each other, as well as additional sources of stimulation such as innate immune signaling molecules (e.g., toll-like receptors), checkpoint receptors (e.g., PD-1, CTLA-4), and homing receptors (e.g., CCL19). Signal 1 (antigen) is the most important stimulus because it not only initiates the adaptive immune response but also maintains it and provides the means of recalling the response upon subsequent exposure to antigen. Antigen is the essence of specificity in the adaptive immuneresponse and the basis for vaccination. Thus, signal 1 is not only the key mediator via the T cell receptors (TCRs) of activation in naive and memory T cells but also stokes the fire of the immune response, once ignited.

[0004] A variety of methods to boost T cell function in ACT, for example in chimeric antigen receptor T cells (CAR-T cells) have been explored. One of the most active areas in CAR-T cell engineering, especially given some of the challenges of cell therapy for solid tumors, involves boosting the performance of immune cells. Some approaches utilize exogenous agents, such as cytokines, lympho-depleting chemotherapeutics, STING agonists, and vaccines that are administered to the patient to enhance immune cell function, including persistence of immune cells in the body.

[0005] Another approach is onboard engineering of boosters. Onboard boosters are especially desirable because they mitigate issues of systemic toxicity, biodistribution, and excretion of certain exogenous agents. A variety of approaches have been explored preclinically and in the clinic, including (i) addition of genes that encode signaling molecules that provide signal 2 (costimulation); (ii) engineering genes that “armor” CAR-Ts via expression of cytokines of various types and in various configurations (e.g., IL-15; Lands et al., 2021 PMID: 33156338 ); (iii) addition of elements of the innate immune response that are regulated by antigen or other means (e.g., MyD88; Collinson-Pautz et al., 2019 PMID: 30816327); (iv) expression of inhibitors of apoptosis (e.g., RNAi directed at FAS, etc.; Santoro et al. J ImmunoTher of Cancer 2021 ;9:doi: 10.1136 / jitc-2021-SITC2021.21); (v) expression of inhibitors of checkpoints (e.g., PD-1 dominant-negative constructs; Liu et al., 2016 PMID: 26979791); and (vi) inhibitors of the TGFbeta pathway (e.g., dominant-negative TGFBR2; Kloss et al., 2018 PMID: 29807781).

[0006] In addition, some efforts have focused on increasing sensitivity of the immune cells to antigen by improvement in ligand-binding domain affinity / sensitivity or structure (Xu et al., 2020 PMID: 32768859; Calderon et al., 2022 PMID: 31707680). However, despite the limited antigen available to T cells infused in the blood to treat solid tumors, few efforts have explored boosting signal 1 directly beyond engineering of the scFvs and ICDs of CARs. Although some have appreciated the possible benefits of tonic (ligand-independent) signaling in T cells (Chen et al., 2023 PMID: 36882513), tonic signaling is viewed as a problematic feature of CARs that must be overcome (Gennert et al., 2021 PMID: 34285077; Calderon et al., 2022 PMID: 31707680). At best, tonic signaling has been viewed as a double-edged sword.

[0007] Some efforts to boost performance of CAR-Ts have centered on downstream elements of TCR signaling (e.g., LAT, SLP76, LCK, etc.; Mock et al., 2022 PMID: 34039676;Tousley et al., 2023 PMID: 36890224). These have in some cases produced new designs for CARs that extend beyond the classical intracellular IT AMs domains of TCR architecture.

[0008] Methods to control T cell response via exogenous molecules that regulate the activation process in cells engineered with proteins that bind such agents include e.g., rapamycin and its analogs (Leung et al., 2019 PMID: 31039141; Foster et al., 2017 PMID: 28697888; Jarjour et al., U.S. Patent Publication No. 2022 / 0025014; Brogdon et al., U.S. Patent Publication No. 2017 / 0274014).

[0009] Bispecific bridging molecules such as TCEs can redirect T cell activation from their natural antigens to specific, selected antigens. TCEs typically comprise bispecific antibodylike molecules with one ligand-binding domain (LBD) directed at a tumor-associated antigen and the other LBD directed at a subunit of the TCR complex (Staerz and Bevin, 1986 PMID: 2869486; Riethmuller and Kufer., 1995 PMID: 7624362). These TCEs broadly activate T cells in an antigen-dependent fashion by bridging the T cell to the tumor cell such that the T cell is activated, leading to cytokine release, proliferation, and / or tumor-cell-specific cytotoxicity. In cases where the tumor-associated antigen is also present on normal tissues, the T cells’ effector functions are typically also stimulated by the target-expressing normal cells; e.g., CD19(+) B cells bind CD19-directed TCEs and together they (B cells and TCEs) activate T cells to secrete cytokines and proliferate (Schlereth et al., 2005 PMID: 16032400; Bargou et al., 2008 PMID: 18703743).

[0010] For solid tumors, tumor-antigen access is a serious limitation. New compositions and methods for the treatment of solid tumors with adoptive immune cells are needed.SUMMARY

[0011] The disclosure relates generally to a combination of therapeutic immune cells that are either adoptively transferred into patients, or engineered in vivo, with exogenous immune cell engager molecules that, without being bound by theory, may trigger immune cell activation by bridging the therapeutic immune cells to normal cells in the body in a manner that stimulates the therapeutic immune cells. In this respect, the exogenous immune cell engager molecules may act like an antigen stimulus of the therapeutic immune cells. The mechanism may, for example, involve a bispecific molecule (for example a T cell engager (TCE) that bridges the therapeutic immune cells to normal cells in the blood (e.g., other immune cells,that express a specific antigen that activates an activator receptor on the therapeutic immune cell, for example a CAR or TCR on the therapeutic immune cells). Importantly, the immune cell engager (e.g., the TCE) may not necessarily recognize the tumor-associated antigen(s) to which the therapeutic cells are directed. Instead, the immune cell engager (e.g., TCE) may serve to boost the activation and / or proliferation of the therapeutic immune cells (e.g., T cells) in the absence of their specific antigen(s). This mechanism may or may not result in the destruction of the normal cells.

[0012] Some embodiments include combination therapy involving: (i) TCEs that recognize antigens expressed on B cells such as CD 19, CD20, and CD22; and (ii) TCEs that recognize TCR complex subunits such as CD3e. Often the TCEs recognize both a B cell antigen and a TCR complex subunit. The TCEs may be used in combination with adoptively transferred T cells that include: (i) TILs; (ii) TCR-Ts; (iii) CAR-Ts; or (iv) Tmod or other logic-gated immune cells (DiAndreth et al., 2022).

[0013] In one aspect, the disclosure provides a method for treating a solid tumor in a subject in need thereof, comprising administering an immune cell engager in an amount effective to activate an exogenous immune cell, wherein the exogenous immune cell or a vector encoding genetic elements capable of generating the exogenous immune cell in vivo is administered to the subject, before, during, or after the administering of the immune cell engager.

[0014] In some embodiments, the exogenous immune cell is an T cell, a NK cell, or a NKT cell.

[0015] In some embodiments, the immune cell engager is a T cell engager and the immune cell is a T cell, optionally a cytolytic T cell, optionally a CD4+ T cell or a CD8+ T cell, or a mixture thereof.

[0016] In some embodiments, the immune cell engager is an NK cell engager and the immune cell is a natural killer (NK) cell or a NKT cell.

[0017] In some embodiments the engager provides a signal 1 to the immune cell; the engager triggers signaling by a receptor in the immune cell; the engager bridges the immune cell to normal endogenous immune cells such that the normal endogenous immune cells activate the immune cell to proliferate; the engager allows tuned stimulation to improve the quality and performance of the immune cell in the subject; and / or the engager bypasses the need for antigen exposure of the immune cell in the subject’s blood.

[0018] In some embodiments, the method comprises administering the exogenous immune cell or the vector in a first pharmaceutical composition apart from a second pharmaceutical composition comprising the engager. In some embodiments, the method comprisesadministering the exogenous immune cell or the vector and the engager in a single pharmaceutical composition.

[0019] In some embodiments, the exogenous immune cell comprises an artificial activator receptor. In some embodiments, the activator receptor is chimeric antigen receptor (CAR).

[0020] In some embodiments, the activator receptor is a T cell receptor (TCR).

[0021] In some embodiments, the exogenous immune cell comprises an artificial inhibitor receptor (e.g., a logic-gated T cell such as a Tmod™ cell).

[0022] In some embodiments, the immune cell engager is a T cell engager and the T cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds a T cell antigen, optionally wherein the T cell antigen is CD3.

[0023] In some embodiments, the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table I.

[0024] In some embodiments, the first antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table II, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

[0025] In some embodiments, the first antibody fragment comprises a polypeptide sequence according to any of the scFvs of Table III, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

[0026] In some embodiments, the immune cell engager is an NK cell engager and the NK cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds an NK cell antigen.

[0027] In some embodiments, the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table IV.

[0028] In some embodiments, the immune cell engager comprises a second ligand-binding domain comprising a second antibody fragment that specifically binds a B cell antigen, optionally wherein the B cell antigen is CD 19, CD20, CD22, or BCMA.

[0029] In some embodiments, the second antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table V.

[0030] In some embodiments, the second antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table VI, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

[0031] In some embodiments, the second antibody fragment comprises a polypeptide sequence according to any of the scFvs of Table VII, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

[0032] In some embodiments, the artificial activator receptor specifically binds an activator antigen present on a solid tumor, optionally wherein the activator antigen is EGFR, MSLN, CEA, or HER2.

[0033] In some embodiments, binding of the activator receptor to the activator antigen on a target cell promotes a cytotoxic response by the exogenous immune cell against the target cell.

[0034] In some embodiments, the artificial inhibitor receptor specifically binds an inhibitor antigen absent from a solid tumor, optionally wherein the inhibitor antigen is an antigen subject to loss of heterozygosity in a solid tumor, optionally wherein the inhibitor antigen is HLA- A*02, HLA-A*03, or HLA-A*11, optionally according to any one of the inhibitor antigens disclosed in Table 14.

[0035] In some embodiments, binding of the inhibitor receptor to an inhibitor antigen on a nontarget cell protects the non-target cell from cytotoxicity mediated by the T cell.

[0036] In some embodiments, the immune cell engager is mosunetuzumab.

[0037] In some embodiments, the immune cell engager is blinatumomab.

[0038] In some embodiments, the exogenous T cell is generated in vivo by administering a vector encoding the artificial receptor or artificial receptors of the T cell, and optionally other genetic elements, wherein the vector transduces endogenous T cells thereby generating the exogenous T cell in vivo.

[0039] In one aspect, the disclosure provides a pharmaceutical composition comprising the engager and the immune cell or the vector of or the vector of the disclosure.

[0040] In one aspect, the disclosure provides a kit comprising a first pharmaceutical composition comprising the engager of any preceding claim and a second pharmaceutical composition comprising the immune cell or the vector of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0042] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0043] FIG. 1 is a diagram of a generic TCE that bridges a normal cell (e.g., a B cell) to a therapeutic T cell, causing activation, cytokine release and proliferation of the T cell. This can serve as a boost to the therapeutic T cell in the absence of direct recognition of its tumor- associated antigen.

[0044] FIG. 2 is a schematic showing the series of events that occurs when the subject of the disclosure, a therapeutic T cell combined with a TCE. (A) The TCE bridges the therapeutic cell to a normal cell; (B) the therapeutic cell is activated to proliferate; (C) the therapeutic cell locates and selectively kills tumor cells.

[0045] FIG. 3 is a schematic of Mosunetuzumab, a type of monoclonal antibody used in the treatment of certain types of cancer, particularly B-cell non-Hodgkin lymphoma. It works by binding to CD20 on the surface of B cells and CD3 on T cells, thereby bringing T cells into close proximity with B cells to induce the immune-mediated killing of the B cells. This bispecific antibody engages the body's immune system to target and destroy malignant B cells.

[0046] FIG. 4 is a schematic of Blinatumomab, a bispecific T-cell engager (BiTE) antibody used for the treatment of certain types of B-cell acute lymphoblastic leukemia (ALL). It targets CD 19 on B cells and CD3 on T cells, facilitating the interaction between T cells and B cells, which leads to the destruction of the malignant B cells by the body's immune system.

[0047] FIG. 5 shows time course plots showing proliferation and transgene(tg)(+) fractions of Tmod-transduced primary T cells cultured in the presence of Mosunetuzumab(Mos) and Raji cells. Transgene(+) was evaluated at each timepoint via flow cytometry.

[0048] FIG. 6 shows a time course plot showing total viable cell counts of Raji cells in the presence of Mosunetuzumab(Mos) and T cells. Raji cell counts were calculated using flow cytometry based on the RFP+ Raji fraction.

[0049] FIG. 7 shows killing of primary B cells with or without Mosunetuzumab. Primary B cell counts were calculated using flow cytometry based on the CD 19+ fraction, which was evaluated every 2-3 days. The data were fit to a sigmoid curve to calculate the time to 50% kill.

[0050] FIG. 8 shows percent activation of the Tmod(+) primary T cells cultured in the presence of A2(+) or A2(-) primary B cells and Mosunetuzumab. Percent CD25+ was calculated via flow cytometry 48 hours after culture.

[0051] FIG. 9 shows time course plots showing proliferation and Tmod(+) fractions of Tmod-transduced primary T cells cultured in the presence of Mosunetuzumab (Mos) and primary B cells. Tmod(+) was evaluated at each timepoint via flow cytometry.

[0052] FIG. 10 shows time course plots showing percent of Tmod(+) T cells that are cycling (defined as phases G2+S+M) in the presence of Mosunetuzumab (Mos) and primary B cells. Tmod(+) and DNA content was evaluated at each timepoint via flow cytometry.DETAILED DESCRIPTION

[0053] The disclosure provides compositions and methods to treat patients suffering from diseases such as cancer which are in some cases poorly treated by other methods. The disclosure is based in part on the discovery that targeting of nonessential normal cells would provide a key potency boost to therapeutic cells targeted to tumor-associated antigen in solid tumors, different from the one used to boost activation in the blood. Mimicking an antigen stimulus with a bispecific molecule (e.g., a bispecific T cell engager, TCE) triggers signaling by the activator receptor (c.g, a CAR or TCR) in the adoptively transferred (or in vivo engineered) immune cells (e.g., T cells). The stimulus to the therapeutic immune cells is provided by the combination of normal cells to which they have access (e.g., normal immune cells) and the bispecific molecule (e.g., a TCE). Thus, the TCE bridges the therapeutic cells to normal immune cells that can activate the therapeutic cells to proliferate, etc. This signal 1 mimetic allows tuned stimulation to improve the quality and performance of the T cell product in patients. The methods bypass the need for antigen exposure in the patient’s blood, a key difference between blood and solid tumor therapy.

[0054] The compositions and methods of the disclosure combine the power of adoptive immune cell transfer or in vivo immune cell engineering with exogenous bispecific molecules that can activate the therapeutic cells in the absence of their tumor antigen. Specifically, the compositions and methods described herein boost the activation and proliferation of therapeutic immune cells in the absence of their tumor-associated target antigens. Thus, the compositions and methods of the disclosure circumvent the requirement that adoptively transferred (or in vivo engineered) immune cells contact tumor cells or tumor antigens to receive signal 1. This in turn boosts their overall potency as anti-cancer agents because they do not depend on receiving the crucial signal 1 stimulus immediately from the tumor. While still resident in the blood, the therapeutic immune cells can receive a signal 1 stimulus from the combination of the immune cell engager (e.g., TCE) and normal cells that enables them to survive, proliferate, and extravasate into the tumor tissue where their tumor-antigen-specific receptors can mediate the destruction of the cancer.

[0055] As described above, the compositions and methods disclosed herein are particularly useful in solid tumors. The compositions and methods are also useful in liquid tumors, for example when additional immune cell activation beyond activation by the tumor cells is needed.Definitions

[0056] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0058] The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or one or more elements.

[0059] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0060] The term “and / or” should be understood to mean either one, or both of the alternatives.

[0061] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements.

[0062] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.

