WO2020028572A2

WO2020028572A2 - ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF

Info

Publication number: WO2020028572A2
Application number: PCT/US2019/044512
Authority: WO
Inventors: Kathleen Mcginness; Brant HERRIN; Charles Wilson
Original assignee: Unum Therapeutics Inc.
Priority date: 2018-08-01
Filing date: 2019-07-31
Publication date: 2020-02-06
Also published as: WO2020028572A3

Abstract

Disclosed herein are genetically engineered hematopoietic cells (e.g., genetically engineered hematopoietic stem cells, or genetically engineered immune cells), which co¬ express one or more co- stimulatory polypeptides with an antibody-coupled T cell receptor (ACTR), and uses thereof for enhancing antibody-dependent cell cytotoxicity (ADCC) in a subject in need of the treatment.

Description

ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of the filing dates of U.S. provisional patent application serial number 62/713,162, entitled“ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF”, filed August 1, 2018 and U.S. provisional patent application serial number 62/756,726, entitled“ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF”, filed November 7, 2018. The entire contents of each of the prior applications are incorporated by reference herein. BACKGROUND OF DISCLOSURE

Cancer immunotherapy, including cell-based therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues. It is a promising option for treating various types of cancer because of its potential to evade genetic and cellular mechanisms of drug resistance, and to target tumor cells while sparing normal tissues.

Cell-based therapy may involve cytotoxic T cells having reactivity skewed toward cancer cells. Eshhar et al., Proc. Natl. Acad. Sci. U. S. A.; 1993; 90(2):720-724; Geiger et al., J Immunol.1999; 162(10):5931-5939; Brentjens et al., Nat. Med.2003; 9(3):279-286; Cooper et al., Blood.2003; 101(4):1637-1644; and Imai et al., Leukemia.2004; 18:676-684. One approach is to express a chimeric receptor having an antigen-binding domain fused to one or more T cell activation signaling domains. Binding of a cancer antigen via the antigen- binding domain results in T cell activation and triggers cytotoxicity. Efficacy of chimeric receptor-expressing autologous T lymphocytes in treating B-cell precursor acute

lymphoblastic leukemia (ALL) has been demonstrated in clinical trials. Pule et al., Nat. Med. 2008;14(11):1264-1270; Porter et al., N Engl J Med; 2011; 25;365(8):725-733; Brentjens et al., Blood.2011;118(18):4817-4828; Till et al., Blood.2012;119(17):3940-3950;

Kochenderfer et al., Blood.2012;119(12):2709-2720; and Brentjens et al., Sci Transl Med. 2013;5(177):177ra138.

Another approach is to express an antibody-coupled T cell Receptor (ACTR) protein in hematopoietic cell (e.g., a hematopoietic stem cell, an immune cell, such as an NK cell or a T cell), the ACTR protein containing an extracellular Fc-binding domain. When the ACTR- expressing hematopoietic cells (e.g., ACTR-expressing T cells, also called“ACTR T cells”) are administered to a subject together with an anti-cancer antibody, they may enhance toxicity against cancer cells targeted by the antibody via their binding to the Fc domain of the antibody. Kudo et al., Cancer Research (2014) 74:93-103.

It is of great interest to develop new strategies to enhance efficacy of cell-based immune therapies. SUMMARY OF DISCLOSURE

The present disclosure is based on the development of strategies to co-express a co-stimulatory peptide and an antibody-coupled T-cell receptor (ACTR) polypeptide for use in cell-based immune therapy (i.e., expressing two separate polypeptides). Modulation of costimulatory pathways may be achieved by expressing (e.g., over-expressing) in hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as T cells or natural killer cells) one or more co-stimulatory polypeptides such as those described herein. In some instances, immune cells that co-express one or more co-stimulatory polypeptides and an ACTR polypeptide would be expected to exhibit superior bioactivities in the presence of a therapeutic antibody, for example, cell proliferation, activation (e.g., increased cytokine production, e.g., IL-2 or IFN-g production), cytotoxicity, and/or in vivo anti-tumor activity.

Accordingly, provided herein are modified (e.g., genetically modified)

hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as T cells or natural killer cells) that have the capacity for modulation of costimulatory pathways relative to the wild-type immune cells of the same type. In some instances, the modified immune cells may express or overly express a co-stimulatory polypeptide. The co- stimulatory polypeptide may be a member of the B7/CD28 superfamily, a member of the tumor necrosis factor (TNF) superfamily or a ligand thereof. Exemplary members of the B7/CD28 superfamily or ligands thereof include, but are not limited to, CD28, CD80, CD86, ICOS, ICOSL, B7-H3, B7-H4, VISTA, TMIGD2, B7-H6, B7-H7, and variants thereof. Exemplary members of the TNF superfamily or ligands thereof include, but are not limited to, 4-1BB, 4-1BBL, BAFF, BAFFR, CD27, CD70, CD30, CD30L, CD40, CD40L, DR3, GITR, GITRL, HVEM, LIGHT, TNF-beta, OX40, OX40L, RELT, TACI, TL1A, TNF-alpha, and TNFRII. Additional examples include BCMA, EDAR2, TROY, LTBR, EDAR, NGFR, OPG, RANK, DCR3, TNFR1, FN14 (TweakR), APRIL, EDA-A2, TWEAK, LTb (TNF-C), NGF, EDA-A1, amyloid precursor protein (APP), TRAIL.

In some embodiments, the member of the B7/CD28 superfamily, member of the tumor necrosis factor (TNF) superfamily, or ligand thereof is a wild type sequence. In some embodiments, the member of the B7/CD28 superfamily, member of the tumor necrosis factor (TNF) superfamily, or ligand thereof is a variant sequence (i.e., comprising one or more insertions, deletions, or mutations in comparison with a wild type sequence). For example, the 4-1BBL may be 4-1BBL Q89A, 4-1BBL L115A, 4-1BBL K127A, or 4- 1BBL Q227A. In some embodiments, the member of the B7/CD28 superfamily, member of the tumor necrosis factor (TNF) superfamily, or ligand thereof may lack a cytoplasmic domain. In an exemplary embodiment, the 4-1BBL lacks a cytoplasmic domain. In some embodiments, the member of the TNF superfamily or ligand thereof is not 4-1BBL.

In some embodiments, the co-stimulatory polypeptide co-expressed with any of the ACTR polypeptides described herein is free of any F506 binding protein (FKBP) such as FKBPv36. In some examples, the co-stimulatory polypeptide is free of a signaling domain derived from MyD88.

In some embodiments, the co-stimulatory polypeptide is 4-1BBL. In some embodiments, the 4-1BBL is 4-1BBL Q89A, 4-1BBL L115A, 4-1BBL K127A, or 4- 1BBL Q227A. In some embodiments, the 4-1BBL lacks a cytoplasmic domain. In some embodiments, the co-stimulatory polypeptide is ICOSL, BAFFR, LIGHT, CD30L, or CD27.

The modified hematopoietic cells (e.g., modified hematopoietic stem cells, or modified immune cells) may further express an ACTR polypeptide, which may comprise (a) an extracellular Fc binding domain; (b) a transmembrane domain; and (c) a

cytoplasmic signaling domain (e.g., a cytoplasmic domain that comprises an

immunoreceptor tyrosine-based activation motif (ITAM)). In some examples, (c) is located at the C-terminus of the ACTR polypeptide. In some instances, the ACTR polypeptide may further comprise at least one co-stimulatory signaling domain. In other instances, the ACTR polypeptide may be free of co-stimulatory signaling domains.

Any of the ACTR polypeptides described herein may further comprise a hinge domain, which is located at the C-terminus of (a) and the N-terminus of (b). In other examples, the ACTR polypeptide may be free of any hinge domain or free of a hinge domain from any non-CD16A receptor. Alternatively or in addition, the ACTR polypeptide further comprises a signal peptide at its N-terminus. In some embodiments, the ACTR polypeptide comprises SEQ ID NO: 57 or SEQ ID NO: 58.

In some examples, the Fc binding domain of (a) can be an extracellular ligand-binding domain of an Fc-receptor, for example, an extracellular ligand-binding domain of an Fc- gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In particular examples, the Fc binding domain is an extracellular ligand-binding domain of CD16A (e.g., F158 CD16A or V158 CD16A), CD32A, or CD64A.

In some examples, the Fc binding domain of (a) can be an antibody fragment that binds the Fc portion of an immunoglobulin. For example, the antibody fragment can be a single chain variable fragment (ScFv), a domain antibody, or a single domain antibody.

In some examples, the Fc binding domain of (a) can be a naturally-occurring protein that binds the Fc portion of an immunoglobulin or an Fc-binding fragment thereof. For example, the Fc binding domain can be Protein A or Protein G, or an Fc-binding fragment thereof.

Further, the Fc binding domain of (a) can be a synthetic polypeptide that binds the Fc portion of an immunoglobulin. Examples include, but are not limited to, a Kunitz peptide, a SMIP, an avimer, an affibody, a DARPin, or an anticalin.

In some embodiments, the transmembrane domain of (b) in any of the ACTR polypeptide can be of a single-pass membrane protein, e.g., CD8a, CD8b, 4-1BB, CD28, CD34, CD4, FceRIg, CD16A, OX40, CD3z, CD3e, CD3g, CD3d, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B. Alternatively, the transmembrane domain of (b) can be a non- naturally occurring hydrophobic protein segment.

In some embodiments, the at least one co-stimulatory signaling domain of the ACTR polypeptides described herein, if applicable, can be of a co-stimulatory molecule, which can be 4-1BB, CD28, CD28_LL^GG variant, OX40, ICOS, CD27, GITR, ICOS, HVEM, TIM1, LFA1, and CD2. In some examples, the at least one co-stimulatory signaling domains is a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain. In some instances, the ACTR polypeptide may comprise two co-stimulatory signaling domains. In some instances, one of the co-stimulatory signaling domains is a CD28 co-stimulatory signaling domain; and the other co-stimulatory domain can be a 4-1BB co-stimulatory signaling domain, an OX40 co-stimulatory signaling domain, a CD27 co-stimulatory signaling domain, or an ICOS co-stimulatory signaling domain. Specific examples include, but are not limited to, CD28 and 4-1BB; or CD28_LL^GG variant and 4-1BB. In some embodiments, the cytoplasmic signaling domain of (c) in any of the ACTR polypeptides described herein can be a cytoplasmic domain of CD3z or FceR1g.

In some embodiments, the hinge domain of any of the ACTR polypeptides described herein, when applicable, can be of CD28, CD16A, CD8a, or IgG. In other examples, the hinge domain is a non-naturally occurring peptide. For example, the non-naturally occurring peptide may be an extended recombinant polypeptide (XTEN) or a (Gly₄Ser)_n polypeptide, in which n is an integer of 3-12, inclusive. In some examples, the hinge domain is a short segment, which may contain up to 60 amino acid residues.

In specific examples, the ACTR polypeptide comprises (i) a CD28 co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. For example, the ACTR polypeptide comprises components (a)-(e) as shown in Table 3. In particular examples, the ACTR polypeptide comprises the amino acid sequence selected from SEQ ID NOs: 1-80.

The hematopoietic cells described herein, expressing the co-stimulatory polypeptide and the ACTR polypeptide, may be a hematopoietic stem cell or a progeny thereof. In some embodiments, the hematopoietic cells can be immune cells such as natural killer cell, monocyte/macrophage, neutrophil, eosinophil, or T cell. The immune cells can be derived from peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs). In some examples, the immune cell is a T cell, in which the expression of an endogenous T cell receptor, an endogenous major

histocompatibility complex, an endogenous beta-2-microglobulin, or a combination thereof has been inhibited or eliminated.

Any of the hematopoietic cells (e.g., HSCs or immune cells) described herein may comprise a nucleic acid or a nucleic acid set, which collectively comprises: (a) a first nucleotide sequence encoding the co-stimulatory polypeptide; and (b) a second nucleotide sequence encoding the antibody-coupled T cell receptor (ACTR) polypeptide. The nucleic acid or the nucleic acid set is an RNA molecule or a set of RNA molecules. In some instances, the immune cell comprises the nucleic acid, which comprises both the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the coding sequence of the co-stimulatory polypeptide is upstream of that of the ACTR polypeptide. In some embodiments, the coding sequence of the ACTR polypeptide is upstream of that of the co-stimulatory polypeptide. Such a nucleic acid may further comprise a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosomal skipping site (e.g., a P2A peptide), an internal ribosome entry site (IRES), or a second promoter.

In some examples, the nucleic acid or the nucleic acid set is comprised within a vector or a set of vectors, which can be an expression vector or a set of expression vectors (e.g., viral vectors such as lentiviral vectors or gammaretroviral vectors). A nucleic acid set or a vector set refers to a group of two or more nucleic acid molecules or two or more vectors, each encoding one of the polypeptides of interest (i.e., the co-stimulatory polypeptide and the ACTR polypeptide). Any of the nucleic acids described herein is also within the scope of the present disclosure.