[0063] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0064] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0065] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0066] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0067] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0068] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a composition to achieve a beneficial or desired therapeutic result, including clinical results.

[0069] A “therapeutically effective amount” of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0070] An “increased” or “enhanced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0071] A “decrease” or “reduced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0072] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0073] In general, “sequence identity” or “sequence homology” refers to an exact nucleotide- to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining theamino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0074] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, or a cell or parts thereof and the like that originate from outside the organism (i.e., not naturally produced by the organism). In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0075] The term “MOI” is used herein to refer to multiplicity of infection, which is the ratio of agents (e.g., viral particles) to infection targets (e.g., cells).

[0076] The term “vector” is used herein to refer to an entity that comprises a nucleic acid encoding for a transgene. Vectors include for example synthetic mRNA, lipid nanoparticles, and viral particles).

[0077] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application,including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0078] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0079] As used herein, a “target cell” refers to cell that is targeted by an adoptive cell therapy. For example, a target cell can be cancer cell, which can be killed by the transplanted T cells of the adoptive cell therapy. Target cells of the disclosure express an activator ligand as described herein, and do not express an inhibitor ligand.Methods

[0080] The disclosure provides methods for treating a solid tumor in a subject in need thereof, comprising administering an immune cell engager in an amount effective to activate an exogenous immune cell. The exogenous immune cell or a vector encoding genetic elements capable of generating the exogenous immune cell in vivo is administered to the subject, before, during, or after the administering of the immune cell engager.

[0081] In some embodiments, the exogenous immune cell (e.g., T cell) is generated in vivo by administering a vector encoding the artificial receptor or artificial receptors of the immune cell (e.g., T cell). In some embodiments, other genetic elements and the vector encoding the artificial receptor or artificial receptors transduces the endogenous immune cells (e.g., T cell)thereby generating the exogenous immune cell (e.g., T cell) in vivo.

[0082] In some embodiments, the disclosure provides for a method for treating a solid tumor in a subject in need thereof, comprising administering an immune cell engager comprising: a first means for binding an immune cell receptor on an immune cell, wherein binding the immune cell receptor activates the immune cell. In some embodiments, the immune cell comprises a first means for binding an activator ligand, wherein binding the activator ligand activates the immune cell. . In some embodiments, the immune cell comprises a second means for binding an inhibitor ligand, wherein binding the inhibitor ligand inhibits the immune cell.

[0083] In some embodiments, the subject in need thereof has cancer. Cancer is a disease in which abnormal cells divide without control and spread to nearby tissue. In some embodiments, the cancer comprises a solid tumor. Exemplary solid tumors include sarcomasand carcinomas. Cancers can arise in virtually an organ in the body, including blood, bone marrow, lung, breast, colon, bone, central nervous system, pancreas, prostate, and ovary. Further cancers that are solid tumors include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell skin cancer, renal cancer, head and neck cancers, throat cancer, squamous carcinomas that form on the moist mucosal linings of the nose, mouth, throat, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the condition treated by the methods described herein is metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, stomach cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, renal cancer cells, head or neck cancer cells, throat cancer cells, squamous carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells. In some embodiments, the cancer comprises a liquid tumor.

[0084] Any cancer wherein a plurality of the cancer cells express one or more activator ligand is envisaged as within the scope of the instant disclosure. For example, CEA positive cancers that can be treated using the methods described herein include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse large B cell cancer or acute myeloid leukemia cancer.

[0085] Treating cancer can result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as “tumor regression”. Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0086] Treating cancer can result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Tumor volume may be measured by any reproducible means of measurement.

[0087] Treating cancer results in a decrease in number of tumors. Preferably, after treatment, tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0088] Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0089] Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0090] Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days;more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0091] Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0092] Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[0093] Treating cancer can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0094] Treating cancer can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.

[0095] Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0096] Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

[0097] Treating or preventing a cell proliferative disorder can result in a decrease in size of an area or zone of cellular proliferation. Preferably, after treatment, size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.

[0098] Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiomorphism.

[0099] In some embodiments, the method increases immune cell activation in a subject. Preferably, after treatment, immune cell activation is increased by 5% or greater relative to the immune cell activation prior to treatment; more preferably, immune cell activation is increased by 10% or greater; more preferably, increased by 20% or greater; more preferably, increased by 30% or greater; more preferably, increased by 40% or greater; even more preferably, increased by 50% or greater; and most preferably, increased by greater than 75% or greater. Immune cell activation may be measured by any reproducible means of measurement, for example by FACS, cytokine release assays, or cytotoxicity assays.

[0100] In some embodiments, the method increases T cell activation in a subject. Preferably, after treatment, T cell activation is increased by 5% or greater relative to the T cell activation prior to treatment; more preferably, T cell activation is increased by 10% or greater; more preferably, increased by 20% or greater; more preferably, increased by 30% or greater; more preferably, increased by 40% or greater; even more preferably, increased by 50% or greater;and most preferably, increased by greater than 75% or greater. T cell activation may be measured by any reproducible means of measurement, for example by FACS, cytokine release assays, or cytotoxicity assays.

[0101] In some embodiments, the method increases immune cell proliferation in a subject. Preferably, after treatment, immune cell proliferation is increased by 5% or greater relative to the immune cell proliferation prior to treatment; more preferably, immune cell proliferation is increased by 10% or greater; more preferably, increased by 20% or greater; more preferably, increased by 30% or greater; more preferably, increased by 40% or greater; even more preferably, increased by 50% or greater; and most preferably, increased by greater than 75% or greater. Immune cell proliferation may be measured by any reproducible means of measurement, for example by FACS, cytokine release assays, or cytotoxicity assays.

[0102] In some embodiments, the method increases T cell proliferation in a subject. Preferably, after treatment, T cell proliferation is increased by 5% or greater relative to the T cell proliferation prior to treatment; more preferably, T cell proliferation is increased by 10% or greater; more preferably, increased by 20% or greater; more preferably, increased by 30% or greater; more preferably, increased by 40% or greater; even more preferably, increased by 50% or greater; and most preferably, increased by greater than 75% or greater. T cell proliferation may be measured by any reproducible means of measurement, for example by FACS, cytokine release assays, or cytotoxicity assays.Immune Cell Engagers

[0103] In one aspect, the disclosure provides immune cell engagers.

[0104] As used herein, an “immune cell engager” refers to a molecule that can activate an immune cell by providing signal 1 to the immune cell.

[0105] In some embodiments, the immune cell engager is a T cell engager and the immune cell is a T cell. In some embodiments, the T cell is a cytolytic T cell, a CD4+ T cell or a CD8+ T cell, or a mixture thereof.

[0106] In some embodiments, the immune cell engager is a NK cell engager and the immune cell is a natural killer (NK) cell or a NKT cell.

[0107] In some embodiments, the immune cell engager (i) provides a signal 1 to the immune cell; (ii) triggers signaling by a receptor in the immune cell; (iii) bridges the immune cell to normal endogenous immune cells such that the normal endogenous immune cell activate the immune cell to proliferate; (iv) allows tuned stimulation to improve the quality and performance of the immune cell in the subject; (v) bypasses the need for antigen exposure of the immune cell in the subject’s blood; or provides a combination of (i)-(v).

[0108] In some embodiments, the immune cell engager is a T cell engager and the T cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds a T cell antigen. In some embodiments, the T cell antigen is a T cell receptor (TCR) component. In some embodiments, the T cell antigen is CD3. In some embodiments, the T cell antigen is CD3gamma, CD3 delta, CD3 epsilon or CD3zeta. In some embodiments, the T cell antigen is TCRalpha or TCRbeta. In some embodiments, the T cell antigen is CD4. In some embodiments, the T cell antigen is CD8. In some embodiments, the T cell antigen is CD5. In some embodiments, the T cell antigen is CD7.

[0109] In some embodiments, the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to TABLE I.TABLE I first ligand-binding domains of CD3 T cell engagers

[0110] In some embodiments, the first antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table II, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.TABLE II first antibody fragment VH sequences and a VL sequences of CD3 T cell engagersaccording to any of the scFvs of Table III, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.TABLE III scFv and Heavy chain or light chain amino acid sequences of exemplary CD3 T cell engagers

[0112] In some embodiments, the immune cell engager is an NK cell engager and the NK cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds an NK cell antigen. In some embodiments, the NK cell antigen is CD 16 or CD16A. In some embodiments, the NK cell antigen is NKG2D, NKp30, or NKp46.

[0113] In some embodiments, the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table IV.TABLE IV first ligand-binding domains of NK cell engagers

[0114] In some embodiments, the immune cell engager comprises a second ligand-binding domain comprising a second antibody fragment that specifically binds a B cell antigen. In some embodiments, the B cell antigen is CD 19, CD20, CD22, or BCMA.

[0115] In some embodiments, the T cell engager comprises a first ligand-binding domain that binds CD3, and a second ligand binding domain that binds CD19. In some embodiments, the T cell engager comprises a first ligand-binding domain that binds CD3, and a second ligand binding domain that binds CD20. In some embodiments, the T cell engager comprises a first ligand-binding domain that binds CD3, and a second ligand binding domain that binds CD22. In some embodiments, the T cell engager comprises a first ligand-binding domain that binds CD3, and a second ligand binding domain that binds BCMA.

[0116] In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD 16 and a second ligand binding domain that binds CD 19. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD 16 and a second ligand binding domain that binds CD20. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD 16 and a second ligand binding domain that binds CD22. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD 16 and a second ligand binding domain that binds BCMA.

[0117] In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD16A and a second ligand binding domain that binds CD 19. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD16A and a second ligand binding domain that binds CD20. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD16A and a second ligand binding domain that binds CD22. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds CD16A and a second ligand binding domain that binds BCMA.

[0118] In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp30 and a second ligand binding domain that binds CD 19. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp30 and a second ligand binding domain that binds CD20. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp30 and a second ligand binding domain that binds CD22. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp30 and a second ligand binding domain that binds BCMA.

[0119] In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp46 and a second ligand binding domain that binds CD 19. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp46 and a second ligand binding domain that binds CD20. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp46 and a second ligand binding domain that binds CD22. In some embodiments, the NK cell engager comprises a first ligand-binding domain that binds NKp46 and a second ligand binding domain that binds BCMA.

[0120] In some embodiments, the second antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table V.TABLE V second ligand-binding domain of immune cell engagers

[0121] In some embodiments, the second antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table VI, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.TABLE VI second antibody fragment VH sequences and a VL sequences of immune cell engagers

[0122] In some embodiments, the second antibody fragment comprises a polypeptide sequence according to any of the scFvs of Table VII, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.TABLE VII scFvs of immune cell engagers

[0123] In some embodiments, the immune cell engager is mosunetuzumab. As used herein, the term “mosunetuzumab” refers to an anti-CD20 / anti-CD3 bispecific antibody having the International Nonproprietary Names for Pharmaceutical Substances (INN) List 117 (WHODrug Information, Vol. 31 , No. 2, 2017, p. 303), or the CAS Registry Number 1905409-39-3. In some embodiments, the immune cell engager comprises the A chain, B chain, C chain, and E chain of SEQ ID NO 513. In some embodiments, the immune cell engager isLunsumio. Generation and characterization of Lunsumio is described in U.S. Patent No.10,174,124, the entirety of which is incorporated here by reference.A chainEVQLVQSGAE VKKPGASVKV SCKASGYTFT NYYMWVRQA PGQGLEWIGW IYPGDGNTKY NEKFKGRATL TADTSTSTAY LELSSLRSED TAVYYCARDS YSNYYFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTC VVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYGS TYRVVSVLTV LHQDWLNGKE YI<CI<VSNI<AL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKB chainDIVMTQSPDS LAVSLGERAT INCKSSQSLL NSRTRKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCTQSFIL RTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGECC chainEVQLVESGGG LVQPGGSLRL SCAASGYTFT SYNMHWVRQA PGKGLEWVGA IYPGNGDTSY NQKFKGRFTI SVDKSKNTLY LQMNSLRAED TAVYYCARVV YYSNSYWYFD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTC VVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YGSTYRVVSV LTVLHQDWLN GKEYKCKVSN K ALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKE chainDIQMTQSPSS LSASVGDRVT ITCRASSSVS YMHWYQQKPG KAPKPLIYAP SNLASGVPSR FSGSGSGTDF TLTISSLQPE DFATYYCQQW SFNPPTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC (SEQ ID NO: 513)

[0124] In some embodiments, the immune cell engager has at least 80%, 90%, 95% or 99% identity to the amino acid sequence SEQ ID NO: 513.

[0125] In some embodiments, the immune cell engager is blinatumomab. In some embodiments, the immune cell engager comprises the amino acid sequence SEQ ID NO: 514. DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNL VSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGS GGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGL EWIGQIWPGDGDTNYNGKFKGKATLT ADES S ST AYMQLS SLASEDS AVYFCARRET TTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGY TFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVD DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASG VPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 514).

[0126] In some embodiments, the immune cell engager has at least 80%, 90%, 95% or 99% identity to the amino acid sequence SEQ ID NO: 514.

[0127] In some embodiments, the immune cell engager is Elranatamab. As used herein, the term “Elranatamab” refers to an anti-BCMA / anti-CD3 bispecific antibody having the CAS Registry Number 2408850-14-4.

[0128] In some embodiments, the immune cell engager comprises the A chain, B chain, C chain, and E chain of SEQ ID NO 515.A chain (Heavy chain) anti-CD3EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMTWVRQA PGKGLEWVAF IRNRARGYTS DHNPSVKGRF TISRDNAKNS LYLQMNSLRA EDTAVYYCAR DRPSYYVLDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKCRVRCPRC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVAVSHED PEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPS SIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS DGSFFLYSRL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKB chain (Heavy chain) anti-BCMAEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMSWVRQA PGKGLEWVSA IGGSGGSLPY ADIVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARYW PMDIWGQGTL VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCEVE CPECPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVAV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TWHQDWLNG KEYKCKVSNK GLPSSIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CEVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KC chain (Light chain)DIVMTQSPDS LAVSLGERAT INCKSSQSLF NVRSRKNYLA WYQQKPGQPP KLLISWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCKQSYDL FTFGSGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGECD chain (Light chain)EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLMY DASIRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYQSWPLTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC (SEQ ID NO: 515)

[0129] In some embodiments, the immune cell engager is Epcoritamab. As used herein, the term “Epcoritamab” refers to an anti-CD20 / anti-CD3 bispecific antibody having the CAS Registry Number 2134641-34-0.Anti-CD3 heavy chainEVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKY NNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVK FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGAnti-CD3 light chainQAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGG TNI<RAPGVPARFSGSLIGDI<AALTITGAQADDESIYFCALWYSNLWVFGGGTI<LTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSAnti-CD20 heavy chainEVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTI SWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYY GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVK FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGAnti-CD20 light chainEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECExogenous Immune Cells

[0130] Provided herein are exogenous immune cells and vectors encoding genetic elements capable of generating the exogenous immune cell in vivo.

[0131] As used herein, the term “immune cell” refers to a cell involved in the innate or adaptive (acquired) immune systems. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages, and mast cells. Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.

[0132] As used herein, a “T-cell” refers to a type of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland. There are several distinct types of T- cells which develop upon migration to the thymus, which include, helper CD4+ T-cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells. Different types of T-cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.

[0133] In some embodiments, the exogenous immune cell is a T cell or an NK cell. In some embodiments, the T cell is a cytolytic T cell, optionally a CD4+ T cell or a CD8+ T cell. In some embodiments, the T cell is a TIL. In some embodiments, the T cell is an engineered TCR T cell. In some embodiments, the T cell is a CAR T cell. In some embodiments, the T cell is a Tmod cell.

[0134] As used herein, a Tmod cell refers to an engineered immune cell, such as a T lymphocyte, that co-expresses:• an activator receptor capable of recognizing and binding to a target antigen (e.g., a tumor-associated antigen), and• an inhibitory receptor capable of recognizing and binding to a distinct antigen (e.g., a self-antigen or tissue-specific antigen), wherein the activator receptor promotes immune cell activation upon antigen engagement, and the inhibitory receptor suppresses or modulates the activation signal in the presence of itscognate antigen. The Tmod cell thereby integrates activating and inhibitory signals to selectively respond to target cells that express the activating antigen but lack the inhibitory antigen, enabling discrimination between diseased and healthy cells.

[0135] The activator and inhibitory receptors may be chimeric receptors, such as chimeric antigen receptors (CARs), synthetic Notch receptors, or other engineered constructs comprising antigen-binding domains (e.g., scFvs), transmembrane domains, and intracellular signaling domains. The inhibitory receptor may include signaling domains derived from immune checkpoint proteins (e.g., LILRB1, PD-1, CTLA-4) or other immunomodulatory molecules.

[0136] In some embodiments, the CAR T cell or TCR T cell targets an antigen on a solid tumor cell. Exemplary CAR T cells and TCR T cells targeting solid tumors that are in clinical trials are listed in NCT04650451, NCT05103631, NCT04377932, NCT03696030, NCT04897321, NCT06198296, NCT04715191, NCT06096038, NCT04732845, NCT02932956, NCT02107963, NCT04995003, NCT05805371, NCT06158139, NCT04119024, NCT05634785, NCT05274451, NCT06347068, NCT04483778, NCT05489991, NCT06305299, NCT06094842, NCT03618381, NCT01822652, NCT02706392, NCT05736731, NCT05022849, NCT02744287, NCT03740256, NCT03907527, NCT04025216, NCT05680922, NCT05120271, NCT06051695, NCT02830724, NCT06236139, NCT03635632, NCT05620342, NCT03721068, NCT05539430, NCT01430390, NCT03818165, NCT05672459, NCT03585764, NCT01460901, NCT01583686, NCT04670068, NCT01218867, NCT06469281, NCT04890236, NCT05319314, NCT06241456, NCT05694364, NCT05371132, NCT00924287, NCT05639972, NCT03648372, and NCT02498912.

[0137] In some embodiments, the immune cell comprises an artificial activator receptor. In some embodiments, the artificial activator receptor is chimeric antigen receptor (CAR). In some embodiments, the activator receptor is a T cell receptor (TCR).

[0138] In some embodiments, the T cell comprises an artificial inhibitor receptor (e.g., a logic-gated T cell such as a Tmod™ cell).

[0139] In some embodiments, the immune cell comprises an artificial activator and / or inhibitor receptor described in, for example U.S. Patent Application Publication No. 2023- 0330227, U.S. Patent Application Publication No. 2024-0173352, U.S. Patent Application Publication No. 2022-0162287, U.S. Patent Application Publication No. 2022-0153807, U.S. Patent Application Publication No. 2023-0029341, U.S. Patent Application Publication No. 2023-0257441, U.S. Patent Application Publication No. 2023-0277590, U.S. PatentApplication Publication No. 2023-0277593, U.S. Patent Application Publication No. 2023- 0172983, U.S. Patent Application Publication No. 2022-0389075, U.S. Patent Application Publication No. 2022-0370498, U.S. Patent Application Publication No. 2022-0315931, U.S. Patent Application Publication No. 2024-0000938, U.S. Patent Application Publication No. 2024-0075067, U.S. Patent Application Publication No. 2024-0180969, International Patent Application Publication No. WO 2023 / 172954, International Patent Application Publication No. WO 2023 / 147019, and International Patent Application Publication No. WO 2024 / 036148 which are incorporated by reference herein.

[0140] Methods for transforming populations of immune cells, such as T cells, with the vectors of the instant disclosure in vitro or in vivo will be readily apparent to the person of ordinary skill in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi®), according to manufacturer’s instructions. T cells can be cultured at a density of 1 x 10A6 cells / mL in X-Vivo 15 media supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1 : 1 cell to bead ratio) and 300 Units / mL of IL-2 (Miltenyi®). After 2 days, T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured in IL-2 or other cytokines such as combinations of IL-7 / 15 / 21 for an additional 5 days prior to enrichment. Methods of isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to the person of ordinary skill in the art. Although this method outlines a potential approach it should be noted that these methodologies are rapidly evolving. For example excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.

[0141] Methods of activating and culturing populations of T cells comprising the engineered TCRs, CARs, Tmods, fusion proteins or vectors encoding the fusion proteins of the instant disclosure, will be readily apparent to the person of ordinary skill in the art.

[0142] Whether prior to or after genetic modification of T cells to express an engineered TCR, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No. 2006 / 0121005.

[0143] In some embodiments, T cells of the instant disclosure are expanded and activated in vitro. Generally, the T cells of the instant disclosure are expanded in vitro by contact with asurface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(l-2):53-63, 1999).

[0144] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In some embodiments, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present disclosure.

[0145] In some embodiments, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigenbinding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In some embodiments, the ratio of CD3 :CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present disclosure, more anti-CD28 antibody is bound tothe particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1.

[0146] Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. In some embodiments, a ratio of 1 : 1 cells to beads is used. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type.

[0147] In further embodiments, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0148] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact the T cells. In one embodiment the cells (for example, CD4+ T cells) and beads (for example, DYNABEADS CD3 / CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer. Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. In some embodiments, cells that are cultured at a density of IxlO6cells / mL are used.

[0149] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the beads and T cells are cultured together for 2-3 days. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFP, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2- mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X- Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. In some embodiments, the media comprises X-VIVO-15 media supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep) and 300 Units / ml of IL-2 (Miltenyi).

[0150] The T cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0151] In some embodiments, the T cells comprising engineered TCRs of the disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from a subject. Immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of T cell lines available in the art, may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation.

[0152] In some embodiments, cells from the circulating blood of an individual are obtained 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 some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art wouldreadily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0153] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 -conjugated beads, for a time period sufficient for positive selection of the desired T cells.

[0154] Enrichment of an immune cell population, such as 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 is cell sorting and / or selection via negative magnetic immune-adherence 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 CD 14, CD20, CD 11b, CD 16, HLA-DR, and CD8.

[0155] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads.

[0156] In some embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C or at room temperature.

[0157] T cells for stimulation, or PBMCs from which immune cells such as T cells are isolated, can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one methodinvolves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.Activators

[0158] The disclosure provides one or more first engineered receptor that is an artificial activator receptor that binds an activator ligand (first ligand).

[0159] The disclosure provides a first engineered receptor comprising an extracellular region, the extracellular region comprising an activator ligand binding domain capable of specifically binding a activator ligand that activates or promotes activation of the receptor, which promotes activation of effector cells expressing the receptor. The disclosure further provides a second engineered receptor comprising a second ligand binding domain capable of binding a second ligand, wherein binding of the second ligand by the second ligand binding domain inhibits or reduces activation of effector cells even in the presence of the first receptor bound to the first ligand.

[0160] As used herein, an “activator” or “activator ligand” refers to a first ligand that binds to a first, activator ligand binding domain (LBD) of an engineered receptor of the disclosure, such as a CAR or TCR, thereby mediating activation of a T cell expressing the engineered receptor. The activator is expressed by target cells, for example cancer cells, and may also be expressed more broadly than just the target cells. For example, the activator can be expressed on some, or all types of normal, non-target cells.

[0161] In some embodiments, the artificial activator receptor specifically binds an activator antigen present on a solid tumor. In some embodiments, the activator antigen is EGFR, MSLN, CEA, or HER2.

[0162] In some embodiments, the activator antigen is an antigen selected from the group consisting of: rbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, BCMA, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, humantelomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, mutated p53, prostein, PSMA, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, MAGE-A1, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFI)-I, IGF-II, IGFI receptor, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, R0R1, Nkp30, NKG2D, tumor stromal antigen, the extra domain A (EDA) of fibronectin, the extra domain B (EDB) of fibronectin, the Al domain of tenascin-C (TnC Al,) fibroblast associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility complex (MHC) molecule, an HIV-specific antigen HIV gpl20, an EBV-specific antigen, a CMV-specific antigen, an HPV-specific antigen, an HBV-specific antigen, an HCV-specific antigen, a Lassa Virus-specific antigen, an Influenza Virus-specific antigen, CD38, CA-125, MUC-1, CD44, surface adhesion molecule, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), epithelial tumor antigen, IL-13R-a2, GD2, GD3, prostate-specific antigen, melanoma-associated antigen, mutated ras, folate binding protein, HIV-1 envelope glycoprotein gp41, CD123, CD23, CD56, c-Met, HERV-K, IL-1 IRalpha, kappa chain, lambda chain, CSPG4, HER1-HER2 in combination, and HER2-HER3 in combination.

[0163] In some embodiments, binding of the activator receptor to the activator antigen on a target cell promotes a cytotoxic response by the exogenous immune cell against the target cell.

[0164] In some embodiments, the first ligand is a peptide ligand from any of the activator targets disclosed herein. In some embodiments, the first ligand is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC, or pMHC), for example an MHC complex comprising human leukocyte antigen A*02 allele (HLA-A*02).

[0165] Target cell-specific first activator ligands comprising peptide antigens complexed with pMHC comprising any of human leukocyte antigen (HLA) HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G are envisaged as within the scope of the disclosure. In some embodiments, the first ligand comprises a pMHC comprising HLA-A. HLA-A receptors are heterodimers comprising a heavy a chain and smaller p chain. The a chain is encoded by a variant of HLA-A, while the P chain (P2-microglobulin) is an invariant. There are several thousand HLA-A gene variants, all of which fall within the scope of the instant disclosure. In some embodiments, the MHC-I comprises a human leukocyte antigen A*02 allele (HLA- A*02).

[0166] In some embodiments, the first activator ligand comprises a pMHC comprising HLA-B. Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLA-B *27).

[0167] In some embodiments, the first activator ligand comprises a pMHC comprising HLA-C. HLA-C belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over one hundred HLA-C alleles are known in the art.

[0168] In some embodiments, the first activator ligand comprises a pMHC comprising HLA- A. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-B. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-C. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-E. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-F. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-G.

[0169] In some embodiments, the first activator ligand comprises HLA-A. In some embodiments, the first activator ligand comprises HLA-B. In some embodiments, the first activator ligand comprises HLA-C. In some embodiments, the first activator ligand comprises HLA-E. In some embodiments, the first activator ligand comprises HLA-F. In some embodiments, the first activator ligand comprises HLA-G. In some embodiments, the first activator ligand comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

[0170] In some embodiments, the first, activator ligand binding domain comprises an ScFv domain.

[0171] In some embodiments, the first, activator ligand binding domain comprises a VP-only ligand binding domain.

[0172] In some embodiments, the first, activator ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR). For example, the first, activator ligand binding domain comprises TCR a and P chain variable domains.

[0173] In some embodiments, the first, activator ligand and the second, inhibitor ligand are not the same.

[0174] In some embodiments, the first, activator ligand is expressed by target cells and is not expressed by non-target cells (i.e. normal cells not targeted by the adoptive cell therapy). In some embodiments, the target cells are cancer cells and the non-target cells are non- cancerous cells.

[0175] In some embodiments, the activator ligand has high cell surface expression on the target cells. This high cell surface expression confers the ability to deliver large activationsignals. Methods of measuring cell surface expression will be known to the person of ordinary skill in the art and include, but are not limited to, immunohistochemistry using an appropriate antibody against the activator ligand, followed by microscopy or fluorescence activated cell sorting (FACS).

[0176] In some embodiments, the activator ligand is encoded by a gene with an essential cellular function. Essential cellular functions are functions required for a cell to live, and include protein and lipid synthesis, cell division, replication, respiration, metabolism, ion transport, and providing structural support for tissues. Selecting activator ligands encoded by genes with essential cellular functions prevents loss of the activator ligand due to aneuploidy in cancer cells, and makes gene encoding the activator ligand less likely to undergo mutagenesis during the evolution of the cancer. In some embodiments, the activator ligand is encoded by a gene that is haploinsufficient, i.e. loss of copies of the gene encoding the activator ligand are not tolerated by the cell and lead to cell death or a disadvantageous mutant phenotype.

[0177] In some embodiments, the activator ligand is present on all target cells. In some embodiments, the target cells are cancer cells.

[0178] In some embodiments, the activator ligand is present on a plurality of target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the activator ligand is present on at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of target cells. In some embodiments, the activator ligand is present on at least 95% target cells. In some embodiments, the activator ligand is present on at least 99% target cells.

[0179] In some embodiments, the activator ligand is present on all cells (ubiquitous activator ligands). Activator ligands can be expressed on all cells, if, for example, the second inhibitor ligand is also expressed on all cells except the target cells.

[0180] In some embodiments, the first, activator ligand is expressed by a plurality of target cells and a plurality of non-target cells. In some embodiments, the plurality of non-target cells expresses both the first, activator ligand and the second inhibitor ligand.

[0181] In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 100 to about 100: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :50 to about 50: 1 of the first ligand to the second ligand. In some embodiments, andthe first, activator ligand and second, inhibitor ligand are present on the plurality of nontarget target cells at a ratio of about 1 :25 to about 25: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 10 to about 10: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :5 to about 5: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :3 to about 3 : 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :2 to about 2: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 1.

[0182] The first, activator ligand is recognized by a first ligand binding domain (sometimes referred to herein as the activator LBD).

[0183] Exemplary activator ligands include ligands selected from the group consisting of cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecule involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, receptors for neurotransmitters and voltage gated ion channels. In some embodiments, the first, activator ligand is transferrin receptor (TFRC) or a peptide antigen thereof. Human transferrin receptor is described in NCBI record No. AAA61153.1, the contents of which are incorporated herein by reference. In some embodiments, TFRC is encoded by a sequence of1 MMDQARSAFS NLFGGE PLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN NTKANVTKPK RCSGS I CYGT IAVIVFFLI G FMI GYLGYCK GVE PKTECER LAGTES PVRE E PGEDFPAAR RLYWDDLKRK LSEKLDSTDF TSTIKLLNEN SYVPREAGSQ KDENLALYVE NQFRE FKLSK VWRDQHFVKI QVKDSAQNSV I IVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK KDFEDLYTPV NGS IVIVRAG KITFAEKVAN AESLNAI GVL IYMDQTKFPI VNAELS FFGHAHLGTGDPYT PGFPS FNHTQ FPPSRSSGLP NI PVQTI SRA AAEKLFGNME GDCPSDWKTDSTCRMVTSES KNVKLTVSNV LKE IKI LNI F GVIKGFVE PD HYVVVGAQRD AWGPGAAKSGVGTALLLKLA QMFSDMVLKD GFQPSRS I I F ASWSAGDFGS VGATEWLEGY LSSLHLKAFT YINLDKAVLG TSNFKVSAS P LLYTLIEKTM QNVKHPVTGQ FLYQDSNWAS KVEKLTLDNA AFPFLAYSGI PAVS FCFCED TDYPYLGTTM DTYKELIERI PELNKVARAAAEVAGQFVIK LTHDVELNLD YERYNSQLLS FVRDLNQYRA DIKEMGLSLQ WLYSARGDFF RATSRLTTDF GNAEKTDRFV MKKLNDRVMR VEYHFLS PYV S PKES PFRHV FWGSGSHTLP ALLENLKLRK QNNGAFNETL FRNQLALATW TIQGAANALS GDVWDIDNEF (SEQ ID NO: 18).