In another aspect, the present disclosure provides a pharmaceutical composition, comprising any of the immune cells described herein, a pharmaceutically acceptable carrier, and optionally an Fc-containing therapeutic agent, which may be a therapeutic antibody or an Fc fusion protein. The Fc-containing therapeutic agent may bind to a target antigen, which can be a tumor antigen or a pathogenic antigen. The Fc-containing therapeutic agent may bind to an immune cell specific to an autoantigen. In some embodiments, the tumor antigen is associated with a hematologic tumor, and optionally wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, Kappa-chain, CD30, CD123, CD33, LeY, CD138, CD5, BCMA, CD7, CD40, and IL-1RAP. In some embodiments, the tumor antigen is associated with a solid tumor, and optionally wherein the tumor antigen is selected from the group consisting of GD2, GPC3, FOLR, HER2, EphA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, Mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD70, MUC16, L1-CAM, and CAIX. The pathogenic antigen can be a bacterial antigen, a viral antigen, or a fungal antigen.

In some examples, the Fc-containing therapeutic agent can be a therapeutic antibody, including, but not limited to, Adalimumab, Ado-Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab, Brentuximab, Canakinumab, Cetuximab,

Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab,

Epratuzumab, Gemtuzumab, Golimumab, hu14.18K322A, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Trastuzumab, Tositumomab, Ustekinumab, and Vedolizumab.

Further, the present disclosure provides a kit, comprising (i) a first pharmaceutical composition that comprises any of the immune cells described herein, which express both the co-stimulatory polypeptide and the ACTR polypeptide and a pharmaceutically acceptable carrier; and (ii) an Fc-containing therapeutic agent as described herein and a pharmaceutically acceptable carrier.

Moreover, provided herein is a method for inhibiting cells expressing a target antigen (e.g., reducing the number of such cells, blocking cell proliferation, and/or suppressing cell activity) in a subject, the method comprising administering to a subject in need thereof a population of the immune cells described herein, which may co-express the co-stimulatory polypeptide and the ACTR polypeptide. The subject (e.g., a human patient such as a human patient suffering from a cancer) may have been treated or is being treating with an Fc- containing therapeutic agent specific to the target antigen, e.g., a tumor antigen or a pathogenic antigen (for example, a bacterial antigen, a viral antigen, or a fungal antigen).

In some examples, the immune cells are autologous. In other examples, the immune cells are allogeneic. In any of the methods described herein, the immune cells can be activated, expanded, or both ex vivo. In some instances, the immune cells comprise T cells, which are activated in the presence of one or more of anti-CD3 antibody, anti-CD28 antibody, IL-2, phytohemagglutinin, and an engineered artificial stimulatory cell or particle. In other instances, the immune cells comprise natural killer cells, which are activated in the presence of one or more of 4-1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, K562 cells, and an engineered artificial stimulatory cell or particle.

In some examples, the subject to be treated by the methods described herein may be a human patient suffering from a cancer, for example, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Additional exemplary target cancer includes, but are not limited to, a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary cancers of B-cell origin are selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin’s lymphoma.

Also within the scope of the present disclosure are co-uses of (a) the immune cells described herein, which co-express a co-stimulatory polypeptide and an ACTR

polypeptide, and (b) an Fc-containing therapeutic agent such as a therapeutic antibody for treating a target disease or disorder such as cancer or an infectious disorder, and uses of (a) and/or (b) for manufacturing a medicament for the intended medical treatment.

Also within the scope of the present disclosure are methods for generating modified immune cells in vivo. In some embodiments, methods comprise administering to a subject in need thereof the nucleic acid or nucleic acid set described herein.

In some embodiments, methods further comprise administering to the subject an Fc- containing therapeutic agent specific to the target antigen.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the detailed description of several embodiments and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 is a set of graphs depicting the level of IL-2 production in T cells expressing an ACTR polypeptide alone or in combination with 4-1BBL. Panel A is a graph depicting IL-2 production from T cells expressing ACTR 1 (SEQ ID NO: 58) alone or ACTR 1 in combination with 4-1BBL (SEQ ID NO: 92), CD70 (SEQ ID NO: 103), or LIGHT (SEQ ID NO: 112) after incubation with FOLRa-expressing OAW42 cells and an anti-FOLRa antibody for 48 hours. Panel B is a graph depicting IL-2 production from T cells expressing ACTR 2 (SEQ ID NO: 57) alone or ACTR 2 in combination with 4-1BBL (SEQ ID NO: 92) after incubation with FOLRa-expressing IGROV-1 cells and an anti- FOLRa antibody for 40 hours.

Figure 2 is a set of graphs depicting the level of IL-2 production in T cells expression an ACTR polypeptide alone or in combination with a 4-1BBL variant. Panel A is a graph depicting IL-2 production as a function of antibody concentration from T cells expressing ACTR 1 in combination with 4-1BBL (SEQ ID NO: 92) or ACTR 1 in combination with 4-1BBL lacking the cytoplasmic domain (4-1BBL-CD; SEQ ID NO: 93) after incubation with FOLRa-expressing OAW42 cells and an anti-FOLRa antibody for 48 hours. Panel B is a graph depicting relative proliferation as a function of T cell variant with T cells expressing ACTR 1 in combination with 4-1BBL (SEQ ID NO: 92) or ACTR 1 in combination with an 4-1BBL polypeptide lacking the cytoplasmic domain (4-1BBL- CD; SEQ ID NO: 93) after incubation with FOLRa-expressing fixed OVCAR8 cells (left) or IGROV-1 cells (right) and an anti- FOLRa antibody for 40-48 hours.

Figure 3 is a graph depicting T cell proliferation (CD3+ cell count) as a function of antibody concentration from T cells expressing ACTR 2 alone or in combination with BAFFR (SEQ ID NO: 101) after incubation with FOLRa-expressing IGROV-1 cells and an anti-FOLRa antibody for 8 days.

Figure 4 is a graph depicting T cell proliferation (CD3+ cell count) from T cells expressing ACTR 2 alone or ACTR in combination with CD40 (SEQ ID NO: 106), OX40 (SEQ ID NO: 115), ICOS (SEQ ID NO: 84), or 4-1BB (SEQ ID NO: 91) after incubation with FOLRa-expressing IGROV-1 cells and an anti- FOLRa antibody for 8 days.

Figure 5 is a graph depicting IL-2 production from T cells co-expressing ACTR 2 (SEQ ID NO: 57) alone or ACTR 2 in combination with ICOSL (SEQ ID NO: 85), CD30L (SEQ ID NO: 105), BAFFR (SEQ ID NO: 101), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115) after incubation with FOLRa-expressing IGROV-1 cells and an anti- FOLRa antibody for 40-48 hours.

Figure 6 is a panel of graphs demonstrating proliferation of T cells expressing ACTR (SEQ ID NO: 57) or ACTR in combination with 4-1BB (SEQ ID NO: 91) in the presence of varying concentrations of solid-tumor-relevant inhibitory molecules PGE₂ (panel A), TGF- beta (panel B), adenosine (panel C), or kynurenine (panel D).

Figure 7 is a panel of graphs demonstrating proliferation of T cells expressing ACTR (SEQ ID NO: 57) or ACTR in combination with 4-1BB (SEQ ID NO: 91), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115) under chronic stimulation conditions with fixed IGROV-1 cells in the absence (panel A) and presence (panel B) of solid-tumor-relevant inhibitory molecules PGE₂ and TGF-beta.

Figure 8 is a panel of graphs showing the effect of regulatory T cells on IFN-gamma production from T cells expressing ACTR alone (parent; SEQ ID NO: 57) in the presence of different ratios of ACTR T cells and regulatory T cells (panel A), and from T cells expressing ACTR alone or co-expressing ACTR and 4-1BB (SEQ ID NO: 91), 4-1BBL (SEQ ID NO: 92), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115) (panel B).

Figure 9 is a graph showing the effect of myeloid-derived suppressor cells on proliferation of T cells expressing ACTR alone (parent; SEQ ID NO: 57) and ACTR co- expressed with 4-1BBL (SEQ ID NO: 92), CD27 (SEQ ID NO: 102), OX40 (SEQ ID NO: 115), or CD40 (SEQ ID NO: 106). DETAILED DESCRIPTION OF DISCLOSURE

Antibody-based immunotherapies are used to treat a wide variety of diseases, including many types of cancer. Such therapies often depend on recognition of cell surface molecules that are differentially expressed on cells for which elimination is desired (e.g., target cells such as cancer cells) relative to normal cells (e.g., non-cancer cells) (Weiner et al. Cell (2012) 148(6): 1081-1084). Several antibody-based immunotherapies have been shown in vitro to facilitate antibody-dependent cell-mediated cytotoxicity of target cells (e.g. cancer cells), and for some it is generally considered that this is the mechanism of action in vivo, as well. ADCC is a cell-mediated innate immune mechanism whereby an effector cell of the immune system, such as natural killer (NK) cells, T cells, monocyte cells, macrophages, or eosinophils, actively lyses target cells (e.g., cancer cells) recognized by specific antibodies.

The present disclosure is based, at least in part, on the development of strategies for enhancing activities of effector immune cells that co-express an ACTR construct. In particular, the present disclosure features methods for imparting the capacity to modulate suitable co-stimulatory pathways by the effector immune cells, thereby enhancing their growth and bioactivity. It has been shown herein that co-expression of an ACTR construct and a co-stimulatory molecule in T cells enhanced T cell proliferation and/or activity, particularly in the presence of solid-tumor relevant inhibitory molecules. The

immunosuppressive features within solid tumors may limit the success of engineered T cell therapies. The approach disclosed herein, involving the co-expression of an ACTR construct and a co-stimulatory polypeptide (which provides a co-stimulation signal in trans), aims at, at least in part, overcoming this key challenge in tumor treatment, particularly solid tumor treatment.

In some instances, the capacity of the effector immune cells to modulate co- stimulatory pathways may be observed in normal cellular environments. In other instances, the capacity of the effector immune cells to modulate co-stimulatory pathways may be observed under conditions that may be found in a tumor microenvironment. The present disclosure provides various approaches to modulate (e.g., to stimulate) co- stimulatory pathways including by, e.g., expressing or overexpressing co-stimulatory polypeptides. The co-stimulatory polypeptides for use in the present disclosure may be members of the B7/CD28 superfamily, members of the tumor necrosis factor (TNF) superfamily or ligands thereof that functional as a co-stimulatory factor in one or more types of immune cells. A co-stimulatory factor refers to a receptor or a ligand thereof, which enhances the primary, antigen-specific signal and fully activates immune cells.

Accordingly, the present disclosure provides modified (e.g., genetically engineered) hematopoietic cells (e.g., HSCs or immune cells) that have the capacity to have modulated (e.g., increased) co-stimulatory pathways. In some embodiments, such modified immune cells may express one or more co-stimulatory polypeptides such as those described herein to impart the capacity to modulate the co-stimulatory pathways, relative to an unmodified immune cell. Such a genetically engineered immune cell may further express an ACTR polypeptide (as a separate polypeptide relative to the co- stimulatory polypeptide). Both the ACTR polypeptide and the co-stimulatory polypeptide expressed in the genetically engineered immune cells are encoded by nucleic acids exogenous to the immune cells (i.e., introduced into immune cells via recombinant technology). They are not encoded by endogenous genes of the immune cells absent of the involved genetic engineering. Also provided herein are uses of the genetically engineered immune cells, in combination with an Fc-containing therapeutic agent, such as a therapeutic antibody for enhancing antibody-dependent cell cytotoxicity (ADCC), improving immune cell proliferation, and/or an inhibition of or decrease in target cells (e.g., target cancer cells) in a subject (e.g., a human cancer patient). The present disclosure also provides pharmaceutical compositions and kits comprising the described genetically engineered immune cells.

The genetically engineered immune cells described herein, expressing (e.g., over- expressing) a co-stimulatory peptide, may confer at least the following advantages. The expression of the co-stimulatory polypeptide would have the capacity to modulate the co- stimulatory pathways. As such, the genetically engineered immune cells may proliferate better, produce more cytokines, exhibit greater anti-tumor cytotoxicity, and/or exhibit greater T cell survival relative to immune cells that do not express (or do not over-express) the co-stimulatory polypeptide, leading to enhanced cytokine production, survival rate, cytotoxicity, and/or anti-tumor activity.