[0184] In some embodiments, the activator ligand is a tumor specific antigen (TSA). In some embodiments, the tumor specific antigen is mesothelin (MSLN), CEA cell adhesion molecule 5 (CEACAM5, or CEA), epidermal growth factor receptor (EGFR) or a peptide antigenthereof. In some embodiments, the TSA is MSLN, CEA, EGFR, delta like canonical Notch ligand 4 (DLL4), mucin 16, cell surface associated (MUC 16 also known as CA125), ganglioside GD2 (GD2), receptor tyrosine kinase like orphan receptor 1 (R0R1), erb-b2 receptor tyrosine kinase 2 (HER2 / NEU) or a peptide antigen thereof. Exemplary mouse and humanized ScFv antigen binding domains targeting TSAs are shown in Table 1 below:

[0185] Table 1. Exemplary ScFv antigen binding domains that target tumor specific antigens (TSAs)

[0186] In some embodiments, the activator ligand is MSLN or a peptide antigen thereof, and the activator ligand binding domain comprises a MSLN binding domain. In some embodiments, the MSLN ligand binding domain comprises an ScFv domain. In some embodiments, the MSLN ligand binding domain comprises a sequence of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 or SEQ ID NO: 518. In some embodiments, the MSLN ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90 SEQ ID NO: 92 or SEQ ID NO: 518. In some embodiments, the MSLN ligand binding domain is encoded by a sequence comprising SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 or SEQ ID NO: 93. In some embodiments, the MSLN ligand binding domain is encoded by asequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 or SEQ ID NO: 93. In some embodiments, the MSLN ligand binding domain comprises a CDR-H1 of SGDYYWS (SEQ ID NO: 519), a CDR-H2 of YIYYSGSTYYNPSLKS (SEQ ID NO: 520), a CDR-H3 of CARED VVKGAFDIW(SEQ ID NO: 521), a CDR-L1 of RASQSISSYLN (SEQ ID NO: 522), a CDR-L2 of AASSLQS (SEQ ID NO: 523)and a CDR-L3 of QQSYSTPLT (SEQ ID NO: 524).

[0187] In some embodiments, the activator ligand is CEA or a peptide antigen thereof, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises an ScFv domain. In some embodiments, the CEA ligand binding domain comprises a sequence of SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284 or SEQ ID NO: 286. In some embodiments, the CEA ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284 or SEQ ID NO: 286. In some embodiments, the CEA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287. In some embodiments, the CEA ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287.

[0188] In some embodiments, the activator ligand is CEA or a peptide antigen thereof, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301), and a CDR- L3 of HQYYTYPLFT (SEQ ID NO: 302) or sequences having at least 85% or at least 95% identity thereto. In some embodiments, a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299),a CDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301) and a CDR- L3 of HQYYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 298), a CDR-L2 of SASYRYS (SEQ ID NO: 300) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ IDNO: 295), a CDR-H3 of WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302).

[0189] In some embodiments, the activator ligand is CEA or a peptide antigen thereof, and the activator receptor is a CEA CAR. In some embodiments, the CEA CAR comprises sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. In some embodiments, the CEA CAR comprises or consists essentially of SEQ ID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. In some embodiments, the CEA CAR is encoded by a sequence comprising or consisting essentially of SEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293. In some embodiments, the CEA CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to SEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293.

[0190] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR binding domain. In some embodiments, the EGFR ligand binding domain comprises an ScFv domain. In some embodiments, the EGFR ligand binding domain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain isencoded by a sequence comprising SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 or SEQ ID NO: 119. In some embodiments, the EGFR ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 or SEQ ID NO: 119.

[0191] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises a VH and / or a VL domain selected from the group disclosed in Table 2 or a sequence having at least 90% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VH domain selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 531. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128 SEQ ID NO: 130 and SEQ ID NO: 53 lor a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VL domain selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131 and SEQ ID NO: 532. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129 SEQ ID NO: 131 and SEQ ID NO: 532or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0192] Table 2. EGFR Variable Heavy (VH) and Variable Light (VL) domains

[0193] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain is an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises complementarity determining region (CDRs) selected from the group of CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises CDRs having at least 95% sequence identity to CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises CDRs selected from SEQ ID NOs: 131-166 and 533-538. In some embodiments,the EGFR ligand binding domain comprises a heavy chain CDR 1 (CDR Hl) selected from the group consisting of SEQ ID NOs: 132-137 and 533. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 2 (CDR H2) selected from the group consisting of SEQ ID NOs: 138-143 and 534. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 3 (CDR H3) selected from the group consisting of SEQ ID NOs: 144-149 and 535. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 1 (CDR LI) selected from the group consisting of SEQ ID NOs: 150-155 and 536. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 2 (CDR L2) selected from the group consisting of SEQ ID NOs: 156-160 and 537. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 3 (CDR L3) selected from the group consisting of SEQ ID NOs: 161-166 and 538. In some embodiments, the EGFR ligand binding domain comprises a CDR Hl selected from SEQ ID NOs: 132-137 and 533, a CDR H2 selected from SEQ ID NOs: 138-143 and 534, a CDR H3 selected from SEQ ID NOs: 144-149 and 535, a CDR LI selected from SEQ ID NOs: 150-155 and 536, a CDR L2 selected from SEQ ID NOs: 156-160 and 537, and a CDR L3 selected from SEQ ID NOs: 161-166 and 538.

[0194] Table 3. EGFR antigen binding domain CDRs.

[0195] In some embodiments, the activator ligand is a pan-HLA ligand, and the activator binding domain is a pan-HLA binding domain, i.e. a binding domain that binds to and recognizes an antigenic determinant shared among products of the HL A A, B and C loci. Various single variable domains known in the art or disclosed herein are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized pan-HLA scFv antibodies. An exemplary pan-HLA ligand is W6 / 32, which recognizes a conformational epitope, reacting with HLA class I alpha3 and alpha2 domains.

[0196] Table 4. pan-HLA ScFv binding domains derived from W6 / 32

[0197] In some embodiments, the activator ligand is pan-HLA ligand, and the activator ligand binding domain comprises a pan-HLA ligand binding domain. In some embodiments, the pan-HLA ligand binding domain comprises an ScFv domain. In some embodiments, the pan-HLA ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan-HLA ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan- HLA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178.

[0198] It will be appreciated by the person of ordinary skill that first, activator ligand binding domains for the first receptor may be isolated or derived from any source known in the art, including, but not limited to, art recognized T cell receptors, chimeric antigen receptors and antibody binding domains. For example, the first ligand binding domain may be derived from any of the antibodies disclosed in Table 5, and bind to a first ligand selected from the antigens described in Table 5. Accordingly, the immune cells comprising the two receptor system described can be used to treat any of the diseases or disorders described in Table 5. Selection of an appropriate first, activator receptor ligand binding domain to treat any the cancers described herein will be apparent to those of skill in the art. Exemplary antibodies, antibody sequences, and activator ligands can be found for example at www.gsrs.ncats.nih.gov / ginas / app / beta / home.Table 5. Exemplary AntibodiesInhibitors

[0199] The disclosure provides and one or more second engineered receptor that is an artificial inhibitor receptor that binds an inhibitor antigen (second ligand).

[0200] The disclosure provides a second engineered receptor comprising an extracellular region, the extracellular region comprising a second ligand binding domain capable of specifically binding to a second ligand that inhibits activation of effector cells expressing thefirst and second receptors, wherein the effector cells are activated by binding of the first ligand to the first engineered receptor.

[0201] As used herein an “inhibitor,” “inhibitor antigen” or “inhibitor ligand,” sometimes called a “blocker,” refers to a second ligand that binds to a second, ligand binding domain (inhibitor LBD) of an engineered receptor of the disclosure, but inhibits activation of an immune cell expressing the engineered receptor. The inhibitor is not expressed by the target cells. The inhibitor ligand is also expressed in a plurality of normal, non-target cells, including normal, non-target cells that express the activator ligand, thereby protecting these cells from the cytotoxic effects of the adoptive cell therapy. Without wishing to be bound by theory, inhibitor ligands can block activation of the effector cells through a variety of mechanisms. For example, binding of the inhibitor ligand to the inhibitor LBD can block transmission of a signal that occurs upon binding of the activator ligand to the activator LBD that would, in the absence of the inhibitor, lead to activation of the immune cell expressing the engineered receptors described herein.

[0202] Alternatively, or in addition, binding of the inhibitor ligand to the second engineered receptor can cause loss of cell surface expression the first, activator receptor from the surface of the immune cells comprising the two receptor system described herein. Without wishing to be bound by theory, it is thought that immune cell engagement of activator and inhibitor ligands on normal cells causes the inhibitor receptor to cause removal of nearby activator receptor molecules from the immune cell surface. This process locally desensitizes the immune cell, reversibly raising its activation threshold. Immune cells that engage only the activator ligand on a target cell cause local activation signals which are unimpeded by signals from the second, inhibitory receptor. This local activation increases until release of cytotoxic granules leads to target cell selective cell death. However, modulation of surface receptor expression levels may not be the only mechanism by which blocker receptors inhibit activation of immune cells by the first activator receptor. Without wishing to be bound by theory, other mechanisms may come into play, including, but not limited to, cross-talk between activator and blocker receptor signaling pathways.

[0203] In some embodiments, the second ligand is not expressed by the target cells, and is expressed by the non-target cells. In some embodiments, the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0204] In some embodiments, the second, inhibitor ligand binding domain comprises an ScFv domain.

[0205] In some embodiments, the second, inhibitor ligand binding domain comprises a VP- only ligand binding domain.

[0206] In some embodiments, the second, inhibitor ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR). For example, the second, inhibitor ligand binding domain comprises TCR a and P chain variable domains.

[0207] In some embodiments, the artificial inhibitor receptor specifically binds an inhibitor antigen absent from a solid tumor. In some embodiments, the inhibitor antigen is an antigen subject to loss of heterozygosity in a solid tumor. In some embodiments the inhibitor antigen is HLA A, HLA-B, HLA-C, HLA-E, HLA-G, or HLA-F. In some embodiments the inhibitor antigen is HLA-A*02, HLA-A*03, or HLA-A*11. In some embodiments, artificial inhibitor receptor binds an inhibitor antigen disclosed in Table 14.Table 14 exemplary inhibitor antigens

[0208] In some embodiments, binding of the inhibitor receptor to an inhibitor antigen on a nontarget cell protects the non-target cell from cytotoxicity mediated by the T cell.Inhibitor Targets

[0209] In some embodiments, the inhibitor ligand comprises a gene with high, homogeneous surface expression across tissues, or a peptide antigen thereof. Without wishing to be bound by theory, high, homogeneous surface expression across tissues allows the inhibitor ligand to deliver a large, even inhibitory signal. Alternatively, or in addition, expression of activatorand inhibitor targets may be correlated, i.e. the two are expressed at similar levels on nontarget cells.

[0210] In some embodiments, the second, inhibitor ligand is a peptide ligand. In some embodiments, the second, inhibitor ligand is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC, or pMHC). Inhibitor ligands comprising peptide antigens complexed with pMHC comprising any of HLA-A, HLA-B or HLA-C are envisaged as within the scope of the disclosure.

[0211] In some embodiment, the inhibitor ligand is encoded by a gene that is absent or polymorphic in many tumors.

[0212] Methods of distinguishing the differential expression of inhibitor ligands between target and non-target cells will be readily apparent to the person or ordinary skill in the art. For example, the presence or absence of inhibitor ligands in non-target and target cells can be assayed by immunohistochemistry with an antibody that binds to the inhibitor ligand, followed by microscopy or FACS, RNA expression profiling of target cells and non-target cells, or DNA sequencing of non-target and target cells to determine if the genomic locus of the inhibitor ligand comprises mutations in either the target or non-target cells.Alleles Lost Due to Loss of Heterozygosity (LOH)

[0213] Homozygous deletions in primary tumors are rare and small, and therefore unlikely to yield target B candidates. For example, in an analysis of 2218 primary tumors across 21 human cancer types, the top four candidates were cyclin dependent kinase inhibitor 2A (CDKN2A), RB transcriptional corepressor 1 (RBI), phosphatase and tensin homolog (PTEN) and N3PB2. However, CDKN2A (Pl 6) was deleted in only 5% homozygous deletion across all cancers. Homozygous HLA-A deletions were found in less than 0.2% of cancers (Cheng et al., Nature Comm. 8: 1221 (2017)). In contrast, deletion of a single copy of a gene in cancer cells due to loss of hemizygosity occurs far more frequently.

[0214] In some embodiments, the second, inhibitor ligand comprises an allele of a gene that is lost in target cells due to loss of heterozygosity. In some embodiments, the target cells comprises cancer cells. Cancer cells undergo frequent genome rearrangements, including duplication and deletions. These deletions can lead to the deletion of one copy of one or more genes in the cancer cells.

[0215] As used herein, “loss of heterozygosity (LOH)” refers to a genetic change that occurs at high frequency in cancers, whereby one of the two alleles is deleted, leaving a single mono-allelic (hemizygous) locus.HLA class I alleles

[0216] In some embodiments, the second, inhibitor ligand comprises an HLA class I allele. The major histocompatibility complex (MHC) class I is a protein complex that displays antigens to cells of the immune system, triggering immune response. The Human Leukocyte Antigens (HLAs) corresponding to MHC class I are HLA- A, HLA-B and HLA-C.

[0217] In some embodiments, the second, inhibitor ligand comprises an HLA class I allele. In some embodiments, the second, inhibitor ligand comprises an allele of HLA class I that is lost in a target cell through LOH. HLA-A is a group of human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) that are encoded by the HLA-A locus. HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer comprising a heavy a chain and smaller p chain. The a chain is encoded by a variant of HLA-A, while the P chain (P2-microglobulin) is invariant. There are several thousand HLA-A variants, all of which fall within the scope of the instant disclosure.

[0218] In some embodiments, the second, inhibitor ligand comprises an HLA-B allele.The HLA-B gene has many possible variations (alleles). Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLA-B27).

[0219] In some embodiments, the second, inhibitor ligand comprises an HLA-C allele. HLA- C belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over one hundred HLA- C alleles have been described.

[0220] In some embodiments, the HLA class I allele has broad or ubiquitous RNA expression.

[0221] In some embodiments, the HLA class I allele has a known, or generally high minor allele frequency.

[0222] In some embodiments, the HLA class I allele does not require a peptide-MHC antigen, for example when the HLA class I allele is recognized by a pan-HLA ligand binding domain.

[0223] In some embodiments, the second inhibitor ligand comprises an HLA-A allele. In some embodiments the HLA-A allele comprises HLA-A*02. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*02 are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind HLA-A*02 in a peptide-independent way shown in Table 6 below (complementarity determining regions underlined):

[0224] Table 6. HLA-A*02 ScFv binding domains

[0225] Exemplary heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for HL A- A* 02 ligand binding domains are shown in table 7 below.Table 7. CDRs corresponding to HLA-A*02 antigen binding domains

[0226] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 41-52, or 525-530. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOS: 41-52 or 525- 530. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 41-52 or 525-520. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 53-64. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 53-64.

[0227] In some embodiments, the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOS: 53-64.

[0228] In some embodiments, the ScFv comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to any one of SEQ ID NOS: 53-64.

[0229] In some embodiments, the second, inhibitory ligand is HLA-A*02, and the inhibitory ligand binding domain comprises an HLA-A*02 ligand binding domain. In some embodiments, the second ligand binding domain binds HLA-A*02 independent of the peptide in a pMHC complex comprising HLA-A*02. In some embodiments, the HLA-A*02 ligand binding domain comprises an ScFv domain. In some embodiments, the HLA-A*02 ligand binding domain comprises a sequence of any one of SEQ ID NOs: 53-64. In someembodiments, the HLA-A*02 ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to a sequence of any one of SEQ ID NOs: 53-64. In some embodiments, the HLA-A*02 ligand binding domain is encoded by a sequence comprising any one of SEQ ID NOs: 179-190. In some embodiments, the HLA-A*02 ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of any one of SEQ ID NOs: 179-190.Minor histocompatibility antigens

[0230] In some embodiments, the second, inhibitor ligand comprises a minor histocompatibility antigen (MiHA). In some embodiments, the second, inhibitor ligand comprises an allele of a MiHA that is lost in a target cell through LOH.

[0231] MiHAs are peptides derived from proteins that contain nonsynonymous differences between alleles and are displayed by common HLA alleles. The non-synonymous differences can arise from SNPs, deletions, frameshift mutations or insertions in the coding sequence of the gene encoding the MiHA. Exemplary MiHAs can be about 9-12 amino acids in length and can bind to MHC class I and MHC class II proteins. Binding of the TCR to the MHC complex displaying the MiHA can activate T cells. The genetic and immunological properties of MiHAs will be known to the person of ordinary skill in the art. Candidate MiHAs are known peptides presented by known HLA class I alleles, are known to elicit T cell responses in the clinic (for example, in graft versus host disease, or transplant rejection, and allow for patient selection by simple SNP genotyping.

[0232] In some embodiments, the MiHA has broad or ubiquitous RNA expression.

[0233] In some embodiments, the MiHA has high minor allele frequency.

[0234] In some embodiments, the MiHA comprises a peptide derived from a Y chromosome gene.

[0235] In some embodiments, the second inhibitor ligand comprises a MiHA selected from the group of MiHAs disclosed in Tables 8 and 9.

[0236] Exemplary, but non-limiting, examples of MiHAs that are envisaged as within the scope of the instant disclosure are disclosed in Table 8 below. Columns in Table 8 indicate, from left to right, the name of the MiHA, the gene which from which it is derived, MHC class I variant which can display the MiHA and the sequences of the peptide variants [A / B variants indicated in brackets).

[0237] Table 8. HLA Class I Autosomal MiHAs.

[0238] Exemplary, but non-limiting, examples of MiHAs that are envisaged as within the scope of the instant disclosure are disclosed in Table 9 below. Columns in Table 9 indicate, from left to right, the name of the MiHA, the gene which from which it is derived, MHC class I variant which can display the MiHA and the sequences of the peptide variants [A / B variants indicated in brackets).

[0239] Table 9. HLA Class I Y linked MiHAs.

[0240] In some embodiments, the MiHA comprises HA-1. HA-1 is a peptide antigen having a sequence of VL[H / R]DDLLEA (SEQ ID NO: 273), and is derived from the Rho GTPase activating protein 45 (HA-1) gene.

[0241] Exemplary ligand binding domains that selectively bind to HA-1 variant H peptide (VLHDDLLEA (SEQ ID NO: 191)) are shown in Table 10 below. TCR alpha and TCR beta sequences in SEQ ID NO: 193 are separated by a P2A self-cleaving polypeptide of sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192) with an N terminal GSG linker.

[0242] Table 10. Ftcr HA-1(H) Inhibitory Receptor Sequences

[0243] In some embodiments, the second, inhibitory ligand comprises HA-1(H). In some embodiments, the second, inhibitory ligand binding is isolated or derived from a TCR. In some embodiments, the second, inhibitory ligand binding domain comprises TCR alpha and TCR beta variable domains. In some embodiments, the TCR alpha and TCR beta variable domains are separated by a self cleaving polypeptide sequence. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self cleaving polypeptide sequence comprise SEQ ID NO: 193. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self cleaving polypeptide sequence comprise SEQ ID NO: 193, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha and TCR beta variable domains are encoded by a sequence ofSEQ ID NO: 194, or a sequence having at least 80% identity, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha variable domain comprises SEQ ID NO: 199 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR beta variable domain comprises SEQ ID NO: 200 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.Loss of Y Chromosome Antigens

[0244] In some embodiments, the second, inhibitor ligand comprises a Y chromosome gene, i.e. peptide encoded by a gene on the Y chromosome. In some embodiments, the second, inhibitor ligand comprises a peptide encoded by a Y chromosome gene that is lost in target cells through loss of Y chromosome (LoY). For example, about a third of the characterized MiHAs come from the Y chromosome. The Y chromosome contains over 200 protein coding genes, all of which are envisaged as within the scope of the instant disclosure.

[0245] As used herein, “loss of Y”, or “LoY” refers a genetic change that occurs at high frequency in tumors whereby one copy of part or all of the Y chromosome is deleted, leading to a loss of Y chromosome encoded gene(s).

[0246] Loss of Y chromosome is known to occur in certain cancers. For example, there is a reported 40% somatic loss of Y chromosome in renal clear cell cancers (Arseneault etal., Sci. Rep. 7: 44876 (2017)). Similarly, clonal loss of the Y chromosome was reported in 5 out of 31 in male breast cancer subjects(Wong et al., Oncotarget 6(42):44927-40 (2015)). Loss of the Y chromosome in tumors from male patients has been described as a “consistent feature” of head and neck cancer patients (el-Naggar et al., Am J Clin Pathol 105(1): 102-8 (1996)). Further, Y chromosome loss was associated with X chromosome disomy in four of seven male patients with gastric cancer (Saal et al., Virchows Arch B Cell Pathol (1993)). Thus, Y chromosome genes can be lost in a variety of cancers, and can be used as inhibitor ligands with the engineered receptors of the instant disclosure targeting cancer cells.Antigen Binding Domains

[0247] The disclosure provides a first ligand binding domain that activates a first engineered receptor, thereby activating immune cells expressing the first engineered receptor, and a second ligand binding domain that activates a second engineered receptor that inhibits activation of immune cells expressing the second engineered receptor, even in the presence of the first engineered receptor bound to the first ligand.

[0248] Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure. In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, ScFv, SdAb, VP-only domains, and TCR antigen binding domains derived from the TCR a and P chain variable domains.

[0249] In some embodiments, the first, activator LBD comprises an antigen binding domain. In some embodiments, the second, inhibitor LBD comprises an antigen binding domain. Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0250] For example, the first, activator LBD and / or the second, inhibitor LBD can comprise an antigen binding domain that can be expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some aspects, the first, activator LBD and / or the second, inhibitor LBD comprises an antigen binding domain that comprises an antibody fragment. In further aspects, the activator LBD comprises an antibody fragment that comprises a scFv or an sdAb. In further aspects, the inhibitor LBD comprises an antibody fragment that comprises a scFv or an sdAb.

[0251] The term “antibody,” as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0252] The terms “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0253] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising avariable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0254] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0255] Unless specified, as used herein a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0256] The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (“K”) and lambda (“ ”) light chains refer to the two major antibody light chain isotypes.

[0257] The term “recombinant antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0258] The term “VP domain”, “VP-only domain”, “P chain variable domain” or “single variable domain TCR (svd-TCR)” refers to an antigen binding domain that consists essentially of a single T Cell Receptor (TCR) beta variable domain that specifically binds to an antigen in the absence of a second TCR variable domain. In some embodiments, the first, activator LBD comprises or consists essentially of a VP-only domain. In some embodiments, the second, inhibitor LBD comprises or consists essentially of a VP-only domain.

[0259] In some embodiments, the VP-only domain may include additional elements besides the TCR variable domain, including additional amino acid sequences, additional protein domains (covalently associated, non- covalently associated or covalently and non-covalently associated with the TCR variable domain), fusion or non-covalent association of the TCR variable domain with other types of macromolecules (for example polynucleotides,polysaccharides, lipids, or a combination thereof), fusion or non-covalent association of the TCR variable domain with one or more small molecules, compounds, or ligands, or a combination thereof. Any additional element, as described, may be combined provided that the TCR variable domain is configured to specifically bind the epitope in the absence of a second TCR variable domain.

[0260] In other embodiments, the VP-only domain as described herein functions independently of an > chain that lacks a VD segment. For example, in some embodiments the one or more VP-only domains are fused to transmembrane (e.g., CD3(^ and CD28) and intracellular domain proteins (e.g., CD3(^, CD28, and / or 4-1BB) that are capable of activating T cells in response to antigen.

[0261] In some embodiments, the VP-only domain engages antigen using complementaritydetermining regions (CDRs). Each s VP-only domain contains three complement determining regions (CDR1, CDR2, and CDR3).

[0262] In some embodiments, the first VP-only domain comprises a TCR VP domain or an antigen-binding fragment thereof.

[0263] In humans, the TCR variable regions of the a and y chains are each encoded by a V and a J segment, whereas the variable region of P and 5 chains are each additionally encoded by a D segment. There are multiple Variable (V), Diversity (D) and Joining (J) gene segments (e.g. 52 VP gene segments, 2 Dp gene segments and 13 jp gene segments) (Janeway et al. (eds.), 2001, Immunobiology: The Immune System in Health and Disease. 5th Edition, New York, Figure 4.13) which can be recombined in different V(D)J arrangements using the enzymes RAG-1 and RAG-2, which recognize recombination signal sequences (RSSs) adjacent to the coding sequences of the V, D and J gene segments. The RSSs consist of conserved heptamers and nonamers separated by spacers of 12 or 23 bp. The RSSs are found at the 3 ' side of each V segment, on both the 5 ' and 3 ' sides of each D segment, and at the 5 ' of each J segment. During recombination, RAG- 1 and RAG-2 cause the formation of DNA hairpins at the coding ends of the joint (the coding joint) and removal of the RSSs and intervening sequence between them (the signal joint). The variable regions are further diversified at the junctions by deletion of a variable number of coding end nucleotides, the random addition of nucleotides by terminal deoxynucleotidyl transferase (TdT), and palindromic nucleotides that arise due to template-mediated fill-in of the asymmetrically cleaved coding hairpins.

[0264] Patent applications WO 2009 / 129247 (herein incorporated by reference in its entirety) discloses an in vitro system, referred to as the HuTarg system, which utilizes V(D)Jrecombination to generate de novo antibodies in vitro. This same system was used to generate the variable regions of the VP-only domain as in patent application WO 2017 / 091905 (herein incorporated by reference in its entirety) by using TCR-specific V, D and J elements. In natural in vivo systems, the nucleic acid sequences which encode CDR1 and CDR2 are contained within the V (a, P, y or 5) gene segment and the sequence encoding CDR3 is made up from portions of V and J segments (for Va or V y) or a portion of the V segment, the entire D segment and a portion of the J segment (for VP or V5), but with random insertions and deletions of nucleotides at the V-J and V-D-J recombination junctions due to action of TdT and other recombination and DNA repair enzymes. The recombined T- cell receptor gene comprises alternating framework (FR) and CDR sequences, as does the resulting T-cell receptor expressed therefrom (i.e. FR1 -CDR1-FR2-CDR2-FR3-CDR3- FR4). Using in vitro V(D)J recombination (i.e. V-J or V-D-J recombination), randomized insertions and deletions may be added in or adjacent to CDR1, CDR2 and / or CDR3 (i.e. not just CDR3), additional insertions may be added using flanking sequences in recombination substrates before and / or after CDR1, CDR2 and / or CDR3, and additional deletions may be made by deleting sequences in recombination substrates in or adjacent to CDR1, CDR2 and / or CDR3.

[0265] In some embodiments, TCR VP chains were identified that specifically bind epitopes in the absence of TCR Va chains. Exemplary CDR3 amino acid sequences that bind epitopes in the absence of TCR Va chains are listed in Table 11 below.

[0266] Table 11. CDR3 amino acid sequences of identified VP domains

[0267] In some embodiments, the VP-only domain specifically binds to an epitope in the absence of a second TCR variable domain, and consists of optional N-terminal and / or C- terminal amino acid sequences (of any length or sequence) flanking a variable domain defined by FR1 -CDR1 - FR2 -CDR2 -FR3 -CDR3 -FR4 regions. FR1, FR2, FR3 and FR4 may be obtained from a natural Va, VP, Vy or V5 domain or encoded by natural Va, VP, Vy or V5 gene segments, but optionally include deletions or insertions of (e.g. 0, 1, 2, 3, 4, 5 or more than 5 amino acids) amino acids independently at one or more of the C-terminus of FR1 , the N-terminus of FR2, the C-terminus of FR2, the N-terminus of FR3, the C-terminus of FR3 and the N-terminus of FR4. CDR1, CDR2 and CDR3 may be obtained from a natural Va, VP, Vy or V5 domain, or encoded by natural Va, VP, Vy or V5 gene segments, but wherein one or more of CDR1, CDR2 and CDR3 independently contains an insertion (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids) and / or a deletion (e.g. 0, 1, 2, 3, 4, 5 or more than 5 amino acids) at the C-terminus, the N- terminus or anywhere within the CDR sequence. In some embodiments, the CDR1 contains an insertion or deletion of amino acids N-terminally, C-terminally or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of natural CDR amino acid residues are retained. In some embodiments, the CDR2 contains an insertion or deletion of amino acids N-terminally, C-terminally or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of natural CDR amino acid residues are retained. In some embodiments, the CDR3 contains an insertion or deletion of amino acids N-terminally, C-terminally or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of natural CDR amino acid residues are retained. Insertions and / or deletions may be produced as a result of in vitro V(D)J recombination methods or from the in-vitro action of TdT and recombination and DNA repair enzymes (e.g. one or more of Artemis nuclease, NDA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), PAXX, and DNA polymerases X and p). Insertion and / or deletion (which includes substitution) may further result from insertions and / or deletions to CDR nucleic acid sequences of the in vitro V(D)J recombination substrates. The VP-only domain may further comprise a TCR constant regionor portion thereof. The VP-only domain may be fused to and / or complexed with additional protein domains. A double stranded break in DNA may be introduced prior to in vitro use of the above recombination and DNA repair enzymes. The VP-only domain may be (or may be incorporated into) a fusion protein. As used herein, the term "fusion protein" means a protein encoded by at least one nucleic acid coding sequence that is comprised of a fusion of two or more coding sequences from separate genes, regardless of whether the organism source of those genes is the same or different.

[0268] In some embodiments, the first, activator LBD comprises an ScFv domain and the second, inhibitor LBD comprises a VP-only domain. In some embodiments, the first, activator LBD comprises a VP-only domain and the second, inhibitor LBD comprises an ScFv domain. In some embodiments, both the first, activator LBD and the second, inhibitor LBD are ScFv domains. In some embodiments, both the first, activator LBD and the second, inhibitor LBD are VP-only domains.

[0269] Additional antigen binding domains used with the activator and / or inhibitor receptors of the disclosure are described in Table 12 below. In table 12, the name of the construct is described as ScFv Inhibitor name [B] / ScFv Activator name [A], In some embodiments, the first or second ligand binding domain comprises a sequence of any one of SEQ ID NO: 210, SEQ ID NO:212, SEQ ID NO: 214, SEQ ID NO:216, SEQ ID NO: 218, SEQ ID NO:220, SEQ ID NO: 222 Or SEQ ID NO:224, or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0270] Table 12. Additional antigen binding domain sequencesEngineered Receptors

[0271] The disclosure provides a first engineered receptor comprising a first activator ligand binding domain and a second engineered receptor comprising a second inhibitor ligand binding domain described herein.Chimeric Antigen Receptors (CARs)

[0272] In some embodiments, the either the first or the second engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the first and second engineered receptors are chimeric antigen receptors. All CAR architectures are envisaged as within the scope of the instant disclosure.Extracellular Domains

[0273] In some embodiments, the first or second ligand binding domain is fused to the extracellular domain of the CAR.Hinge Region

[0274] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl .

[0275] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 1). In some embodiments, the CD8a hinge comprises SEQ ID NO: 1. In some embodiments, the CD8a hinge consists essentially of SEQ ID NO: 1. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 2).

[0276] In some embodiments, the CD8a hinge is encoded by SEQ ID NO: 2.

[0277] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 3. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofTGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATG GAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGG ACCTTCTAAGCCC (SEQ ID NO: 4).

[0278] In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 4.Transmembrane Domain

[0279] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. 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.

[0280] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0281] In some embodiments of the CARs of the disclosure, the CARs comprise a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 5). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 5. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence havingat least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGA CAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 6).

[0282] In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 6.

[0283] In some embodiments of the CARs of the disclosure, the CARs comprise an IL- 2Rbeta transmembrane domain. In some embodiments, the IL-2Rbeta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 7). In some embodiments, the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 7. In some embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofATTCCGTGGC TCGGCCACCT CCTCGTGGGC CTCAGCGGGG CTTTTGGCTT CATCATCTTA GTGTACTTGC TGATC (SEQ ID NO: 8).

[0284] In some embodiments, the IL-2Rbeta transmembrane domain is encoded by SEQ ID NO: 8.Cytoplasmic Domain

[0285] The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the disclosure is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Effector functions of a regulatory T cell, for example, include the suppression or downregulation of induction or proliferation of effector T cells. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can becombined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0286] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) 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.Accordingly, the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In some embodiments, the at least one cytoplasmic activation is a CD247 molecule (CD3Q activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP 12) activation domain. In some embodiments, the CD3(^ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 9).