For example, immune cells (e.g., T cells) co-expressing an ACTR construct (e.g., an ACTR construct comprising a CD28 co-stimulatory domain) and an exogenous co- stimulatory polypeptide (e.g., 4-1BBL, CD70, LIGHT, BAFFR, ICOSL, CD30L, and CD27) showed enhanced T cell function, as measured by IL-2 release, relative to T cells that expressed ACTR alone in the presence of target cells and a cognate targeting antibody. In another example, immune cells (e.g., T cells) co-expressing an ACTR construct (e.g., an ACTR construct comprising a CD28 co-stimulatory domain) and an exogenous co-stimulatory polypeptide (e.g., BAFFR, CD40, OX40, ICOS, and 4-1BB separated by a P2A ribosomal skip sequence) showed enhanced T cell function, as measured by proliferation, relative to T cells that expressed ACTR alone in the presence of target cells and a cognate targeting antibody. In another example, immune cells (e.g., T cells) co-expressing an ACTR construct (e.g., an ACTR construct comprising a CD28 co- stimulatory domain) and 4-1BBL lacking the cytoplasmic domain enhanced T cell function, as measured by IL-2 release and as measured by proliferation, relative to immune cells that expressed ACTR and full-length 4-1BBL in the presence of target cells and a cognate targeting antibody. I. Co-Stimulatory Polypeptides

As used herein, a co-stimulatory polypeptide refers to a polypeptide that has the capacity to modulate (e.g., stimulate) a co-stimulatory pathway. Such a polypeptide may modulate (e.g., increase) the co-stimulatory pathway via any mechanism. In some examples, the co-stimulatory polypeptide may comprise a co-stimulatory receptor or the co-stimulatory signaling domain thereof. In other examples, the co-stimulatory polypeptide may comprise a ligand of a co-stimulatory receptor or a signaling domain thereof where applicable. Such a ligand may trigger a co-stimulatory signaling pathway upon binding to the cognate co-stimulatory receptor. Alternatively, the co-stimulatory polypeptide may be a non-naturally occurring polypeptide that mimics the activity of a naturally-occurring ligand to any of the co-stimulatory receptors disclosed herein. Such a non-naturally occurring polypeptide may be a single-chain agonistic antibody specific to a co-stimulatory receptor, e.g., an scFv specific to 4-1BB and mimics the activity of 4- 1BBL.

Exemplary co-stimulatory polypeptides may include, but are not limited to, members of the B7/CD28 superfamily, members of the tumor necrosis factor (TNF) superfamily or ligands thereof (e.g., CD28, CD80, CD86, ICOS, ICOSL, B7-H3, B7-H4, VISTA, TMIGD2, B7-H6, B7-H7, 4-1BB, 4-1BBL, BAFF, BAFFR, CD27, CD70, CD30, CD30L, CD40, CD40L, DR3, GITR, GITRL, HVEM, LIGHT, TNF-beta, OX40, OX40L, RELT, TACI, TL1A, TNF-alpha, or TNFRII). Additional examples include BCMA, EDAR2, TROY, LTBR, EDAR, NGFR, OPG, RANK, DCR3, TNFR1, FN14 (TweakR), APRIL, EDA-A2, TWEAK, LTb (TNF-C), NGF, EDA-A1, amyloid precursor protein (APP), TRAIL. Any such polypeptide from any suitable species (e.g., a mammal such as a human) may be contemplated for use with the compositions and methods described herein. In some embodiments, the co-stimulatory polypeptides do not comprise the combination of CD40 and MyD88.

As used herein, a co-stimulatory polypeptide that is a member of the B7/CD28 superfamily or a member of the TNF superfamily refers to a member of either superfamily that plays co-stimulatory roles in activation of any type of immune cells. Such a member may be a naturally-occurring receptor or ligand of either superfamily. Alternatively, such a member may be a variant of the naturally-occurring receptor or ligand. The variant may have increased or decreased activity relative to the native counterpart. In some examples, the variant lacks the cytoplasmic domain or a portion thereof relative to the native counterpart. Described below are exemplary co-stimulatory polypeptides that can be used in the present disclosure.

CD28 (Cluster of Differentiation 28) is a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. It is the receptor for CD80 and CD86 proteins, and is the only B7 receptor constitutively expressed on naïve T cells. The amino acid sequence of an exemplary human CD28 is provided below: CD28 (SEQ ID NO: 81)

MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLD SAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRS CD80 (Cluster of Differentiation 80; B7-1) is a protein found on dendritic cells, activated B cells, and monocytes. It provides a co-stimulatory signal necessary for T cell activation and survival. CD80 is a ligand of both CD28 and CTLA-4. The amino acid sequence of an exemplary human CD80 is provided below: CD80 (SEQ ID NO: 82)

MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQK EKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTL SVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKL DFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCR ERRRNERLRRESVRPV CD86 (Cluster of Differentiation 86; B7-2) is a type I membrane protein that is a member of the immunoglobulin superfamily. CD86 is expressed on antigen-presenting cells that provide co-stimulatory signals necessary for T cell activation and survival.

CD86 is a ligand of both CD28 and CTLA-4. The amino acid sequence of an exemplary human CD86 is provided below: CD86 (SEQ ID NO: 83)

MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLN EVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSV LANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFC LILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF ICOS (CD278; Inducible T cell co-stimulator; or CVID1) is a member of the CD28-superfamily. ICOS is expressed on activated T cells. The amino acid sequence of an exemplary human ICOS is provided below: ICOS (SEQ ID NO: 84)

MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLT KTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYE SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT L ICOSL (ICOSLG; B7-H2; CD275) is a protein that is a ligand for T cell specific protein ICOS. ICOSL acts as a co-stimulatory signal for T cell proliferation and cytokine secretion. The amino acid sequence of an exemplary human ICOSL is provided below: ICOSL (SEQ ID NO: 85)

MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIP QNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAA NFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLR IARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVA IGWVCRDRCLQHSYAGAWAVSPETELTGHV B7-H3 (CD276; Cluster of Differentiation 276) is a member of the immunoglobulin superfamily that is thought to participate in the regulation of T cell- mediated immune response. The amino acid sequence of an exemplary human B7-H3 is provided below: B7-H3 (SEQ ID NO: 86)

MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQ LTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSL QVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDV HSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSP EPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTC FVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNV TTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVC LIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA VISTA (V-domain Ig suppressor of T cell activation; B7-H5; PD-1H) is a Type I transmembrane protein that functions as an immune checkpoint. VISTA co-stimulates T cells via TMIGD2 (CD28H). The amino acid sequence of an exemplary human VISTA is provided below:

VISTA (SEQ ID NO: 87)

MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTF YKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNL TLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVG ILCLPLILLLVYKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPS ESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI TMIGD2 (Transmembrane and immunoglobulin domain containing 2; CD28H) is a TMIGD2 is thought to enhance T cell proliferation and cytokine production via an AKT- dependent signaling cascade. The amino acid sequence of an exemplary human TMIGD2 is provided below: TMIGD2 (SEQ ID NO: 88)

MGSPGMVLGLLVQIWALQEASSLSVQQGPNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQ PYITNGSLSLGVCGPQGRLSWQAPSHLTLQLDPVSLNHSGAYVCWAAVEIPELEEAEGNITRLFVDPD DPTQNRNRIASFPGFLFVLLGVGSMGVAAIVWGAWFWGRRSCQQRDSGNSPGNAFYSNVLYRPRGAPK KSEDCSGEGKDQRGQSIYSTSFPQPAPRQPHLASRPCPSPRPCPSPRPGHPVSMVRVSPRPSPTQQPR PKGFPKVGEE B7-H6 (NCR3LG1; Natural Killer Cell Cytotoxicity Receptor 3 Ligand 1) is a member of the B7 family selectively expressed on tumor cells. B7-H6 interacts with NKp30, resulting in natural killer (NK) cell activation and cytotoxicity. The amino acid sequence of an exemplary human B7-H6 is provided below: B7-H6 (SEQ ID NO: 89)

MTWRAAASTCAALLILLWALTTEGDLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWK SLTFDKEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQ LEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNMD GTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFSIHWWPISFIG VGLVLLIVLIPWKKICNKSSSAYTPLKCILKHWNSFDTQTLKKEHLIFFCTRAWPSYQLQDGEAWPPE GSVNINTIQQLDVFCRQEGKWSEVPYVQAFFALRDNPDLCQCCRIDPALLTVTSGKSIDDNSTKSEKQ TPREHSDAVPDAPILPVSPIWEPPPATTSTTPVLSSQPPTLLLPLQ B7-H7 (HHLA2; HERV-H LTR-Associating 2) is a protein ligand found on the surface of monocytes. B7-H7 is thought to regulate cell-mediated immunity through binding a receptor on T lymphocytes and inhibiting proliferation in the same. The amino acid sequence of an exemplary human B7-H7 is provided below: B7-H7 (SEQ ID NO: 90)

MKAQTALSFFLILITSLSGSQGIFPLAFFIYVPMNEQIVIGRLDEDIILPSSFERGSEVVIHWKYQDS YKVHSYYKGSDHLESQDPRYANRTSLFYNEIQNGNASLFFRRVSLLDEGIYTCYVGTAIQVITNKVVL KVGVFLTPVMKYEKRNTNSFLICSVLSVYPRPIITWKMDNTPISENNMEETGSLDSFSINSPLNITGS NSSYECTIENSLLKQTWTGRWTMKDGLHKMQSEHVSLSCQPVNDYFSPNQDFKVTWSRMKSGTFSVLA YYLSSSQNTIINESRFSWNKELINQSDFSMNLMDLNLSDSGEYLCNISSDEYTLLTIHTVHVEPSQET ASHNKGLWILVPSAILAAFLLIWSVKCCRAQLEARRSRHPADGAQQERCCVPPGERCPSAPDNGEENV PLSGKV 4-1BB (CD137; TNFRSF9) is a tumor necrosis factor (TNF) superfamily member that is expressed by activated T cells. Crosslinking of 4-1BB enhances T cell

proliferation, IL-2 secretion, survival, and cytolytic activity. The amino acid sequence of an exemplary human 4-1BB is provided below: 4-1BB (SEQ ID NO: 91)

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQC KGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPW TNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLF FLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

4-1BBL (TNFSF9; 4-1BB ligand) is a Type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily. 4-1BBL is expressed on activated T

Lymphocytes and binds to 4-1BB. The amino acid sequence of certain exemplary human 4-1BBL polypeptides (including native and variants) are provided below: 4-1BBL (SEQ ID NO: 92)

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASP RLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL-CD (lacking cytoplasmic domain; SEQ ID NO: 93)

MRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQ GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL Q89A (SEQ ID NO: 94)

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASP RLREGPELSPDDPAGLLDLRAGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL Q89A/CD (lacking cytoplasmic domain) (SEQ ID NO: 95)

MRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRAGM FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSG SVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQ LTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL L115A (SEQ ID NO: 96)

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASP RLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGAAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL L115A/CD (SEQ ID NO: 97)

MRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQ GMFAQLVAQNVLLIDGPLSWYSDPGAAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL K127A (SEQ ID NO: 98)

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASP RLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYAEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

4-1BBL Q227A (SEQ ID NO: 99)

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASP RLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWALTQGATVLGLFRVTPEIPAGLPSPRSE BAFF (B-cell activating factor; TNFSF13B) is a member of the TNF ligand family and serves as a ligand for receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and

TNFRSF13C/BAFF-R. BAFF is a potent B cell activator and plays an important role in B cell proliferation and differentiation. The amino acid sequence of an exemplary human BAFF is provided below: BAFF (SEQ ID NO: 100)

MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCCLTVVSFYQ VAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAVQ GPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDK TYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISL DGDVTFFGALKLL BAFFR (B-cell activating factor receptor; TNFRSF13C) is a membrane protein of the TNF receptor superfamily and acts as a receptor for BAFF. BAFFR enhances B cell survival and is a regulator of the peripheral B-cell population. The amino acid sequence of an exemplary human BAFFR is provided below: BAFFR (SEQ ID NO: 101)

MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAG EAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGDKDAPEPLDKVIILSPG ISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ CD27 (TNFRSF7) is a member of the TNF receptor superfamily and is required for generation and long-term maintenance of T cell immunity. CD27 binds to CD70 and also plays a role in regulation of B-cell activation and immunoglobulin synthesis. The amino acid sequence of an exemplary human CD27 is provided below: CD27 (SEQ ID NO: 102)

MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPG VSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQAL SPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFT LAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP CD70 (CD27LG; TNFSF7) is a protein expressed on highly activated lymphocytes. CD70 acts as a ligand for CD27. The amino acid sequence of an exemplary human CD70 is provided below: CD70 (SEQ ID NO: 103)

MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQ QDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPA SRSISLLRLSFHQGCTIVSQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP CD30 (TNFRSF8) is a member of the TNF receptor superfamily that is expressed by activated T cells and B cells. CD30 is a cell membrane protein that has been shown to interact with CD30L, TRAF1, TRAF2, TRAF3, and TRAF5. The amino acid sequence of an exemplary human CD30 is provided below: CD30 (SEQ ID NO: 104)

MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQ CEPDYYLDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMI VKFPGTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEA ASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVSCSRDDLVEKTP CAWNSSRTCECRPGMICATSATNSCARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPE NGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCH RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVG AAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPA EPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK CD30L (CD30LG; TNFSF8) is a member of the TNF receptor superfamily.