[0287] In some embodiments, the CD3(^ activation domain comprises or consists essentially of SEQ ID NO: 9. In some embodiments, the CD3(^ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 10).

[0288] In some embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 10.

[0289] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0290] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the IT AM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL) (SEQ ID NO: 21).

[0291] In some embodiments, the cytoplasmic domain contains 1, 2, or 3 ITAMs. In some embodiments, the cytoplasmic domain contains 1 IT AM. In some embodiments, the cytoplasmic domain contains 2 ITAMs. In some embodiments, the cytoplasmic domain contains 3 ITAMs. In some embodiments, the cytoplasmic domain contains 4 ITAMs. In some embodiments, the cytoplasmic domain contains 5 ITAMs.

[0292] In some embodiments, the cytoplasmic domain is a CD3(^ activation domain. In some embodiments, CD3(^ activation domain comprises a single IT AM. In some embodiments, CD3(^ activation domain comprises two ITAMs. In some embodiments, CD3(^ activation domain comprises three ITAMs.

[0293] In some embodiments, the CD3(^ activation domain comprising a single IT AM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3(^ activation domain comprises SEQ ID NO: 11. In some embodiments, the CD3(^ activation domain comprising a single IT AM consists essentially of an amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3(^ activation domain comprising a single IT AM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofAGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGCACAT GCAGGCCGTGCCCCCTCGC (SEQ ID NO: 12).

[0294] In some embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 12.

[0295] Further examples of IT AM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR^, FcRy, FcRp, CD3y, CD35, CD3s, CD3< CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the instant disclosure comprises a cytoplasmic signaling sequence derived from CD3(^.Co-Stimulatory Domain

[0296] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3(^ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3(^ chain portion and a costimulatory domain. The co-stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include the costimulatory domain is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

[0297] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0298] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 13).

[0299] In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 13. In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGG AGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCG CCGCCTACCGGAGC (SEQ ID NO: 14).

[0300] In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 14.

[0301] In some embodiments, the intracellular domain of the CARs of the instant disclosure comprises an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2Rbeta domain is truncated. In some embodiments, the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs. In some embodiments, the CAR comprises one or more STAT5-recruitment motifs outside the IL- 2Rbeta cytoplasmic domain.

[0302] In some embodiments, the IL-2-Rbeta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofNCRNTGPWLKKVLKCNTPDP SKFF SQLS SEHGGD VQKWL S SPFP S S SF SPGGLAPEIS PLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 15).

[0303] In some embodiments, the IL2R-beta intracellular domain comprises or consists essentially of SEQ ID NO: 15. In some embodiments, the IL-2R-beta intracellular domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0304] AACTGCAGGAACACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACA CCCCAGACCCCTCGAAGTTCTTTTCCCAGCTGAGCTCAGAGCATGGAGGCGACGT CCAGAAGTGGCTCTCTTCGCCCTTCCCCTCATCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGACAAGGTGACGCAGCTGC TCCCCCTGAACACTGATGCCTACTTGTCTCTCCAAGAACTCCAGGGTCAGGACCC AACTCACTTGGTG (SEQ ID NO: 16).

[0305] In some embodiments, the IL-2R-beta intracellular domain is encoded by SEQ ID NO: 16.

[0306] In an embodiment, the IL-2R-beta cytoplasmic domain comprises one or more STAT5-recruitment motifs. Exemplary STAT5-recruitment motifs are provided by Passerini et al. (2008) STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells. International Immunology, Vol. 20, No. 3, pp. 421-431, and by Kagoya et al. (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nature Medicine doi: 10.1038 / nm.4478.

[0307] In some embodiments, the STAT5-recruitment motif(s) consists of the sequence Tyr- Leu-Ser-Leu (SEQ ID NO: 17).Inhibitory domains

[0308] In some embodiments, for example in the second engineered receptors of the disclosure which provide an inhibitory signal, the inhibitory signal is transmitted through the intracellular domain of the receptor. In some embodiments, the engineered receptor comprises an inhibitory intracellular domain. In some embodiments, the second engineered receptor is a CAR comprising an inhibitory intracellular domain (an inhibitory CAR).

[0309] In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.

[0310] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0311] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof. In some embodiments, the inhibitory domain comprises an immunoreceptor tyrosinebased inhibitory motif (ITIM). In some embodiments, the inhibitory domain comprising anITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.

[0312] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In some embodiments, the TRAIL receptor comprises TR10A, TRI OB or TR10D.

[0313] Endogenous TRAIL is expressed as a 281 -amino acid type II trans-membrane protein, which is anchored to the plasma membrane and presented on the cell surface. TRAIL is expressed by natural killer cells, which, following the establishment of cell-cell contacts, can induce TRAIL-dependent apoptosis in target cells. Physiologically, the TRAIL-signaling system was shown to be essential for immune surveillance, for shaping the immune system through regulating T-helper cell 1 versus T-helper cell 2 as well as "helpless" CD8+ T-cell numbers, and for the suppression of spontaneous tumor formation.

[0314] In some embodiments, the inhibitory domain comprises an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot ref: Q8TD46) represents another example of an inhibitory intracellular domain of the present disclosure. This inhibitory receptor for the CD200 / OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS) in response to selected stimuli.

[0315] In some embodiments, the engineered receptor comprises an inhibitory domain isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor Bl (LIR1, also called LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor KI (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI K369A (kinase inactive ZAP70).

[0316] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0317] In some embodiments, the second, inhibitory receptor comprises a cytoplasmic domain and transmembrane domain isolated or derived from the same protein, for example an ITIM containing protein. In some embodiments, the second, inhibitory receptor comprises acytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITIM containing protein. In some embodiments, the second, inhibitory receptor comprises a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and / or transmembrane domain, for example an ITIM containing protein.

[0318] In some embodiments, the second, inhibitory engineered receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory engineered receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the second engineered receptor is a CAR comprising an inhibitory domain (an inhibitory CAR). In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of a CAR. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of a CAR.T Cell Receptors (TCRs)

[0319] In some embodiments, the first or second engineered receptor is a T Cell Receptor (TCR). In some embodiments, the first and second engineered receptors are a T Cell Receptors (TCR).

[0320] As used herein, a “TCR”, sometimes also called a “TCR complex” or “TCR / CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha / beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma for a heterodimer, and two CD3 zeta form a homodimer.Extracellular Domains

[0321] The disclosure provides a first engineered receptor comprising a first extracellular ligand binding domain and a second engineered receptor comprising a second extracellular ligand binding domain. Either the first engineered receptor, the second engineered receptor,or both, may be a TCR. Any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the engineered TCRs described herein.

[0322] In some embodiments, the first and / or second ligand binding domain is fused to an extracellular domain of a TCR subunit. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. In some embodiments, both the first and second ligand binding domains are fused to the same TCR subunit in different TCR receptors. In some embodiments, the first and second ligand binding domains are fused to different TCR subunits in different TCR receptors. In some embodiments, the first, activator ligand binding domain is fused to a first TCR subunit in a first engineered receptor and the second, inhibitor ligand binding domain is fused to a second TCR subunit in a second engineered receptor. In some embodiments, the first and second TCR subunits are not the same subunit. In some embodiments, the first and second TCR subunits are the same subunit. For example, the first ligand binding domain can be fused to TCR alpha, and the second ligand binding domain can be fused to TCR beta. As a further example, the first ligand binding is fused to TCR beta and the second ligand binding domain used fused to TCR alpha.

[0323] In some embodiments, the first, activator LBD comprises an ScFv domain and the second, inhibitor LBD comprises a VP-only domain. In some embodiments, the first, activator LBD comprises a VP-only domain and the second, inhibitor LBD comprises an ScFv domain. In some embodiments, both the first, activator LBD and the second, inhibitor LBD are ScFv domains. In some embodiments, both the first, activator LBD and the second, inhibitor LBD are VP-only domains.

[0324] In some embodiments, the first engineered TCR of the disclosure comprises an extracellular domain comprising a VP-only domain, a transmembrane domain and an intracellular domain. In some embodiments, the intracellular domain comprises one or more exogenous domains.

[0325] In some embodiments, the first engineered TCR of the disclosure comprises an extracellular domain comprising an ScFv domain, a transmembrane domain and an intracellular domain. In some embodiments, the intracellular domain comprises one or more exogenous domains.

[0326] In some embodiments, the second engineered TCR of the disclosure comprises an extracellular domain comprising a VP-only domain, a transmembrane domain and an inhibitory intracellular domain.

[0327] In some embodiments, the second engineered TCR of the disclosure comprises an extracellular domain comprising an ScFv domain, a transmembrane domain and an inhibitory intracellular domain.

[0328] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta or CD3 epsilon, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta or CD3 epsilon, or fragments or derivative thereof, can be fused to an inhibitory intracellular domain of the disclosure.

[0329] In some embodiments, for example those embodiments wherein the first engineered receptor or second engineered receptor comprises a first and a second polypeptide, the antigen binding domain is isolated or derived from a T cell receptor (TCR) extracellular domain or an antibody.

[0330] In some embodiments, the first engineered receptor and second engineered receptor comprise a first antigen binding domain and a second antigen binding domain. The antigen-binding domain or domains of the engineered receptor may be provided on the same or a different polypeptide as the intracellular domain.

[0331] In some embodiments, the antigen-binding domain of the first and / or second engineered receptor comprises a single chain variable fragment (scFv).

[0332] In some embodiments, the first and / or second engineered receptor comprises a second polypeptide. The disclosure provides receptors having two polypeptides each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa / p- or Fab-based LBD). The disclosure further provides receptors having two polypeptides, each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa / p- or Fab-based LBD) and one part of the ligand binding domain is fused to a hinge or transmembrane domain, while the other part of the ligand binding domain has no intracellular domain. Further variations include receptors where each polypeptide has a hinge domain, and where each polypeptide has a hinge and transmembrane domain. In some embodiments, the hinge domain is absent. In other embodiments, the hinge domain is a membrane proximal extracellular region (MPER), such as the LILRB1 D3D4 domain.

[0333] In some embodiments, for example those embodiments where the first and / or second engineered receptor comprises at least two polypeptides, the first polypeptidecomprises a first chain of an antibody and the second polypeptide comprise a second chain of said antibody.

[0334] In some embodiments, the receptor comprises a Fab fragment of an antibody. In embodiments, a first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody and an intracellular domain, and a second polypeptide comprises an antigenbinding fragment of the light chain of the antibody. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody and the intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody.

[0335] In some embodiments, the first and / or second engineered receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, a first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR and the intracellular domain, and a second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, a first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR and the intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.TCRs comprising VP-only domains

[0336] Certain embodiments of present disclosure relate to engineered TCRs comprising a TCR variable domain, the TCR variable domain specifically binding to an antigen in the absence of a second TCR variable domain (a VP-only domain).

[0337] In some embodiments, the engineered TCR comprises additional elements besides the TCR variable domain, including additional amino acid sequences, additional protein domains (covalently associated, non- covalently associated or covalently and non-covalently associated with the TCR variable domain), fusion or non-covalent association of the TCR variable domain with other types of macromolecules (for example polynucleotides, polysaccharides, lipids, or a combination thereof), fusion or non-covalent association of the TCR variable domain with one or more small molecules, compounds, or ligands, or a combination thereof. Any additional element, as described, may be combined provided that the TCR variable domain is configured to specifically bind the epitope in the absence of a second TCR variable domain.

[0338] An engineered TCR comprising a VP-only domain as described herein may comprise a single TCR chain (e.g. a, P, y, or 5 chain), or it may comprise a single TCR variable domain (e.g. of a, P, y, or 5 chain). If the engineered TCR is a single TCR chain, then the TCR chaincomprises a transmembrane domain, a constant (or C domain) and a variable (or V domain), and does not comprise a second TCR variable domain. The engineered TCR may therefore comprise or consist of a TCR a chain, a TCR P chain, a TCR y chain or a TCR 5 chain. The engineered TCR may be a membrane bound protein. The engineered TCR may alternatively be a membrane-associated protein.

[0339] In some embodiments, the engineered TCR as described herein utilizes a surrogate a chain that lacks a V segment, which forms activation-competent TCRs complexed with the six CD3 subunits.

[0340] In other embodiments, the engineered TCR as described herein functions independently of a surrogate a chain that lacks a Va segment. For example, in some embodiments the one or more engineered TCRs are fused to transmembrane e.g., CD3(^ and CD28) and intracellular domain proteins (e.g., CD3(^, CD28, and / or 4-1BB) that are capable of activating T cells in response to antigen.

[0341] In some embodiments, the engineered TCR comprises one or more single TCR chains fused to the VP-only domain described herein. For example, the engineered TCR may comprise, or consist essentially of single a TCR chain, a single P TCR chain, a single y TCR chain, or a single 5 TCR chain fused to one or more VP-only domains.

[0342] In some embodiments, the engineered TCR engages antigen using complementaritydetermining regions (CDRs). Each engineered TCR contains three complement determining regions (CDR1, CDR2, and CDR3).

[0343] The first and / or second ligand binding VP-only domain may be a human TCR variable domain. Alternatively, the first and / or second VP-only domain may be a non-human TCR variable domain. The first and / or second VP-only domain may be a mammalian TCR variable domain. The first and / or second VP-only domain may be a vertebrate TCR variable domain.

[0344] In embodiments where VP-only domain is incorporated into a fusion protein, for example a fusion protein comprising a TCR subunit, and optionally, an additional stimulatory intracellular domain. The fusion protein may comprise a VP-only domain and any other protein domain or domains.Transmembrane Domains

[0345] The disclosure provide a first fusion protein comprising a first, activator LBD and a second fusion protein comprising a second, inhibitor LBD and an inhibitor intracellulardomain. In some embodiments, the first and second fusion proteins comprise transmembrane domains.

[0346] The disclosure provides polypeptides comprising a transmembrane domain, and an intracellular domain capable of providing a stimulatory signal or an inhibitory signal. In some embodiments, the engineered TCR comprises multiple intracellular domains capable of providing a stimulatory signal.

[0347] A “transmembrane domain”, as used herein, refers to a domain of a protein that spans membrane of the cell. Transmembrane domains typically consist predominantly of non-polar amino acids, and may traverse the lipid bilayer once or several times. Transmembrane domains usually comprise alpha helices, a configuration which maximizes internal hydrogen bonding.

[0348] Transmembrane domains isolated or derived from any source are envisaged as within the scope of the fusion proteins of the disclosure.

[0349] In some embodiments, the transmembrane domain is one that is associated with one of the other domains of the fusion protein, or isolated or derived from the same protein as one of the other domains of the fusion protein. In some embodiments, the transmembrane domain and the second intracellular domain are from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. In some embodiments, the extracellular domain (svd-TCR), the transmembrane domain and the second intracellular domain are from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. In other embodiments, the extracellular domain (comprising one or more ligand binding domains, such as VP-only domain and ScFv domains), the transmembrane domain and the intracellular domain(s) are from different proteins. For example, in some embodiments the engineered svd-TCR comprises a CD28 transmembrane domain with a CD28, 4-1BB and CD3(^ intracellular domain.

[0350] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0351] In some embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target. A transmembrane domain may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0352] In some embodiments, the transmembrane domain can be attached to the extracellular region of the fusion protein, e.g., the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

[0353] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 1). In some embodiments, the CD8a hinge comprises SEQ ID NO: 1. In some embodiments, the CD8a hinge consists essentially of SEQ ID NO: 1. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of:ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 2).

[0354] In some embodiments, the CD8a hinge is encoded by SEQ ID NO: 2.

[0355] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3. In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 3. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofTGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATG GAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGG ACCTTCTAAGCCC (SEQ ID NO: 4).

[0356] In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 4.

[0357] In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:26). In some embodiments, the TCR alpha transmembrane domain comprises, or consists essentially of, SEQ ID NO: 26. In some embodiments, the TCR alpha transmembrane domain is encoded by a sequence ofGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGA CGCTGCGGCTGTGG (SEQ ID NO: 27).

[0358] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 28). In some embodiments, the TCR beta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 28. In some embodiments, the TCR beta transmembrane domain is encoded by a sequence ofACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCA GTGCCCTCGTGCTG (SEQ ID NO: 20).

[0359] In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain. In some embodiments, the CD3 zeta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 29). In some embodiments, the CD3 zeta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 29.

[0360] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).

[0361] In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains ofthe same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0362] When present, the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without limitation, a natural or artificial transmembrane domain may comprise a hydrophobic a-helix of about 20 amino acids, often with positive charges flanking the transmembrane segment. The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Prediction of transmembrane domains / segments may be made using publicly available prediction tools (e.g. TMHMM, Krogh et al. Journal of Molecular Biology 2001 ; 305(3):567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166). Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence) and the like.Intracellular Domain

[0363] The disclosure provides fusion proteins comprising an intracellular domain. An “intracellular domain,” as the term is used herein, refers to an intracellular portion of a protein.

[0364] In some embodiments, the intracellular domain comprises one or more domains capable of providing a stimulatory signal to a transmembrane domain. In some embodiments, the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain comprises a first, second and third intracellular domain capable of providing a stimulatory signal. The intracellular domains capable of providing a stimulatory signal are selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lek) domain, a TNF receptor superfamily member 9 (4- IBB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

[0365] In some embodiments, an intracellular domain comprises at least one intracellular signaling domain. An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T-cell. In some embodiments, the intracellular domain of the fusion proteins of the disclosure includes at least one intracellular signaling domain. For example, the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.

[0366] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

[0367] Examples of intracellular domains for use in the fusion proteins of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and the intracellular signaling 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 recombinant sequence that has the same functional capability.

[0368] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.

[0369] An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the fusion protein has been introduced. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function.

[0370] While in some cases the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire intracellular signaling domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0371] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain. In some embodiments, the CD3 delta intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence ofGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKGGSRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30).

[0372] In some embodiments, the CD3 delta intracellular domain comprises or consists essentially of, SEQ ID NO: 30. In some embodiments, the CD3 delta intracellular domain is encoded by a sequence of

[0373] GGACATGAGACTGGAAGGCTGTCTGGGGCTGCCGACACACAAGCTCTGTT GAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAG CCACCTTGGAGGAAACTGGGCTCGGAACAAGGGCGGAAGCAGGAGCAAGCGGA GCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCA CCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAG CTA(SEQ ID NO: 31).

[0374] In some embodiments, the intracellular domain comprises a CD3 epsilon intracellular domain. In some embodiments, the CD3 epsilon intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIG GSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 32). In some embodiments, the CD3 epsilon intracellular domain comprises or consists essentially of, SEQ ID NO: 32. In some embodiments, the CD3 epsilon intracellular domain is encoded by a sequence ofAAGAATAGAA AGGCCAAGGC CAAGCCTGTG ACACGAGGAG CGGGTGCTGG CGGCAGGCAAAGGGGACAAA ACAAGGAGAG GCCACCACCT GTTCCCAACC CAGACTATGA GCCCATCCGGAAAGGCCAGC GGGACCTGTA TTCTGGCCTG AATCAGCGCA GAATCGGCGG AAGCAGGAGCAAGCGGAGCA GACTGCTGCA CAGCGACTAC ATGAACATGA CCCCCCGGAG GCCTGGCCCCACCCGGAAGC ACTACCAGCC CTACGCCCCT CCCAGGGATT TCGCCGCCTA CCGGAGCTAG (SEQ ID NO: 19).

[0375] In some embodiments, the intracellular domain comprises a CD3 gamma intracellular domain. In some embodiments, the CD3 gamma intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence ofGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNGGSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 33).

[0376] In some embodiments, the CD3 gamma intracellular domain comprises, or consists essentially of, SEQ ID NO: 33. In some embodiments, the CD3 gamma intracellular domain is encoded by a sequence ofGGACAGGATG GAGTTCGCCA GTCGAGAGCT TCAGACAAGC AGACTCTGTT GCCCAATGAC CAGCTCTACC AGCCCCTCAA GGATCGAGAA GATGACCAGT ACAGCCACCT TCAAGGAAAC CAGTTGAGGA GGAATGGCGG AAGCAGGAGC AAGCGGAGCA GACTGCTGCA CAGCGACTAC ATGAACATGA CCCCCCGGAG GCCTGGCCCC ACCCGGAAGC ACTACCAGCC CTACGCCCCT CCCAGGGATT TCGCCGCCTA CCGGAGCTAG (SEQ ID NO: 22).

[0377] In some embodiments, the intracellular domain comprises a CD3 zeta intracellular domain. In some embodiments, the CD3 zeta intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 9) or a subsequence thereof.

[0378] In some embodiments, the CD3 zeta intracellular domain comprises, or consists essentially of, SEQ ID NO: 9.

[0379] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, a TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, a TCR alpha intracellular domain is encoded by a sequence of TCCAGC.

[0380] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or is identical to a sequence of: MAMVKRKDSR (SEQ ID NO: 35). In some embodiments, the TCR beta intracellular domain comprises, or consists essentially of SEQ ID NO: 35. In some embodiments, the TCR beta intracellular domain is encoded by a sequence of ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 36).

[0381] In some embodiments, the intracellular signaling domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma andCD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co- stimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0382] Exemplary co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation.

[0383] The term “co-stimulatory molecule” refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T-cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors. Co-stimulatory molecules and their ligands are required for an efficient immune response. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP 10, DAP12, CD30, LIGHT, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CDl la / CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecule (CD28).

[0384] A “co-stimulatory domain”, sometimes referred to as “a co-stimulatory intracellular signaling domain” can be the intracellular portion of a co-stimulatory protein. A co- stimulatory domain can be a domain of a co-stimulatory protein that transduces the co- stimulatory signal. A co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like. The co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0385] In some embodiments, the stimulatory domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a CD28 or 4-1BB co-stimulatory domain. CD28 and 4- IBB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain. Likewise, inclusion of co-stimulatory domains, for example CD28 and 4- 1BB domains, in engineered TCR can increase T cell effector function and specifically allowco-stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells.

[0386] In some embodiments, the stimulatory domain comprises a CD28 intracellular domain. In some embodiments, the CD28 intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37). In some embodiments, the CD28 intracellular domain comprises, or consists essentially of, RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37). In some embodiments, a CD28 intracellular domain is encoded by a nucleotide sequence comprising:AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGG AGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCG CCGCCTACCGGAGC (SEQ ID NO: 38).

[0387] In some embodiments, the stimulatory domain comprises a 4-1BB intracellular domain. In some embodiments, the 4- IBB intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 39). In some embodiments, the 4-1BB intracellular domain comprises, or consists essentially of, KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 39). In some embodiments, a 4-1BB intracellular domain is encoded by a nucleotide sequence comprising: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG (SEQ ID NO: 40).Inhibitory domains

[0388] The disclosure provides inhibitory intracellular domains which can be fused to the transmembrane or intracellular domain of any of the TCR subunits to generate an inhibitory TCR.

[0389] In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, theinhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.

[0390] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0391] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof. In some embodiments, the inhibitory domain comprises an immunoreceptor tyrosinebased inhibitory motif (ITIM). In some embodiments, the inhibitory domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.

[0392] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In some embodiments, the TRAIL receptor comprises TR10A, TRI 0B or TR10D.

[0393] Endogenous TRAIL is expressed as a 281 -amino acid type II trans-membrane protein, which is anchored to the plasma membrane and presented on the cell surface. TRAIL is expressed by natural killer cells, which, following the establishment of cell-cell contacts, can induce TRAIL-dependent apoptosis in target cells. Physiologically, the TRAIL-signaling system was shown to be essential for immune surveillance, for shaping the immune system through regulating T-helper cell 1 versus T-helper cell 2 as well as "helpless" CD8+ T-cell numbers, and for the suppression of spontaneous tumor formation.

[0394] In some embodiments, the inhibitory domain comprises an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot ref: Q8TD46) represents another example of an inhibitory intracellular domain of the present disclosure. This inhibitory receptor for the CD200 / OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS) in response to selected stimuli.

[0395] In some embodiments, the engineered receptor comprises an inhibitory domain isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and longcytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor Bl (LIR1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor KI (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI_K369A(kinase inactive ZAP70).

[0396] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0397] In some embodiments, the second, inhibitory receptor comprises a cytoplasmic domain and transmembrane domain isolated or derived from the same protein, for example an ITIM containing protein. In some embodiments, the second, inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITIM containing protein. In some embodiments, the second, inhibitory receptor comprises a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and / or transmembrane domain, for example an ITIM containing protein.

[0398] In some embodiments, the second engineered receptor is a TCR comprising an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory TCR comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon or a portion thereof a TCR. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

[0399] In some embodiments, the second engineered receptor is a TCR comprising an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory domain is isolated or derived from LILRB1.LILRB 1 Inhibitory receptors

[0400] The disclosure provides a second, inhibitory receptor comprising a LILRB 1 inhibitory domain, and optionally, a LILRB 1 transmembrane and / or hinge domain, or functional variants thereof. The inclusion of the LILRB 1 transmembrane domain and / or the LILRB 1 hinge domain in the inhibitory receptor may increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domains. The second, inhibitory receptorcomprising the LILRB1 inhibitory domain may be a CAR or TCR, as described herein. Any suitable ligand binding domain, as described herein, may be fused to the LILRB1 -based second, inhibitory receptors.

[0401] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as Leukocyte immunoglobulin-like receptor Bl, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2 LIR-1, MIR-7 and PIR-B, is a member of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB1 is thought to regulate inflammatory responses, as well as cytotoxicity, and to play a role in limiting auto-reactivity. Multiple transcript variants encoding different isoforms of LILRB1 exist, all of which are contemplated as within the scope of the instant disclosure.

[0402] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRBl. In some embodiments of the receptors having one or more domains isolated or derived from LILRBl, the one or more domains of LILRBl comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRBl comprise an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRBl consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRBl consist of an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 65.

[0403] In some embodiments of the receptors having one or more domains isolated or derived from LILRBl, the one or more domains of LILRBl are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 66.

[0404] In some embodiments of the receptors having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to a sequence or subsequence of SEQ ID NO: 66.

[0405] In various embodiments, an inhibitory receptor is provided, comprising a polypeptide, wherein the polypeptide comprises one or more of: an LILRB 1 hinge domain or functional fragment or variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0406] As used herein an “immunoreceptor tyrosine-based inhibitory motif’ or “ITIM” refers to a conserved sequence of amino acids with a consensus sequence of S / I / V / LxYxxI / V / L (SEQ ID NO: 274), or the like, that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol-phosphatase called SHIP.

[0407] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0408] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one ITIM selected from the group of ITIMs consisting of NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0409] In further particular embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0410] In some embodiments, the intracellular domain comprises both ITIMs NLYAAV (SEQ ID NO: 67) and VTYAEV (SEQ ID NO: 68). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 71. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 71.

[0411] In some embodiments, the intracellular domain comprises both ITIMs VTYAEV (SEQ ID NO: 68) and VTYAQL (SEQ ID NO: 69). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 72. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 72.

[0412] In some embodiments, the intracellular domain comprises both ITIMs VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 70). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 73. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 73.

[0413] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), and VTYAQL (SEQ ID NO: 69). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 74. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 74.

[0414] In some embodiments, the intracellular domain comprises the ITIMs VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 75. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 75.

[0415] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70). In embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 76. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 76.

[0416] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 81). In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 81).

[0417] LILRB1 intracellular domains or functional variants thereof of the disclosure can have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7, or at least 8 ITIMs. In some embodiments, the LILRB 1 intracellular domain or functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.I l l

[0418] In particular embodiments, the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITEM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0419] In particular embodiments, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0420] In particular embodiments, the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0421] In particular embodiments, the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0422] In particular embodiments, the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0423] In particular embodiments, the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0424] In particular embodiments, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0425] The LILRB1 protein has four immunoglobulin (Ig) like domains termed DI, D2, D3 and D4. In some embodiments, the LILRB1 hinge domain comprises an LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB 1 D3D4 domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to SEQ ID NO: 77. In some embodiments, the LILRB 1 D3D4 domain comprises or consists essentially of SEQ ID NO: 77.

[0426] In some embodiments, the polypeptide comprises the LILRB1 hinge domain or functional fragment or variant thereof. In embodiments, the LILRB 1 hinge domain or functional fragment or variant thereof comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to SEQ ID NO: 84, SEQ ID NO: 77, or SEQ ID NO: 78. In embodiments, the LILRB 1 hinge domain or functional fragment or variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 84, SEQ ID NO: 77, or SEQ ID NO: 78.

[0427] In some embodiments, the LILRB 1 hinge domain comprises a sequence identical to SEQ ID NO: 84, SEQ ID NO: 77, or SEQ ID NO: 78.

[0428] In some embodiments, the LILRB 1 hinge domain consists essentially of a sequence identical to SEQ ID NO: 84, SEQ ID NO: 77, or SEQ ID NO: 78.

[0429] In some embodiments, the transmembrane domain is a LILRB 1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% to SEQ ID NO: 85. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 85. In some embodiments, the LILRB 1 transmembrane domain comprises a sequence identical to SEQ ID NO: 85. In embodiments, the LILRB 1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 85.

[0430] In some embodiments, the transmembrane domain can be attached to the extracellular region of the second, inhibitory receptor, e.g., the antigen binding domain or ligand binding domain, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge or an LILRB 1 hinge.

[0431] In some embodiments, the second, inhibitory receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated or derived from LILR1B.Inhibitory Receptors Comprising Combinations of LILRB 1 Domains

[0432] In some embodiments, the LILRB 1-based inhibitory receptors of the disclosure comprise more than one LILRB 1 domain or functional equivalent thereof. For example, in some embodiments, the inhibitory receptor comprises an LILRB 1 transmembrane domainand intracellular domain, or an LILRB1 hinge domain, transmembrane domain and intracellular domain.

[0433] In particular embodiments, the inhibitory receptor comprises an LILRB1 hinge domain or functional fragment or variant thereof, and the LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 79. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 79. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 79.

[0434] In further embodiments, the inhibitory receptor comprises: the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70). In some embodiments, the polypeptide comprises the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIM, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0435] In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and intracellular domain. In some embodiments, the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 80. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 80. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 80. In some embodiments, the inhibitory receptor comprises the LILRB1 transmembrane domain and intracellular domain of SEQ ID NO: 80 fused to an extracellular ligand binding domain. In some embodiments, the inhibitory receptor comprises a first polypeptide comprising SEQ ID NO: 80 fused to a TCR alpha variable domain, and a second polypeptide comprising SEQ ID NO: 80 fused to a TCR beta variable domain.

[0436] In preferred embodiments, the inhibitory receptor comprises: an LILRB1 hinge domain or functional fragment or variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs),wherein each ITEM is independently selected from LYAAV (SEQ ID NO: 67), VTYAE (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70).

[0437] In some embodiments, the inhibitory receptor comprises a sequence at least 95% identical to SEQ ID NO: 82 or SEQ ID NO: 83, or at least 99% identical to SEQ ID NO: 82 or SEQ ID NO: 83, or identical to SEQ ID NO: 82 or SEQ ID NO: 83.

[0438] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 79, or at least 99% identical to SEQ ID NO: 79, or identical to SEQ ID NO: 79.

[0439] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 80, or at least 99% identical to SEQ ID NO: 80, or identical to SEQ ID NO: 80.

[0440] Table 13. Polypeptide sequences for illustrative LILRB1 -based inhibitory receptorsLinkers

[0441] In some embodiments, the engineered receptors comprise a linker linking two domains of the engineered receptor. Provided herein are linkers that, in some embodiments, can be used to link domains of the engineered receptors described herein.

[0442] The terms “linker” and “flexible polypeptide linker” as used in the context of linking protein domains, for example intracellular domains or domains within an scFv, refers to apeptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link two domains together.