CD30L acts as a ligand of CD30, and is expressed on induced T cells and

monocytes/macrophages. The amino acid sequence of an exemplary human CD30L is provided below: CD30L (SEQ ID NO: 105)

MDPGLQQALNGMAPPGDTAMHVPAGSVASHLGTTSRSYFYLTTATLALCLVFTVATIMVLVVQRTDSI PNSPDNVPLKGGNCSEDLLCILKRAPFKKSWAYLQVAKHLNKTKLSWNKDGILHGVRYQDGNLVIQFP GLYFIICQLQFLVQCPNNSVDLKLELLINKHIKKQALVTVCESGMQTKHVYQNLSQFLLDYLQVNTTI SVNVDTFQYIDTSTFPLENVLSIFLYSNSD CD40 (TNFRSF5) is a cell surface receptor expressed on the surface of B cells, monocytes, dendritic cells, endothelial cells, and epithelial cells. CD40 has been demonstrated to have involvement in T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. The amino acid sequence of an exemplary human CD40 is provided below: CD40 (SEQ ID NO: 106)

MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFL DTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIAT GVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGI LFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRIS VQERQ CD40L (CD40LG; TRAP; TNFSF5) is a member of the TNF superfamily expressed on B lymphocytes, epithelial cells, and some carcinoma cells. CD40L is a transmembrane protein that is known to interact with CD40 in order to mediate B cell proliferation, adhesion, and differentiation. The amino acid sequence of an exemplary human CD40L is provided below: CD40L (SEQ ID NO: 107)

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKT IQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTS VLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERI LLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL DR3 (TNFR25; APO3; TRAMP; LARD; WSL-1,) is a TNF receptor superfamily member expressed in lymphocytes. DR3 is thought to be the receptor responsible for TL1A-induced T cell co-stimulation. The amino acid sequence of an exemplary human DR3 is provided below: DR3 (SEQ ID NO: 108)

MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGNS TCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADTRCGCKPGWFVECQVSQCVSSSPF YCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVCGWRQMFWVQ VLLAGLVVPLLLGATLTYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKIC TVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMD AVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVED LRSRLQRGP GITR (Glucocorticoid-induced TNFR-related protein; AITR; TNFRSF18) is a member of the TNF receptor superfamily and is expressed in several cells and tissues including T lymphocytes, NK cells and antigen-presenting cells. GITR interaction with its ligand (GITRL) induces a co-activating signal. The amino acid sequence of an exemplary human GITR is provided below: GITR (SEQ ID NO: 109)

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCS EWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQ FGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQL LLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLW GITRL (TNFSF18) is a cytokine belonging to the TNF ligand family and acts as a receptor for GITR. GITR interaction with its ligand (GITRL) induces a co-activating signal and has been shown to modulate T lymphocyte survival in peripheral tissues. The amino acid sequence of an exemplary human GITRL is provided below: GITRL (SEQ ID NO: 110)

MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIF IFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFE VRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS HVEM (Herpesvirus entry mediator; TNFRSF14; CD270) is a cell surface receptor and a member of the TNF receptor superfamily. HVEM provides a stimulatory signal to T cells following engagement with LIGHT (TNFSF14); or an inhibitory signal to T cells when it binds the B and T lymphocyte attenuator (BTLA), a ligand member of the

Immunoglobulin (Ig) superfamily. The amino acid sequence of an exemplary human HVEM is provided below: HVEM (SEQ ID NO: 111)

MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACG ELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCA ACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWW FLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEET IPSFTGRSPNH LIGHT (TNFSF14; CD258; HVEML) is a member of the TNF ligand family that functions as a co-stimulatory factor along with HVEM. LIGHT has been demonstrated to stimulate the proliferation of T cells and trigger apoptosis of various tumor cells. The amino acid sequence of an exemplary human LIGHT is provided below: LIGHT (SEQ ID NO: 112)

MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEM VTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVT KAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLG GVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV TNF-alpha (TNFSF2) is a member of the TNF ligand superfamily known to be secreted by, for example, macrophages and activated CD4-positive T cells. TNF-alpha is known to induce certain co-stimulatory molecules such as B7h and TNFRII. The amino acid sequence of an exemplary human TNF-alpha is provided below: TNF-alpha (SEQ ID NO: 113)

MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLS LISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK GDRLSAEINRPDYLDFAESGQVYFGIIAL TNF-beta (TNFSF1; Lymphotoxin alpha) is a member of the TNF superfamily involved in the regulation of cell survival, proliferation, differentiation, and apoptosis.

The amino acid sequence of an exemplary human TNF-beta is provided below: TNF-beta (SEQ ID NO: 114)

MTPPERLFLPRVCGTTLHLLLLGLLLVLLPGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLI GDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQ LFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFA L OX40 (TNFRSF4; CD134) is a member of the TNF receptor superfamily. OX40 binds to OX40L and contributes to T cell expansion, survival, and cytokine production. The amino acid sequence of an exemplary human OX40 is provided below: OX40 (SEQ ID NO: 115)

MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCG PGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPG DNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPST RPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHS TLAKI OX40L (TNFSF4; CD252) is a member of the TNF ligand superfamily and is expressed, for example, on activated CD4 and CD8 T cells as well as a number of other lymphoid and non-lymphoid cells. OX40L interacts with OX40 in order to regulate, for example, T cell expansion, survival, and cytokine production. The amino acid sequence of an exemplary human OX40L is provided below: OX40L (SEQ ID NO: 116)

MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTE YKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVN SLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL RELT (TNFRSF19L) is a member of the TNF receptor superfamily. RELT is a type I transmembrane glycoprotein and is thought to be capable of co-stimulating T cell proliferation in the presence of CD3 signaling. The amino acid sequence of an exemplary human RELT is provided below: RELT (SEQ ID NO: 117)

MKPSLLCRPLSCFLMLLPWPLATLTSTTLWQCPPGEEPDLDPGQGTLCRPCPPGTFSAAWGSSPCQPH ARCSLWRRLEAQVGMATRDTLCGDCWPGWFGPWGVPRVPCQPCSWAPLGTHGCDEWGRRARRGVEVAA GASSGGETRQPGNGTRAGGPEETAAQYAVIAIVPVFCLMGLLGILVCNLLKRKGYHCTAHKEVGPGPG GGGSGINPAYRTEDANEDTIGVLVRLITEKKENAAALEELLKEYHSKQLVQTSHRPVSKLPPAPPNVP HICPHRHHLHTVQGLASLSGPCCSRCSQKKWPEVLLSPEAVAATTPVPSLLPNPTRVPKAGAKAGRQG EITILSVGRFRVARIPEQRTSSMVSEVKTITEAGPSWGDLPDSPQPGLPPEQQALLGSGGSRTKWLKP PAENKAEENRYVVRLSESNLVI TACI (Transmembrane activator and CAML interactor; TNFRSF13B; CD267) is a TNF receptor superfamily member that is found, for example, on the surface of B cells. TACI is known to interact with ligands BAFF and APRIL. The amino acid sequence of an exemplary human TACI is provided below: TACI (SEQ ID NO: 118)

MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRS LSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRRQRSGEVENNSDNSGRY QGLEHRGSEASPALPGLKLSADQVALVYSTLGLCLCAVLCCFLVAVACFLKKRGDPCSCQPRSRPRQS PAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPDPTCAGRWGCHTRTTVLQPCP HIPDSGLGIVCVPAQEGGPGA TL1A (TNFSF15) is a member of the TNF ligand superfamily that is known to bind to DR3. TL1A can act to enhance T cell proliferation and cytokine production of T cells. The amino acid sequence of an exemplary human TL1A is provided below: TL1A (SEQ ID NO: 119)

MAEDLGLSFGETASVEMLPEHGSCRPKARSSSARWALTCCLVLLPFLAGLTTYLLVSQLRAQGEACVQ FQALKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTN KFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEV GSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL TNFRII (TNFRSF1B) is a TNF receptor superfamily member that binds to TNF- alpha. TNFRII has been shown to act as a co-stimulatory receptor for T cells and as a critical factor for the development of regulatory T cells (Treg) and myeloid suppressor cells. The amino acid sequence of an exemplary human TNFRII is provided below: TNFRII (SEQ ID NO: 120)

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCT KTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRL CAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTS PTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALG LLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDR RAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDT DSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS BCMA is a cell surface receptor of the TNF receptor superfamily, and binds to the tumor necrosis factor superfamily, member 13b (TNFSF13B), leading to NF-kappaB and MAPK8/JNK activation. It is preferentially expressed on mature B lymphocytes and plays a pivotal role in B cell development, function, and regulation. The amino acid sequence of an exemplary human BCMA is provided below: BCMA (SEQ ID NO: 121)

MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISL AVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKP KVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR EDA2R is a type III transmembrane protein of the TNFR (tumor necrosis factor receptor) superfamily and contains 3 cysteine-rich repeats and one transmembrane domain. It binds to the EDA-A2 isoform of the ectodysplasin, playing an important role in maintaining hair and teeth. The amino acid sequence of an exemplary human EDA2R is provided below: EDA2R (SEQ ID NO: 122)

MDCQENEYWDQWGRCVTCQRCGPGQELSKDCGYGEGGDAYCTACPPRRYKSSWGHHRCQSCITCAVIN RVQKVNCTATSNAVCGDCLPRFYRKTRIGGLQDQECIPCTKQTPTSEVQCAFQLSLVEADTPTVPPQE ATLVALVSSLLVVFTLAFLGLFFLYCKQFFNRHCQRGGLLQFEADKTAKEESLFPVPPSKETSAESQV SENIFQTQPLNPILEDDCSSTSGFPTQESFTMASCTSESHSHWVHSPIECTELDLQKFSSSASYTGAE TLGGNTVESTGDRLELNVPFEVPSP TROY or TNFR (tumor necrosis factor receptor) superfamily member 19 is a type 1 cell surface receptor that is highly expressed in the embryonic and adult CNS and developing hair follicles. It activates the JNK signaling pathway when overexpressed in cells, interacts with TRAF family members, and can induce apoptosis by a caspace-independent mechanism. The amino acid sequence of an exemplary human TROY is provided below: TROY (SEQ ID NO: 123)

MALKVLLEQEKTFFTLLVLLGYLSCKVTCESGDCRQQEFRDRSGNCVPCNQCGPGMELSKECGFGYGE DAQCVTCRLHRFKEDWGFQKCKPCLDCAVVNRFQKANCSATSDAICGDCLPGFYRKTKLVGFQDMECV PCGDPPPPYEPHCASKVNLVKIASTASSPRDTALAAVICSALATVLLALLILCVIYCKRQFMEKKPSW SLRSQDIQYNGSELSCFDRPQLHEYAHRACCQCRRDSVQTCGPVRLLPSMCCEEACSPNPATLGCGVH SAASLQARNAGPAGEMVPTFFGSLTQSICGEFSDAWPLMQNPMGGDNISFCDSYPELTGEDIHSLNPE LESSTSLDSNSSQDLVGGAVPVQSHSENFTAATDLSRYNNTLVESASTQDALTMRSQLDQESGAVIHP ATQTSLQVRQRLGSL LTBR or tumor necrosis factor receptor superfamily member 3 (TNFRSF3) is a cell surface receptor that binds to the lymphotoxin membrane form (a complex of lymphotoxin- alpha and lymphtoxin-beta). It plays a role in apoptosis, lipid metabolism, and the development and organization of lymphoid tissue and transformed cells. The amino acid sequence of an exemplary human LTBR is provided below: LTBR (SEQ ID NO: 124)

MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYV SAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWA LECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTA QSDTTCKNPLEPLPPEMSGTMLMLAVLLPLAFFLLLATVFSCIWKSHPSLCRKLGSLLKRRPQGEGPN PVAGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLDLTREPQLEPGEQSQ VAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGK AWHLAETEHCGATPSNRGPRNQFITHD EDAR (Ectodysplasin A receptor) is a cell surface receptor for ectodysplasin A and plays a pivotal role in embryonic development, as well as the development of hair, teeth, and other ectodermal derivatives. It can activate the nuclear factor-kappaB, JNK, and caspase- independent cell death pathways. The amino acid sequence of an exemplary human EDAR is provided below:

EDAR (SEQ ID NO: 125)

MAHVGDCTQTPWLPVLVVSLMCSARAEYSNCGENEYYNQTTGLCQECPPCGPGEEPYLSCGYGTKDED YGCVPCPAEKFSKGGYQICRRHKDCEGFFRATVLTPGDMENDAECGPCLPGYYMLENRPRNIYGMVCY SCLLAPPNTKECVGATSGASANFPGTSGSSTLSPFQHAHKELSGQGHLATALIIAMSTIFIMAIAIVL IIMFYILKTKPSAPACCTSHPGKSVEAQVSKDEEKKEAPDNVVMFSEKDEFEKLTATPAKPTKSENDA SSENEQLLSRSVDSDEEPAPDKQGSPELCLLSLVHLAREKSATSNKSAGIQSRRKKILDVYANVCGVV EGLSPTELPFDCLEKTSRMLSSTYNSEKAVVKTWRHLAESFGLKRDEIGGMTDGMQLFDRISTAGYSI PELLTKLVQIERLDAVESLCADILEWAGVVPPASQPHAAS NGFR (Nerve Growth Factor Receptor) is a low affinity cell surface receptor for the neurotrophins, which are protein growth factors that stimulate neuronal cell survival and differentiation. NGFR also binds pro-neurotrophins and functions as a co-receptor with other receptor partners, including SORT1 (Sortilin), LINGO1, and RTN4R. It has broad expression in the spleen, adrenal, and brain, among other tissues. The amino acid sequence of an exemplary human NGFR is provided below: NGFR (SEQ ID NO: 126)

MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCL DSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVF SCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEG SDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAF KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPHTQTASGQALKGDGGLYSSLP PAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPVRALLASWATQDSATLDALLAALRRI QRADLVESLCSESTATSPV OPG (osteoprotegerin) is a cytokine receptor of tumor necrosis factor (TNF) receptor superfamily encoded by the TNFRSF11B gene that binds to TNF-related apoptosis-inducing ligand (TRAIL) and inhibits TRAIL-induced apoptosis of specific cells, including tumour cells. It functions as a negative regulator of bone resorption and plays an important role in osteoclast development, tumor growth and metastasis, heart disease, immune system development and signaling, mental health, diabetes, and the prevention of pre-eclampsia and osteoporosis during pregnancy. The amino acid sequence of an exemplary human OPG is provided below: OPG (SEQ ID NO: 127)

MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKTVCAPCPDH YYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGVVQAGTP ERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNATHDNICSGNSESTQKCGIDVTLCEE AFFRFAVPTKFTPNWLSVLVDNLPGTKVNAESVERIKRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQ DIDLCENSVQRHIGHANLTFEQLRSLMESLPGKKVGAEDIEKTIKACKPSDQILKLLSLWRIKNGDQD TLKGLMHALKHSKTYHFPKTVTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL RANK (Receptor activator of nuclear factor k B) is the receptor for RANK-Ligand (RANKL) and part of the RANK/RANKL/OPG signaling pathway that regulates osteoclast differentiation and activation. It is an important regulator of the interaction between T cells and dendritic cells and it plays an important role in bone remodeling and repair, immune cell function, lymph node development, thermal regulation, and mammary gland development. The amino acid sequence of an exemplary human RANK is provided below: RANK (SEQ ID NO: 128)

MAPRARRRRPLFALLLLCALLARLQVALQIAPPCTSEKHYEHLGRCCNKCEPGKYMSSKCTTTSDSVC LPCGPDEYLDSWNEEDKCLLHKVCDTGKALVAVVAGNSTTPRRCACTAGYHWSQDCECCRRNTECAPG LGAQHPLQLNKDTVCKPCLAGYFSDAFSSTDKCRPWTNCTFLGKRVEHHGTEKSDAVCSSSLPARKPP NEPHVYLPGLIILLLFASVALVAAIIFGVCYRKKGKALTANLWHWINEACGRLSGDKESSGDSCVSTH TANFGQQGACEGVLLLTLEEKTFPEDMCYPDQGGVCQGTCVGGGPYAQGEDARMLSLVSKTEIEEDSF RQMPTEDEYMDRPSQPTDQLLFLTEPGSKSTPPFSEPLEVGENDSLSQCFTGTQSTVGSESCNCTEPL CRTDWTPMSSENYLQKEVDSGHCPHWAASPSPNWADVCTGCRNPPGEDCEPLVGSPKRGPLPQCAYGM GLPPEEEASRTEARDQPEDGADGRLPSSARAGAGSGSSPGGQSPASGNVTGNSNSTFISSGQVMNFKG DIIVVYVSQTSQEGAAAAAEPMGRPVQEETLARRDSFAGNGPRFPDPCGGPEGLREPEKASRPVQEQG GAKA DCR3 (Decoy receptor 3) is a soluble protein of the tumor necrosis factor receptor superfamily which plays a regulatory role in suppressing FasL- and LIGHT-mediated cell death and is a decoy receptor that competes with death receptors for ligand binding. It is overexpressed in gastrointestinal tract tumors. The amino acid sequence of an exemplary human DCR3 is provided below: DCR3 (SEQ ID NO: 129)

MRALEGPGLSLLCLVLALPALLPVPAVRGVAETPTYPWRDAETGERLVCAQCPPGTFVQRPCRRDSPT TCGPCPPRHYTQFWNYLERCRYCNVLCGEREEEARACHATHNRACRCRTGFFAHAGFCLEHASCPPGA GVIAPGTPSQNTQCQPCPPGTFSASSSSSEQCQPHRNCTALGLALNVPGSSSHDTLCTSCTGFPLSTR VPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPEGWGPTPRAGRAALQLKLRRRLTELLGAQDGAL LVRLLQALRVARMPGLERSVRERFLPVH TNFR1 (Tumor necrosis factor receptor 1) is a ubiquitous membrane receptor that binds tumor necrosis factor-alpha (TNFa), which can activate the transcription factor NF-kB, mediate apoptosis, and function as a regulator of inflammation. The amino acid sequence of an exemplary human TNFR1 is provided below: TNFR1 (SEQ ID NO: 130)

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYL YNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYW SENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKG TEDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPS FSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQK WEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWR RRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR FN14 (Fibroblast growth factor-inducible 14) is induced in a variety of cell types in situations of tissue injury and is activated by TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF ligand family that controls many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis and inflammation. NFAT1 regulates the expression of FN14 and its ligand TWEAK with lipocalin 2 to increase breast cancer cell invasion. The amino acid sequence of an exemplary human FN14 is provided below: FN14 (SEQ ID NO: 131)

MARGSLRRLLRLLVLGLWLALLRSVAGEQAPGTAPCSRGSSWSADLDKCMDCASCRARPHSDFCLGCA AAPPAPFRLLWPILGGALSLTFVLGLLSGFLVWRRCRRREKFTTPIEETGGEGCPAVALIQ APRIL (A proliferation-inducing ligand) is a ligand for TNFRSF17/BCMA, a member of the TNF receptor family. Both APRIL and its receptor are important for B cell development. It is expressed at low levels in lymphoid tissue and is over-expressed by a number of tumors. The amino acid sequence of an exemplary human APRIL is provided below: APRIL (SEQ ID NO: 132)

MPASSPFLLAPKGPPGNMGGPVREPALSVALWLSWGAALGAVACAMALLTQQTELQSLRREVSRLQGT GGPSQNGEGYPWQSLPEQSSDALEAWENGERSRKRRAVLTQKQKKQHSVLHLVPINATSKDDSDVTEV MWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHP DRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKL EDA-A2 is a type II transmembrane protein that is a member of the TNF Superfamily (TNFSF) and acts as a homotrimer that may be involved in cell-cell signaling during the development of ectodermal organs. Defects in this gene are a cause of ectodermal dysplasia, anhidrotic, which is also known as X-linked hypohidrotic ectodermal dysplasia. The amino acid sequence of an exemplary human EDA-A2 is provided below: EDA-A2 (SEQ ID NO: 133)

MGYPEVERRELLPAAAPRERGSQGCGCGGAPARAGEGNSCLLFLGFFGLSLALHLLTLCCYLELRSEL RRERGAESRLGGSGTPGTSGTLSSLGGLDPDSPITSHLGQPSPKQQPLEPGEAALHSDSQDGHQMALL NFFFPDEKPYSEEESRRVRRNKRSKSNEGADGPVKNKKKGKKAGPPGPNGPPGPPGPPGPQGPPGIPG IPGIPGTTVMGPPGPPGPPGPQGPPGLQGPSGAADKAGTRENQPAVVHLQGQGSAIQVKNDLSGGVLN DWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQVYYINFTDFASYEVVVDEKPFLQCTRSIETGKTN YNTCYTAGVCLLKARQKIAVKMVHADISINMSKHTTFFGAIRLGEAPAS TWEAK (TNF-related weak inducer of apoptosis) is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family and a ligand for the FN14/TWEAKR receptor. It has overlapping signaling functions with TNF, but displays a much wider tissue distribution. It plays an important role in apoptosis, proliferation and migration of endothelial cells, and angiogenesis. The amino acid sequence of an exemplary human TWEAK is provided below: TWEAK (SEQ ID NO: 134)

MAARRSQRRRGRRGEPGTALLVPLALGLGLALACLGLLLAVVSLGSRASLSAQEPAQEELVAEEDQDP SELNPQTEESQDPAPFLNRLVRPRRSAPKGRKTRARRAIAAHYEVHPRPGQDGAQAGVDGTVSGWEEA RINSSSPLRYNRQIGEFIVTRAGLYYLYCQVHFDEGKAVYLKLDLLVDGVLALRCLEEFSATAASSLG PQLRLCQVSGLLALRPGSSLRIRTLPWAHLKAAPFLTYFGLFQVH LTA (Lymphotoxin-alpha) is a cytokine produced by lymphocytes, and exists in both a membrane bound and soluble state. It forms heterotrimers with lymphotoxin-beta which anchor lymphotoxin-alpha to the cell surface, is involved in the formation of secondary lymphoid organs, and mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. The amino acid sequence of an exemplary human LTA is provided below: LTB (SEQ ID NO: 135)

MGALGLEGRGGRLQGRGSLLLAVAGATSLVTLLLAVPITVLAVLALVPQDQGGLVTETADPGAQAQQG LGFQKLPEEEPETDLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLY CLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTS VGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG NGF (Nerve growth factor) is a neurotrophic factor and neuropeptide primarily involved in the regulation of growth, maintenance, proliferation, and survival of certain target neurons. More specifically, NGF is critical for the survival of the sympathetic and sensory neurons. The amino acid sequence of an exemplary human NGF is provided below: NGF (SEQ ID NO: 136)

MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTR NITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVC DSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTT HTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA EDA-A1 is a type II transmembrane protein belonging to the TNF superfamily that acts as a homotrimer and may be involved in cell-cell signaling during the development of ectodermal organs. The attachment of EDA-A1 to the ectodysplasin A receptor triggers a series of chemical signals that affect cell activities such as division, growth, and maturation. The amino acid sequence of an exemplary human EDA-A1 is provided below:

EDA-A1 (SEQ ID NO: 137)

MGYPEVERRELLPAAAPRERGSQGCGCGGAPARAGEGNSCLLFLGFFGLSLALHLLTLCCYLELRSEL RRERGAESRLGGSGTPGTSGTLSSLGGLDPDSPITSHLGQPSPKQQPLEPGEAALHSDSQDGHQMALL NFFFPDEKPYSEEESRRVRRNKRSKSNEGADGPVKNKKKGKKAGPPGPNGPPGPPGPPGPQGPPGIPG IPGIPGTTVMGPPGPPGPPGPQGPPGLQGPSGAADKAGTRENQPAVVHLQGQGSAIQVKNDLSGGVLN DWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQVEVYYINFTDFASYEVVVDEKPFLQCTRSIETGK TNYNTCYTAGVCLLKARQKIAVKMVHADISINMSKHTTFFGAIRLGEAPAS APP (amyloid precursor protein) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It is expressed in many tissues, including the brain and spinal cord, and metabolized in a rapid and highly complex fashion by a series of sequential proteases, including the intramembranous g-secretase complex, which also process other key regulatory molecules. The amino acid sequence of an exemplary human APP is provided below: APP (SEQ ID NO: 138)

MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEG ILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFL HQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEED DSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSI ATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAV CGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQ VMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENY ITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQS LSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGE FSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSG YEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERH LSKMQQNGYENPTYKFFEQMQN TRAIL (TNF-related apoptosis-inducing ligand) is a cytokine that induces apoptosis. It binds to two death receptors DR4 (TRAIL-RI) and DR5 (TRAIL-RII), and two decoy receptors DcR1 and DcR2. TRAIL functions by binding to the death receptors, recruiting the FAS-associated death domain, and activating caspases 8 and 10, which results in apoptosis. The amino acid sequence of an exemplary human TRAIL is provided below: TRAIL (SEQ ID NO: 139)

MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPN DEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTL SSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEA SFFGAFLVG B7-H4, also known as V-set domain-containing T-cell activation inhibitor 1 (VTCN1) is a member of the B7 family. This protein is found to be expressed on the surface of antigen-presenting cells and to interact with ligands such as CD28 or MIM 186760 on T cells. The amino acid sequence of an exemplary human B7-H4 is provided below: B7-H4 (SEQ ID NO :140):

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIV IQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSK GKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSE NVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWAL LPLSPYLMLK The co-stimulatory polypeptide may be a naturally-occurring polypeptide from a suitable species, for example, a mammalian co-stimulatory polypeptide such as those derived from human or a non-human primate. Such naturally-occurring polypeptides are known in the art and can be obtained, for example, using any of the above-noted amino acid sequences as a query to search a publicly available gene database, for example GenBank. The co- stimulatory polypeptide for use in the instant disclosure may share a sequence identity of at least 85% (e.g., 90%, 95%, 97%, 98%, 99%, or above) with any of the exemplary proteins noted above. In some embodiments, the member of the B7/CD28 superfamily, member of the tumor necrosis factor (TNF) superfamily, or ligand thereof may lack a cytoplasmic domain. In an exemplary embodiment, the 4-1BBL lacks a cytoplasmic domain. In some embodiments, the member of the TNF superfamily or ligand thereof is not 4-1BBL.