[0443] Any linker may be used and many fusion protein linker formats are known. For example, the linker may be flexible or rigid. Non-limiting examples of rigid and flexible linkers are provided in Chen et al. (Adv Drug Deliv Rev. 2013; 65(10): 1357-1369).

[0444] The antigen-binding domains described herein may be linked to each other in a random or specified order.

[0445] The antigen-binding domains described herein may be linked to each other in any orientation of N to C terminus.

[0446] Optionally, a short oligo- or polypeptide linker, for example, between 2 and 40 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between the domains.

[0447] In some embodiments, the linker is a peptide of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acid residues. Non-limiting examples of amino acids found in linkers include Gly, Ser, Glu, Gin, Ala, Leu, Iso, Lys, Arg, Pro, and the like. In some embodiments, the linker is [(Gly)nlSer]n2, where nl and n2 may be any number (e.g. nl and n2 may independently be 1, 2, 4, 5, 6, 7, 8, 9, 10 or more than 10). In some embodiments, nl is 4.

[0448] In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Ser), (Gly-Gly-Gly-Ser, SEQ ID NO: 231), or (Gly-Gly-Gly-Gly-Ser, SEQ ID NO: 226) which can be repeated n times, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In some embodiments, the flexible polypeptide linkers include, but are not limited to, GGS, GGGGS (SEQ ID NO: 226), GGGGS GGGGS (SEQ ID NO: 227), GGGGS GGGGS GGGGS (SEQ ID NO: 228), GGGGS GGGGS GGGGS GG (SEQ ID NO: 229) or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230).

[0449] In some embodiments, the linkers include multiple repeats of (Gly Gly Ser), (Gly Ser) or (Gly Gly Gly Ser (SEQ ID NO: 231)). Also included within the scope of the disclosure are linkers described in WO2012 / 138475 (incorporated herein by reference).

[0450] In some embodiments, the linker sequence comprises a long linker (LL) sequence. In some embodiments, the long linker sequence comprises GGGGS (SEQ ID NO: 226), repeated four times. In some embodiments, a GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230) is used to link intracellular domains in a TCR alpha fusion protein of the disclosure.

[0451] In some embodiments, the long linker sequence comprises GGGGS (SEQ ID NO: 226), repeated three times. In some embodiments, a GGGGS GGGGS GGGGS (SEQ ID NO: 228) is used to link intracellular domains in a TCR beta fusion protein of the disclosure.

[0452] In some embodiments, the linker sequence comprises a short linker (SL) sequence. In some embodiments, the short linker sequence comprises GGGGS (SEQ ID NO: 226).

[0453] In some embodiments, a glycine-serine doublet can be used as a suitable linker.

[0454] In some embodiments, domains are fused directly to each other via peptide bonds without use of a linker.Polynucleotides

[0455] The disclosure provides polynucleotides encoding the sequence(s) of the engineered receptors described herein.

[0456] In some embodiments, the sequence of the first and / or second fusion protein is operably linked to a promoter. In some embodiments, the sequence encoding the first fusion protein is operably linked to a first promoter, and the sequence encoding a second fusion protein is operably linked to a second promoter.

[0457] The disclosure provides vectors comprising the polynucleotides described herein.

[0458] The disclosure provides vectors encoding the coding sequence or sequences of any of the engineered receptors described herein. In some embodiments, the sequence of the first and / or second fusion protein is operably linked to a promoter. In some embodiments, the sequence encoding the first fusion protein is operably linked to a first promoter, and the sequence encoding a second fusion protein is operably linked to a second promoter.

[0459] In some embodiments, the first engineered receptor is encoded by a first vector and the second engineered receptor is encoded by second vector. In some embodiments, both engineered receptors are encoded by a single vector.

[0460] In some embodiments, the first and second receptors are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and / or selfcleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A selfcleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 271). In some embodiments, theP2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192). In some embodiments, the E2A self-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 272). In some embodiments, the F2A selfcleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 539).

[0461] In some embodiments, the vector is an expression vector, i.e. for the expression of the fusion protein in a suitable cell.

[0462] Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0463] The expression of natural or synthetic nucleic acids encoding fusion proteins is typically achieved by operably linking a nucleic acid encoding the fusion protein or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0464] The polynucleotides encoding the fusion proteins can be cloned into a number of types of vectors. For example, the polynucleotides can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0465] Further, the expression vector may be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0466] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0467] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0468] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EFla). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0469] In order to assess the expression of a fusion protein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0470] Reporter genes are used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0471] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0472] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0473] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, havebecome the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0474] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0475] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.Pharmaceutical Compositions

[0476] The disclosure provides pharmaceutical compositions comprising the engager and / or the exogenous immune cell or the vector of the disclosure, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0477] Such compositions may 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; and preservatives.Kits and Articles of Manufacture

[0478] The disclosure provides kits and articles of manufacture comprising the exogenous immune cells and immune cell engagers of the described herein. In some embodiments, the kit comprises a first pharmaceutical composition comprising the engager of the disclosure and a second pharmaceutical composition comprising the immune cell or the vector. In some embodiments, the kit comprises articles such as vials, syringes, and instructions for use.

[0479] In some embodiments, the second pharmaceutical composition comprises a plurality of immune cells comprising an engineered receptor as described herein. In some embodiments, the plurality of immune cells comprises a plurality of T cells or NK cells.EXAMPLESExample 1: CD3 - CD20 bispecific engager

[0480] This example describes the determination of the effect of a CD3-CD20 bispecific engager on T cell proliferation.

[0481] Mosunetuzumab is a type of monoclonal antibody used in the treatment of certain types of cancer, particularly B-cell non-Hodgkin lymphoma. It works by binding to CD20 on the surface of B cells and CD3 on T cells, thereby bringing T cells into close proximity with B cells to induce the immune-mediated killing of the B cells. This bispecific antibody engages the body's immune system to target and destroy malignant B cells.

[0482] Briefly, Raji cells (a model for B cell lymphoma) were maintained as suggested by ATCC. RFP+ and HLA-A*02+ Raji cells were made by transducing Raji cells with HLA- A*02 lentivirus (custom lentivirus, Alstem) and / or RLuc-2A-RFP lentivirus (Biosettia) at a MOI of 5. Raji cells were sorted using a FACSMelody Cell Sorter (BD).Primary T cell transduction, expansion

[0483] T cells were transduced with a construct encoding a CEA activating receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA silencing module and were cryopreserved after 12 days in culture. After thawing in 37°C water bath, 4e6 cells were seeded for each condition and optionally supplemented with le6 Raji cells and lug / mL or O.lug / mL Mosunetuzumab. At each timepoint (every 2-3 days), cells were counted, sampled by flow cytometry, media was refreshed and Mosunetuzumab was added. Cells were stained with anti-HLA-A,B,C and anti-HLA*02-blocker antibodies and evaluated by flow cytometry to assess transgene(+) T cell fraction and Raji (RFP+) fraction.

[0484] The results show that in the presence of CD20+ Raji cells, addition of Mosunetuzumab induces proliferation of the transgene+ T cells (FIG. 5). Similarly, the cocultured Raji cells are killed in the presence of the T cells and Mosunetuzumab (FIG. 6). In the presence of Mosunetuzumab (especially the higher dose) + Raji(CD20+), Tmod drift is diminished and total Tg(+) proliferate and are stimulated.Example 2: CD3 - CD19 bispecific engager

[0485] This example describes the determination of the effect of a CD3-CD19 bispecific engager on T cell proliferation.

[0486] Blinatumomab is a bispecific T-cell engager (BiTE) antibody used for the treatment of certain types of B-cell acute lymphoblastic leukemia (ALL). It targets CD 19 on B cells and CD3 on T cells, facilitating the interaction between T cells and B cells, which leads to the destruction of the malignant B cells by the body's immune system.

[0487] Briefly, Raji cells (a model for B cell lymphoma) are maintained as suggested by ATCC. RFP+ and HL A- A* 02+ Raji cells are made by transducing Raji cells with HL A- A* 02 lentivirus (custom lentivirus, Alstem) and / or RLuc-2A-RFP lentivirus (Biosettia) at a MOI of 5. Raji cells are sorted using a FACSMelody Cell Sorter (BD).Primary T cell transduction, expansion

[0488] T cells are transduced with a construct encoding a CEA activating receptor, an HLA- A*02 inhibitory receptor, and a B2M shRNA silencing module and are cryopreserved after 12 days in culture. After thawing in 37°C water bath, 4e6 cells are seeded for each condition and optionally supplemented with le6 Raji cells and lug / mL or O.lug / mL Blinatumomab. At each timepoint (every 2-3 days), cells are counted, sampled by flow cytometry, media is refreshed and Blinatumomab is added. Cells are stained with anti-HLA-A,B,C and anti- HLA*02-blocker antibodies and evaluated by flow cytometry to assess transgene(+) T cell fraction and Raji (RFP+) fraction.

[0489] The results show that in the presence of CD19+ Raji cells, addition of Blinatumomab induces proliferation of the transgene+ T cells. Similarly, the co-cultured Raji cells are killed in the presence of the T cells and Blinatumomab.Example 3: CD3 - CD20 bispecific engager

[0490] This example describes the determination of the effect of a CD3-CD20 bispecific engager on T cell proliferation when co-cultured with primary B cells.

[0491] Primary B cells were isolated from peripheral blood mononuclear cells (PBMCs) using a Pan B cell isolation kit (Miltenyi).Primary T cell transduction and expansion

[0492] T cells from three different donors were transduced with a construct encoding a CEA, MSLN or EGFR activating receptor (SEQ ID NOs: 292, 519 and 391, respectively), an HLA- A*02 inhibitory receptor (SEQ ID NO: 57), and a B2M shRNA silencing module and werecryopreserved after 12 days in culture. Untransduced (UTD) T cells were used as a control in this experiment and were also cryopreserved after 12 days in culture. After thawing in 37°Cwater bath, 4e6 T cells were seeded for each condition with 300IU / mL IL2 and optionally supplemented with le6 primary B cells and lug / mL Mosunetuzumab. At each timepoint (every 2-3 days), cells were counted, sampled by flow cytometry, and media was refreshed with IL2. Cells were stained with anti-HLA-A,B,C, anti-HLA*02-blocker, antiCD 19, and anti-CD25 antibodies and evaluated by flow cytometry to assess Tmod(+) T cell fraction. For cell cycle analysis cells were stained with anti-HLA-A,B,C and anti-HLA*02- blocker antibodies then fixed with paraformaldehyde, stained with Hoechst then analyzed for DNA content.

[0493] The results show that Tmod(+) T cells, across different donors and constructs, kill primary B cells equivalently to untransduced (UTD) T cells regardless of B cell HLA-A*02 status with the addition of Mosunetuzumab (FIG. 7). Similarly, in the presence of primary B cells, addition of Mosunetuzumab induces rapid activation (FIG. 8), proliferation (FIG. 9) and cycling (FIG. 10) of the Tmod(+) T cells.

Claims

1. CLAIMSWhat is claimed is:

1. A method for treating a solid tumor in a subj ect in need thereof, comprising administering an immune cell engager in an amount effective to activate an exogenous immune cell, wherein the exogenous immune cell or a vector encoding genetic elements capable of generating the exogenous immune cell in vivo is administered to the subject, before, during, or after the administering of the immune cell engager.

2. The method of claim 1, wherein the exogenous immune cell is an T cell, a NK cell, or a NKT cell.

3. The method of claim 1 or claim 2, wherein the immune cell engager is a T cell engager and the immune cell is a T cell, optionally a cytolytic T cell, optionally a CD4+ T cell or a CD8+ T cell, or a mixture thereof.

4. The method of claim 1 or claim 2, wherein the immune cell engager is an NK cell engager and the immune cell is a natural killer (NK) cell or a NKT cell.

5. The method of any one of claims 1 to 4, wherein: the immune cell engager provides a signal 1 to the immune cell; the immune cell engager triggers signaling by a-receptor in the immune cell; the immune cell engager bridges the immune cell to normal endogenous immune cells such that the normal endogenous immune cells activate the immune cell to proliferate; the immune cell engager allows tuned stimulation to improve the quality and performance of the immune cell in the subject; and / or the immune cell engager bypasses the need for antigen exposure of the immune cell in the subject’s blood.

6. The method of any preceding claim, wherein the method comprises administering the exogenous immune cell or the vector in a first pharmaceutical composition apart from a second pharmaceutical composition comprising the immune cell engager.

7. The method of any one of claims 1 to 5, wherein the method comprises administering the exogenous immune cell or the vector and the immune cell engager in a single pharmaceutical composition.

8. The method of any preceding claim, wherein the exogenous immune cell comprises an artificial activator receptor.

9. The method of claim 8, wherein the activator receptor is chimeric antigen receptor (CAR).

10. The method of claim 8, wherein the activator receptor is a T cell receptor (TCR).

11. The method of any preceding claim, wherein the exogenous immune cell comprises an artificial inhibitor receptor (e.g., a logic-gated T cell such as a Tmod™ cell).

12. The method of any preceding claim, wherein the immune cell engager is a T cell engager and the T cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds a T cell antigen, optionally wherein the T cell antigen is CD3.

13. The method of claim 12, wherein the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table I.

14. The method of claim 12, wherein the first antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table II, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

15. The method of claim 12, wherein the first antibody fragment comprises a polypeptide sequence according to any of the scFvs of Table III, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

16. The method of any one of claims 1-10, wherein the immune cell engager is an NK cell engager and the NK cell engager comprises a first ligand-binding domain comprising a first antibody fragment that specifically binds an NK cell antigen.

17. The method of claim 16, wherein the first antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table IV.

18. The method of any preceding claim, wherein the immune cell engager comprises a second ligand-binding domain comprising a second antibody fragment that specifically binds a B cell antigen, optionally wherein the B cell antigen is CD 19, CD20, CD22, or BCMA.

19. The method of claim 18, wherein the second antibody fragment comprises a set of complementarity determining regions (CDRs) according to Table V.

20. The method of claim 18, wherein the second antibody fragment comprises a VH sequence and a VL sequence according to any of the VH / VL pairs Table VI, or polypeptide sequences at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

21. The method of claim 18, wherein the second antibody fragment comprises a polypeptide sequence according to any of the scFvs of Table VII, or a polypeptide sequence at least 80%, at least 90%, at least 95%, or at least 98% identical thereto.

22. The method of any one of claims 8-21, wherein the artificial activator receptor specifically binds an activator antigen present on a solid tumor, optionally wherein the activator antigen is EGFR, MSLN, CEA, or HER2.

23. The method of claim 22, wherein binding of the activator receptor to the activator antigen on a target cell promotes a cytotoxic response by the exogenous immune cell against the target cell.

24. The method of any one of claims 11-23, wherein the artificial inhibitor receptor specifically binds an inhibitor antigen absent from a solid tumor, optionally wherein the inhibitor antigen is an antigen subject to loss of heterozygosity in a solid tumor,optionally wherein the inhibitor antigen is HLA-A*02, HLA-A*03, or HLA-A*11, optionally according to any one of the inhibitor antigens disclosed in Table 14.

25. The method of claim 24, wherein binding of the inhibitor receptor to an inhibitor antigen on a non-target cell protects the non-target cell from cytotoxicity mediated by the T cell.

26. The method of any one of claims 1-25, wherein the immune cell engager is mosunetuzumab.

27. The method of any one of claims 1-25, wherein the immune cell engager is blinatumomab.

28. A method according to any one of claims 1 - 27 wherein the exogenous T cell is generated in vivo by administering a vector encoding the artificial receptor or artificial receptors of the T cell, and optionally other genetic elements, wherein the vector transduces endogenous T cells thereby generating the exogenous T cell in vivo.

29. A pharmaceutical composition comprising the immune cell engager and the immune cell or the vector of any one of claims 1-5 or 7-22.

30. A kit comprising a first pharmaceutical composition comprising the immune cell engager of any preceding claim and a second pharmaceutical composition comprising the immune cell or the vector of any one of claims 1-6 and 8-22.

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