The“percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol.215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997.

When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Alternatively, the co-stimulatory polypeptide may be a functional variant of a native counterpart. Such a functional variant may contain one or more mutations within the functional domain(s) (e.g., within the active site of an enzyme) of the native counterpart. Such a functional variant may contain one or more mutations outside the functional domain(s) of the native counterpart. Functional domains of a native co-stimulatory polypeptide may be known in the art or can be predicted based on its amino acid sequence. Mutations outside the functional domain(s) would not be expected to substantially affect the biological activity of the protein. In some instances, the functional variant may have the capacity to modulate (i.e., stimulate) co-stimulatory pathways relative to the native counterpart.

Alternatively or in addition, the functional variant may contain a conservative mutation(s) at one or more positions in the native counterpart (e.g., up to 20 positions, up to 15 positions, up to 10 positions, up to 5, 4, 3, 2, 1 position(s)). As used herein, a

“conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

The co-stimulatory polypeptide described herein may not require chemical induced (e.g., rimiducid-induced) dimerization to regulate the activity of the immune cells expressing such. For example, the co-stimulatory polypeptide may be free of a F506 binding protein (FKBP) or a fragment thereof (e.g., theFKBPv36 domain), which allows for dimerization induced by rimiducid. II. ACTR Polypeptides

As used herein, an ACTR polypeptide (a.k.a., an ACTR construct) refers to a non- naturally occurring molecule that can be expressed on the surface of a host cell and comprises an extracellular domain with binding affinity and specificity for the Fc portion of an immunoglobulin (“Fc binder” or“Fc binding domain”), a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, the ACTR polypeptides described herein may further include at least one co-stimulatory signaling domain. The ACTR polypeptides are configured such that, when expressed on a host cell, the extracellular ligand- binding domain is located extracellularly for binding to a target molecule that contains the Fc portion and the cytoplasmic signaling domain. The optional co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling. In some embodiments, an ACTR polypeptide as described herein may comprise, from N-terminus to C-terminus, the Fc binding domain, the transmembrane domain, and the cytoplasmic signaling domain. In some embodiments, an ACTR polypeptide as described herein comprises, from N-terminus to C-terminus, the Fc binding domain, the transmembrane domain, at least one co-stimulatory signaling domain, and the cytoplasmic signaling domain. In other embodiments, an ACTR polypeptide as described herein comprises, from N-terminus to C-terminus, the Fc binding domain, the transmembrane domain, the cytoplasmic signaling domains, and at least one co-stimulatory signaling domain.

Exemplary ACTR constructs for use with the methods and compositions described herein may be found, for example, in the instant description and figures or may be found in WO2016040441A1, WO2017/161333, and PCT Application No.:

PCT/US2018/015999, each of which is incorporated by reference herein for this purpose and the subject matter referenced herein.

The ACTR polypeptides described herein may comprise a CD16A extracellular domain with binding affinity and specificity for the Fc portion of an IgG molecule, a transmembrane domain, and a CD3z cytoplasmic signaling domain. In some

embodiments, the ACTR polypeptides may further include one or more co-stimulatory signaling domains, one of which may be a CD28 co-stimulatory signaling domain or a 4- 1BB co-stimulatory signaling domain. The ACTR polypeptides are configured such that, when expressed on a host cell, the extracellular ligand-binding domain is located

extracellularly for binding to a target molecule and the CD3z cytoplasmic signaling domain. The co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling. In some embodiments, an ACTR polypeptide as described herein may comprise, from N-terminus to C-terminus, the Fc binding domain such as a CD16A extracellular domain, the transmembrane domain, the optional one or more co-stimulatory domains (e.g., a CD28 co-stimulatory domain, a 4- 1BB co-stimulatory signaling domain, an OX40 co-stimulatory signaling domain, a CD27 co-stimulatory signaling domain, or an ICOS co-stimulatory signaling domain), and the CD3z cytoplasmic signaling domain.

Alternatively or in addition, the ACTR polypeptides described herein may contain two or more co-stimulatory signaling domains, which may link to each other or be separated by the cytoplasmic signaling domain. The extracellular Fc binder, transmembrane domain, optional co-stimulatory signaling domain(s), and cytoplasmic signaling domain in an ACTR polypeptide may be linked to each other directly, or via a peptide linker. In some embodiments, any of the ACTR polypeptides described herein may comprise a signal sequence at the N-terminus.

As used herein, the phrase“a protein X transmembrane domain” (e.g., a CD8 transmembrane domain) refers to any portion of a given protein, i.e., transmembrane- spanning protein X, that is thermodynamically stable in a membrane.

As used herein, the phrase“a protein X cytoplasmic signaling domain,” for example, a CD3z cytoplasmic signaling domain, refers to any portion of a protein (protein X) that interacts with the interior of a cell or organelle and is capable of relaying a primary signal as known in the art, which lead to immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein differs from a co-stimulatory signaling domain, which relays a secondary signal for fully activating immune cells.

As used herein, the phrase“a protein X co-stimulatory signaling domain,” e.g., a CD28 co-stimulatory signaling domain, refers to the portion of a given co-stimulatory protein (protein X, such as CD28, 4-1BB, OX40, CD27, or ICOS) that can transduce co- stimulatory signals (secondary signals) into immune cells (such as T cells), leading to fully activation of the immune cells.

In some embodiments, ACTR polypeptides described herein may further comprise a hinge domain, which may be located at the C-terminus of the Fc binding domain and the N- terminus of the transmembrane domain. The hinge may be of any suitable length. In other embodiments, the ACTR polypeptide described herein may have no hinge domain at all. In yet other embodiments, the ACTR polypeptide described herein may have a shortened hinge domain (e.g., including up to 25 amino acid residues).

As used in this specification and the appended claims, the singular forms“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise. A. Fc binding domains

The ACTR polypeptides described herein comprise an extracellular domain that is an Fc binding domain, i.e., capable of binding to the Fc portion of an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey). Suitable Fc binding domains may be derived from naturally occurring proteins such as mammalian Fc receptors or certain bacterial proteins (e.g., protein A, protein G). Additionally, Fc binding domains may be synthetic polypeptides engineered specifically to bind the Fc portion of any of the antibodies described herein with high affinity and specificity. For example, such an Fc binding domain can be an antibody or an antigen-binding fragment thereof that specifically binds the Fc portion of an

immunoglobulin. Examples include, but are not limited to, a single-chain variable fragment (scFv), a domain antibody, or a single domain antibody. Alternatively, an Fc binding domain can be a synthetic peptide that specifically binds the Fc portion, such as a Kunitz domain, a small modular immunopharmaceutical (SMIP), an adnectin, an avimer, an affibody, a DARPin, or an anticalin, which may be identified by screening a peptide combinatory library for binding activities to Fc.

In some embodiments, the Fc binding domain is an extracellular ligand-binding domain of a mammalian Fc receptor. As used herein, an“Fc receptor” is a cell surface bound receptor that is expressed on the surface of many immune cells (including B cells, dendritic cells, natural killer (NK) cells, macrophage, neutrophils, mast cells, and eosinophils) and exhibits binding specificity to the Fc domain of an antibody. Fc receptors are typically comprised of at least two immunoglobulin (Ig)-like domains with binding specificity to an Fc (fragment crystallizable) portion of an antibody. In some instances, binding of an Fc receptor to an Fc portion of the antibody may trigger antibody dependent cell-mediated cytotoxicity (ADCC) effects. The Fc receptor used for constructing an ACTR polypeptide as described herein may be a naturally-occurring polymorphism variant (e.g., the CD16 V158 variant), which may have increased or decreased affinity to Fc as compared to a wild-type counterpart. Alternatively, the Fc receptor may be a functional variant of a wild-type counterpart, which carry one or more mutations (e.g., up to 10 amino acid residue substitutions including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) that alter the binding affinity to the Fc portion of an Ig molecule. In some instances, the mutation may alter the glycosylation pattern of the Fc receptor and thus the binding affinity to Fc.

The table below lists a number of exemplary polymorphisms in Fc receptor extracellular domains (see, e.g., Kim et al., J. Mol. Evol.53:1-9, 2001) which may be used in any of the methods or constructs described herein:

Table 1. Exemplary Polymorphisms in Fc Receptors

Fc receptors are classified based on the isotype of the antibody to which it is able to bind. For example, Fc-gamma receptors (FcgR) generally bind to IgG antibodies, such as one or more subtype thereof (i.e., IgG1, IgG2, IgG3, IgG4); Fc-alpha receptors (FcaR) generally bind to IgA antibodies; and Fc-epsilon receptors (FceR) generally bind to IgE antibodies. In some embodiments, the Fc receptor is an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. Examples of Fc-gamma receptors include, without limitation, CD64A, CD64B, CD64C, CD32A, CD32B, CD16A, and CD16B. An example of an Fc-alpha receptor is FcaR1/CD89. Examples of Fc-epsilon receptors include, without limitation, FceRI and FceRII/CD23. The table below lists exemplary Fc receptors for use in constructing the ACTR polypeptides described herein and their binding activity to corresponding Fc domains: Table 2. Exemplary Fc Receptors

Selection of the ligand binding domain of an Fc receptor for use in the ACTR polypeptides described herein will be apparent to one of skill in the art. For example, it may depend on factors such as the isotype of the antibody to which binding of the Fc receptor is desired and the desired affinity of the binding interaction.

In some examples, the Fc binding domain is the extracellular ligand-binding domain of CD16, which may incorporate a naturally occurring polymorphism that may modulate affinity for Fc. In some examples, the Fc binding domain is the extracellular ligand-binding domain of CD16 incorporating a polymorphism at position 158 (e.g., valine or phenylalanine). In some embodiments, the Fc binding domain is produced under conditions that alter its glycosylation state and its affinity for Fc.

The amino acid sequences of human CD16A F158 and CD16A V158 variants are provided below with the F158 and V158 residue highlighted in bold/face and underlined (signal peptide italicized): CD16A F158 (SEQ ID NO: 141):

MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLIS SQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTAL HKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFP PGYQ CD16A V158 (SEQ ID NO: 142):

MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESL ISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWK NTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVST ISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK In some embodiments, the Fc binding domain is the extracellular ligand-binding domain of CD16 incorporating modifications that render the ACTR polypeptide specific for a subset of IgG antibodies. For example, mutations that increase or decrease the affinity for an IgG subtype (e.g., IgG1) may be incorporated.

Any of the Fc binding domains described herein may have a suitable binding affinity for the Fc portion of a therapeutic antibody. As used herein,“binding affinity” refers to the apparent association constant or K_A. The K_A is the reciprocal of the dissociation constant, KD. The extracellular ligand-binding domain of an Fc receptor domain of the ACTR polypeptides described herein may have a binding affinity K_d of at least 10^-5, 10^-6, 10^-7, 10^-8, 10^-9, 10^-10 M or lower for the Fc portion of antibody. In some embodiments, the Fc binding domain has a high binding affinity for an antibody, isotype(s) of antibodies, or subtype(s) thereof, as compared to the binding affinity of the Fc binding domain to another antibody, isotype(s) of antibodies, or subtypes(s) thereof. In some embodiments, the extracellular ligand-binding domain of an Fc receptor has specificity for an antibody, isotype(s) of antibodies, or subtype(s) thereof, as compared to binding of the extracellular ligand-binding domain of an Fc receptor to another antibody, isotype(s) of antibodies, or subtypes(s) thereof.

Other Fc binding domains as known in the art may also be used in the ACTR constructs described herein including, for example, those described in WO2015058018A1 and PCT Application No.: PCT/US2018/015999, the relevant disclosures of each of which are incorporated by reference for the purpose and subject matter referenced herein. B. Transmembrane domain

The transmembrane domain of the ACTR polypeptides described herein can be in any form known in the art. As used herein, a“transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain compatible for use in the ACTR polypeptides used herein may be obtained from a naturally occurring protein.

Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi-pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).

Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane- spanning segments and may be further sub-classified based on the number of

transmembrane segments and the location of N- and C-termini.

In some embodiments, the transmembrane domain of the ACTR polypeptide described herein is derived from a Type I single-pass membrane protein. Single-pass membrane proteins include, but are not limited to, CD8a, CD8b, 4-1BB/CD137, CD27, CD28, CD34, CD4, FceRIg, CD16, OX40/CD134, CD3z, CD3e, CD3g, CD3d, TCRa, TCRb, TCRz, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B. In some embodiments, the transmembrane domain is from a membrane protein selected from the following: CD8a, CD8b, 4- 1BB/CD137, CD28, CD34, CD4, FceRIg, CD16, OX40/CD134, CD3z, CD3e, CD3g, CD3d, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B. In some examples, the transmembrane domain is of CD8 (e.g., the transmembrane domain is of CD8a). In some examples, the transmembrane domain is of 4-1BB/CD137. In other examples, the transmembrane domain is of CD28. In some cases, the ACTR polypeptide described herein may be free of a hinge domain from any non-CD16A receptor. In some instances, such an ACTR polypeptide may be free of any hinge domain. In other examples, the transmembrane domain is of CD34. In yet other examples, the transmembrane domain is not derived from human CD8a. In some embodiments, the transmembrane domain of the ACTR polypeptide is a single-pass alpha helix.

Transmembrane domains from multi-pass membrane proteins may also be compatible for use in the ACTR polypeptides described herein. Multi-pass membrane proteins may comprise a complex alpha helical structure (e.g., at least 2, 3, 4, 5, 6, 7 or more alpha helices) or a beta sheet structure. Preferably, the N-terminus and the C- terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side. Either one or multiple helix passes from a multi-pass membrane protein can be used for constructing the ACTR polypeptide described herein.

Transmembrane domains for use in the ACTR polypeptides described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No.7,052,906 B1 and PCT Publication No. WO

2000/032776 A2, the relevant disclosures of each of which are incorporated by reference herein.

In some embodiments, the amino acid sequence of the transmembrane domain does not comprise cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises one cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises two cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises more than two cysteine residues (e.g., 3, 4, 5, or more). The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence.

The hydropathy, hydrophobic or hydrophilic characteristics of a protein or protein segment, can be assessed by any method known in the art including, for example, the Kyte and Doolittle hydropathy analysis. C. Co-stimulatory signaling domains

Many immune cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. In some embodiments, the ACTR polypeptides described herein comprise at least one co-stimulatory signaling domain. In certain embodiments, the ACTR polypeptides may contain a CD28 co-stimulatory signaling domain or a 4-1BB (CD137) co-stimulatory signaling domain. The term“co-stimulatory signaling domain,” as used herein, refers to at least a fragment of a co-stimulatory signaling protein that mediates signal transduction within a cell to induce an immune response such as an effector function (a secondary signal). As known in the art, activation of immune cells such as T cells often requires two signals: (1) the antigen specific signal (primary signal) triggered by the engagement of T cell receptor (TCR) and antigenic peptide/MHC complexes presented by antigen presenting cells, which typically is driven by CD3z as a component of the TCR complex; and (ii) a co-stimulatory signal (secondary signal) triggered by the interaction between a co-stimulatory receptor and its ligand. A co- stimulatory receptor transduces a co-stimulatory signal (secondary signal) as an addition to the TCR-triggered signaling and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

Activation of a co-stimulatory signaling domain in a host cell (e.g., an immune cell) may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. The co- stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the ACTR polypeptides described herein. The type(s) of co-stimulatory signaling domain is selected based on factors such as the type of the immune cells in which the ACTR polypeptides would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC). Examples of co- stimulatory signaling domains for use in the ACTR polypeptides may be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, members of the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD- 1, PD-L2/B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB/

TNFRSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF

R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5,

DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40

Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, and TNF RII/TNFRSF1B); members of the SLAM family (e.g., 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3,

CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and

SLAM/CD150); and any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA Class I, HLA- DR, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, TIM-1/KIM-1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen- 1 (LFA-1), and NKG2C. In some embodiments, the co-stimulatory signaling domain is of 4-1BB, CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1(CD11a) or CD2, or any variant thereof.

Also within the scope of the present disclosure are variants of any of the co- stimulatory signaling domains described herein, such that the co-stimulatory signaling domain is capable of modulating the immune response of the immune cell. In some embodiments, the co-stimulatory signaling domains comprises up to 10 amino acid residue mutations (e.g., 1, 2, 3, 4, 5, or 8) such as amino acid substitutions, deletions, or additions as compared to a wild-type counterpart. Such co-stimulatory signaling domains comprising one or more amino acid variations (e.g., amino acid substitutions, deletions, or additions) may be referred to as variants.

Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co- stimulatory signaling domains that do not comprise the mutation. For example, mutation of residues 186 and 187 of the native CD28 amino acid sequence may result in an increase in co-stimulatory activity and induction of immune responses by the co-stimulatory domain of the ACTR polypeptide. In some embodiments, the mutations are substitution of a lysine at each of positions 186 and 187 with a glycine residue of the CD28 co- stimulatory domain, referred to as a CD28_LL®GG variant. Additional mutations that can be made in co-stimulatory signaling domains that may enhance or reduce co-stimulatory activity of the domain will be evident to one of ordinary skill in the art. In some embodiments, the co-stimulatory signaling domain is of 4-1BB, CD28, OX40, or

CD28_LL®GG variant.

In some embodiments, the ACTR polypeptides may contain a single co-stimulatory domain such as, for example, a CD27 co-stimulatory domain, a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, an ICOS co-stimulatory domain, or an OX40 co- stimulatory domain.

In some embodiments, the ACTR polypeptides may comprise more than one co- stimulatory signaling domain (e.g., 2, 3, or more). In some embodiments, the ACTR polypeptide comprises two or more of the same co-stimulatory signaling domains, for example, two copies of the co-stimulatory signaling domain of CD28. In some embodiments, the ACTR polypeptide comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. Selection of the type(s) of co-stimulatory signaling domains may be based on factors such as the type of host cells to be used with the ACTR polypeptides (e.g., T cells or NK cells) and the desired immune effector function. In some embodiments, the ACTR polypeptide comprises two co-stimulatory signaling domains, for example, two copies of the co-stimulatory signaling domain of CD28. In some embodiments, the ACTR polypeptide may comprise two or more co-stimulatory signaling domains from different co-stimulatory receptors, such as any two or more co-stimulatory receptors described herein, for example, CD28 and 4-1BB, CD28 and CD27, CD28 and ICOS, CD28_LL®GG variant and 4-1BB, CD28 and OX40, or CD28_LL®GG variant and OX40. In some embodiments, the two co-stimulatory signaling domains are CD28 and 4- 1BB. In some embodiments, the two co-stimulatory signaling domains are CD28_LL®GG variant and 4-1BB. In some embodiments, the two co-stimulatory signaling domains are CD28 and OX40. In some embodiments, the two co-stimulatory signaling domains are CD28_LL®GG variant and OX40. In some embodiments, the ACTR constructs described herein may contain a combination of a CD28 and ICOSL. In some embodiments, the ACTR construct described herein may contain a combination of CD28 and CD27. In certain embodiments, the 4-1BB co-stimulatory domain is located N-terminal to the CD28 or CD28LL®GG variant co-stimulatory signaling domain.

In some embodiments, the ACTR polypeptides described herein do not comprise a co-stimulatory signaling domain. D. Cytoplasmic signaling domain

Any cytoplasmic signaling domain can be used to create the ACTR polypeptides described herein. Such a cytoplasmic domain may be any signaling domain involved in triggering cell signaling (primary signaling) that leads to immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein is not a co-stimulatory signaling domain, which, as known in the art, relays a co-stimulatory or secondary signal for fully activating immune cells.

The cytoplasmic domain described herein may comprise an immunoreceptor tyrosine-based activation motif (ITAM) domain (e.g., at least one ITAM domain, at least two ITAM domains, or at least three ITAM domains) or may be ITAM free. An“ITAM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprises two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix_(6-8)YxxL/I.

ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways.

In some examples, the cytoplasmic signaling domain is of CD3z or FceR1g. In other examples, cytoplasmic signaling domain is not derived from human CD3z. In yet other examples, the cytoplasmic signaling domain is not derived from an Fc receptor, when the extracellular ligand-binding domain of the same ACTR polypeptide is derived from CD16A.

In one specific embodiment, several signaling domains can be fused together for additive or synergistic effect. Non-limiting examples of useful additional signaling domains include part or all of one or more of TCR Zeta chain, CD28, OX40/CD134, 4-1BB/CD137, FceRIy, ICOS/CD278, IL2R-beta/CD122, IL-2R-gamma/CD132, and CD40.

In other embodiments, the cytoplasmic signaling domain described herein is free of the ITAM motif. Examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, Toll-interleukin receptor (TIR), and tyrosine kinase. E. Hinge domain

In some embodiments, the ACTR polypeptides described herein further comprise a hinge domain that is located between the extracellular ligand-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular ligand-binding domain of an Fc receptor relative to the transmembrane domain of the ACTR polypeptide can be used.

Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the ACTR polypeptides described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the ACTR polypeptide. In some embodiments, the hinge domain is of CD8. In some embodiments, the hinge domain is a portion of the hinge domain of CD8, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8. In some embodiments, the hinge domain is of CD28. In some embodiments, the hinge domain is a portion of the hinge domain of CD28, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28.

In some embodiments, the hinge domain is of CD16A receptor, for example, the whole hinge domain of a CD16A receptor or a portion thereof, which may consists of up to 40 consecutive amino acid residues of the CD16A receptor (e.g., 20, 25, 30, 35, or 40). Such an ACTR construct may contain no hinge domain from a different receptor (a non- CD16A receptor).

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the ACTR polypeptides described herein. In some

embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as hinge domains for the ACTR polypeptides described herein. In some embodiments, the hinge domain between the C- terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (Gly_xSer)_n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. In some embodiments, the hinge domain is (Gly₄Ser)_n (SEQ ID NO: 153), wherein n can be an integer between 3 and 60, including 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In certain embodiments, n can be an integer greater than 60. In some embodiments, the hinge domain is (Gly₄Ser)₃ (SEQ ID NO: 143). In some embodiments, the hinge domain is (Gly4Ser)6 (SEQ ID NO: 144). In some embodiments, the hinge domain is (Gly₄Ser)₉ (SEQ ID NO: 145). In some embodiments, the hinge domain is (Gly₄Ser)₁₂ (SEQ ID NO: 146). In some embodiments, the hinge domain is (Gly₄Ser)₁₅ (SEQ ID NO: 147). In some embodiments, the hinge domain is (Gly₄Ser)₃₀ (SEQ ID NO: 148). In some embodiments, the hinge domain is (Gly4Ser)45 (SEQ ID NO: 149). In some embodiments, the hinge domain is (Gly₄Ser)₆₀ (SEQ ID NO: 150).

In other embodiments, the hinge domain is an extended recombinant polypeptide (XTEN), which is an unstructured polypeptide consisting of hydrophilic residues of varying lengths (e.g., 10-80 amino acid residues). Amino acid sequences of XTEN peptides will be evident to one of skill in the art and can be found, for example, in U.S. Patent No.8,673,860, the relevant disclosures of which are incorporated by reference herein. In some embodiments, the hinge domain is an XTEN peptide and comprises 60 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 30 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 45 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 15 amino acids.

Any of the hinge domains used for making the ACTR polypeptide as described herein may contain up to 250 amino acid residues. In some instances, the ACTR polypeptide may contain a relatively long hinge domain, for example, containing 150-250 amino acid residues (e.g., 150-180 amino acid residues, 180-200 amino acid residues, or 200-250 amino acid residues). In other instances, the ACTR polypeptide may contain a medium sized hinge domain, which may contain 60-150 amino acid residues (e.g., 60-80, 80-100, 100-120, or 120-150 amino acid residues). Alternatively, the ACTR polypeptide may contain a short hinge domain, which may contain less than 60 amino acid residues (e.g., 1-30 amino acids or 31-60 amino acids). In some embodiments, an ACTR construct described herein contains no hinge domain or no hinge domain from a non-CD16A receptor. F. Signal peptide

In some embodiments, the ACTR polypeptide also comprises a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal sequences are peptide sequences that target a polypeptide to the desired site in a cell. In some embodiments, the signal sequence targets the ACTR polypeptide to the secretory pathway of the cell and will allow for integration and anchoring of the ACTR polypeptide into the lipid bilayer. Signal sequences including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences that are compatible for use in the ACTR polypeptides described herein will be evident to one of skill in the art. In some embodiments, the signal sequence from CD8a. In some embodiments, the signal sequence is from CD28. In other embodiments, the signal sequence is from the murine kappa chain. In yet other embodiments, the signal sequence is from CD16. G. Examples of ACTR polypeptides

Table 3 provides exemplary ACTR polypeptides described herein. These exemplary constructs have, from N-terminus to C-terminus in order, the signal sequence, the Fc binding domain (e.g., an extracellular domain of an Fc receptor), the hinge domain, and the transmembrane, while the positions of the optional co-stimulatory domain and the cytoplasmic signaling domain can be switched. Table 3: Exemplary Components of ACTR polypeptides.

Amino acid sequences of the example ACTR polypeptides are provided below (signal sequence italicized). SEQ ID NO: 1:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQ ASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNG KGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPA PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 2:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRG KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 3:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSK KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 4:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT} PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLIALVTSGALLAVLGITGYFLMNRKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 5:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT ^{PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLLAALLALLAALLALLAALLARSKKRGRKK} LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 6:

MALPVTALLLPLALLLHAARPQAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPT HTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTF FQNGKSQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP ^{FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE} MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 7:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 8:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCALYLLR RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 9:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 10:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTIYIWAPLAGTCGVLLLSLVITLYCKRGRKK ^{LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD} KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 12:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR GGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 13:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR GGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM ^{AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR} SEQ ID NO: 14:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFCVRKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO: 15:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFCVRRSKRS RGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 16:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT} PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLCYILDAILFLYGIVLTLLYCRLKKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 17:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLLLILLGVLAGVLATLAALLARSKKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 18:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDITLGLLVAGVLVLLVSLGVAIHLCKRGRKK ^{LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD} KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 19:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVSFCLVMVLLFAVDTGLYFSVKTNKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 20:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 21:

M^{ALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI} SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLCYLLDGILFIYGVILTALFLRVKKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 22:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVMSVATIVIVDICITGGLLLLVYYWSKNRK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR SEQ ID NO: 23:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDGFLFAEIVSIFVLAVGVYFIAGQDKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA ^LPPR SEQ ID NO: 24:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDGIIVTDVIATLLLALGVFCFAGHETKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO: 25:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVIGFRILLLKVAGFNLLMTLRLWKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 26:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIVAVVIATAVAAIVAAVVALIYCRKKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR SEQ ID NO: 27:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVLFYLAVGIMFLVNTVLWVTIRKEKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 28:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIILVGTAVIAMFFWLLLVIILRTKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 29:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLGWLCLLLLPIPLIVWVKRKKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 30:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIAIYCIGVFLIACMVVTVILCRMKKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 31:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 32:

MALPVTALLLPLALLLHAARPQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTS TPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYY RNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLG LQLPTPVWFHIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 33:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 34:

M^{ALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI} SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPK SCDKTHTCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR SEQ ID NO: 35:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPK SCDKTHTCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 36:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEAFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 37:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE ^{EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK} MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 38:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGG GSGGGGSGGGGSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 39:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 40:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 41:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSIYIWAPLAGTCGVLL LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR SEQ ID NO: 42:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAIYIWAPLAGTCGVLL LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR SEQ ID NO: 43:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGS} PAGSPTSTEEGTSESATPESGPGTSTEIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 44:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGS PAGSPTSTEEGTIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 45:

MLRLLLALNLFPSIQVTGGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQ ASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYL QNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R SEQ ID NO: 46:

METDTLLLWVLLLWVPGSTGDGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 47:

MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQAS SYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQN GKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 48:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKK KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 49:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQRRKYR SNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 50:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQLGLHI ^{WQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVRVKFSRSADAPAYQQGQNQL} YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR SEQ ID NO: 51:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCVKRRK PRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNHRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR SEQ ID NO: 52:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT ^{PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKYFFK} KEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATDRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 53:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCYKVGFF KRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGKDRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR SEQ ID NO: 54:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRKKQR SRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPP APSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR SEQ ID NO: 55:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPQLCYILDAILFLYGIVLTLLYCRLKIQVR KAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL SEQ ID NO: 56:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT} PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLK HEKPPQ SEQ ID NO: 57:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIEV} MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 58:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA ^{PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR} GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 59:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQE DYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 60:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 61:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 62:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 63:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R SEQ ID NO:64:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQRRKYR SNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ^ALHMQALPPR SEQ ID NO:65:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO:66:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKKYSS SVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:67:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV ^{TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT} PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRL PPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:68:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:69:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:70:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVK ^{FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG} MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 71:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL ^{YQGLSTATKDTYDALHMQALPPR} SEQ ID NO: 72:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI ^{SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV} TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYI WAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 73:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 74:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQFAC ^{DIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR} SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 75:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR SEQ ID NO: 76:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQKSN GTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP ^{TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN} PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 77

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGKH LCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 78

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQFWV LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 79:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLI SSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ ID NO: 80:

MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQ ASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNG KGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKPIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR III. Hematopoietic Cells Expressing Co-Stimulatory Polypeptides and Optionally

ACTR polypeptides

Provided herein are genetically engineered host cells (e.g., hematopoietic cells such as HSCs and immune cells, e.g., T cells or NK cells) expressing one or more of the co-stimulatory polypeptides as described herein and optionally also an ACTR

polypeptides (ACTR-expressing cells, e.g., ACTR T cells) as also described herein. In some embodiments, the host cells are hematopoietic cells or a progeny thereof. In some embodiments, the hematopoietic cells can be hematopoietic stem cells. In other

embodiments, the host cells are immune cells, such as T cells or NK cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In other embodiments, the immune cells can be established cell lines, for example, NK-92 cells.

In some instances, the co-stimulatory polypeptide to be introduced into the host cells is identical to an endogenous protein of the host cell. Introducing additional copies of the coding sequences of the co-stimulatory polypeptide into the host cell would enhance the expression level of the polypeptide (i.e., over-express) as relative to the native counterpart. In some instances, the co-stimulatory polypeptide to be introduced into the host cells is heterologous to the host cell, i.e., does not exist or is not expressed in the host cell. Such a heterologous co-stimulatory polypeptide may be a naturally-occurring protein not expressed in the host cell in nature (e.g., from a different species). Alternatively, the heterologous co-stimulatory polypeptide may be a variant of a native protein, such as those described herein. In some examples, the exogenous (i.e., not native to the host cells) copy of the coding nucleic acid may exist extrachromosomally. In other examples, the exogenous copy of the coding sequence may be integrated into the chromosome of the host cell, and may be located at a site that is different from the native loci of the

endogenous gene.

Such genetically engineered host cells have the capacity to have a modulated co- stimulatory pathway. The genetically engineered cells, when expressing an ACTR, can recognize and inhibit target cells bound by Fc-containing therapeutic agents such asanti- tumor antibodies. Given their expected high proliferation rate, bioactivity, and/or survival rate, the genetically engineered cells such as T cell and NK cells would be expected to have higher therapeutic efficacy as relative to ACTR T cells that do not express or express a lower level or less active form of the co-stimulatory polypeptide.

The population of immune cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as spleen, lymph node, thymus, stem cells, or tumor tissue. Alternatively, the immune cell

population may be derived from stem cells, for example, hematopoietic stem cells and induced pluripotent stem cells (iPSCs). A source suitable for obtaining the type of host cells desired would be evident to one of skill in the art. In some embodiments, the population of immune cells is derived from PBMCs, which may be obtained from a patient (e.g., a human patient) who needs the treatment described herein. The type of host cells desired (e.g., T cells, NK cells, or T cells and NK cells) may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules. As a non-limiting example, anti-CD3 and anti-CD28 antibodies may be used for expansion of T cells.

To construct the immune cells that express any of the co-stimulatory polypeptides and optionally the ACTR polypeptide described herein, expression vectors for stable or transient expression of the co-stimulatory polypeptides and/or the ACTR polypeptide may be created via conventional methods as described herein and introduced into immune host cells. For example, nucleic acids encoding the co-stimulatory polypeptides and/or the ACTR polypeptides may be cloned into one or two suitable expression vectors, such as a viral vector or a non-viral vector in operable linkage to a suitable promoter. In some instances, each of the coding sequences for the ACTR polypeptide and the co-stimulatory polypeptide are on two separate nucleic acid molecules and can be cloned into two separate vectors, which may be introduced into suitable host cells simultaneously or sequentially. Alternatively, the coding sequences for the ACTR polypeptide and the co-stimulatory polypeptide are on one nucleic acid molecule and can be cloned into one vector. The coding sequences of the ACTR polypeptide and the co-stimulatory polypeptide may be in operable linkage to two distinct promoters such that the expression of the two polypeptides is controlled by different promoters. Alternatively, the coding sequences of the ACTR polypeptide and the co- stimulatory polypeptide may be in operable linkage to one promoter such that the expression of the two polypeptides is controlled by a single promoter. Suitable sequences may be inserted between the coding sequences of the two polypeptides so that two separate polypeptides can be translated from a single mRNA molecule. Such sequences, for example, IRES or ribosomal skipping site, are well known in the art. Additional descriptions are provided below.

The nucleic acids and the vector(s) may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding the co-stimulatory polypeptides and/or the ACTR polypeptides. The synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/plasmids/viral vectors would depend on the type of host cells for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides, but should be suitable for integration and replication in eukaryotic cells.

A variety of promoters can be used for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides described herein, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, the human EF1-alpha promoter, or herpes simplex tk virus promoter.

Additional promoters for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within an immune cell.

Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene or the kanamycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; intron sequences of the human EF1-alpha gene; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyomavirus origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or“suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase or an inducible caspase such as iCasp9), and reporter gene for assessing expression of the co-stimulatory polypeptides and/or the ACTR polypeptide. In one specific embodiment, such vectors also include a suicide gene. As used herein, the term“suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of the preparation of vectors for expression of co-stimulatory polypeptides and/or ACTR polypeptides can be found, for example, in US2014/0106449, herein incorporated in its entirety by reference.

Any of the vectors comprising a nucleic acid sequence that encodes a co- stimulatory polypeptide and/or an ACTR polypeptide described herein is also within the scope of the present disclosure. Such a vector, or the sequence encoding a co-stimulatory polypeptide and/or an ACTR polypeptide contained therein, may be delivered into host cells such as host immune cells by any suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction such as lentiviral transduction).

In some embodiments, the vectors for expression of the co-stimulatory

polypeptides and/or the ACTR polypeptides are delivered to host cells by viral

transduction (e.g., retroviral transduction such as lentiviral or gamma-retroviral

transduction). Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; and WO 91/02805; U.S. Pat. Nos.5,219,740 and 4,777,127; GB Patent No.2,200,651; and EP Patent No.0345242), alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see, e.g., PCT

Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO

95/11984; and WO 95/00655). In some embodiments, the vectors for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides are retroviruses. In some embodiments, the vectors for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides are lentiviruses.

Examples of references describing retroviral transduction include Anderson et al., U.S. Pat. No.5,399,346; Mann et al., Cell 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No.4,980,289; Markowitz et al., J. Virol.62:1120 (1988); Temin et al., U.S. Pat. No.5,124,263; International Patent Publication No. WO 95/07358, published Mar.16, 1995, by Dougherty et al.; and Kuo et al., Blood 82:845 (1993). WO 95/07358 describes high efficiency transduction of primary B lymphocytes. See also WO2016040441A1, which is incorporated by reference herein for the purpose and subject matter referenced herein.

In examples in which the vectors encoding co-stimulatory polypeptides and/or ACTR polypeptides are introduced to the host cells using a viral vector, viral particles that are capable of infecting the immune cells and carry the vector may be produced by any method known in the art and can be found, for example in WO 1991/002805A2, WO 1998/009271 A1, and U.S. Patent 6,194,191. The viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with the immune cells.

In some embodiments, RNA molecules encoding any of the co-stimulatory polypeptides and/or the ACTR polypeptides as described herein may be prepared by a conventional method (e.g., in vitro transcription) and then introduced into suitable host cells, e.g., those described herein, via known methods, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035.

In some instances, the nucleic acid encoding a co-stimulatory polypeptide and the nucleic acid encoding a suitable ACTR polypeptide may be cloned into separate expression vectors, which may be introduced into suitable host cells concurrently or sequentially. For example, an expression vector (or an RNA molecule) for expressing the co-stimulatory polypeptide may be introduced into host cells first and transfected host cells expressing the co-stimulatory polypeptide may be isolated and cultured in vitro. An expression vector (or an RNA molecule) for expressing a suitable ACTR polypeptide can then introduced into the host cells that express the co-stimulatory polypeptide and transfected cells expressing both polypeptides can be isolated. In another example, expression vectors (or RNA molecules) each for expressing the co-stimulatory polypeptide and the ACTR polypeptide can be introduced into host cells simultaneously and transfected host cells expressing both polypeptides can be isolated via routine methodology.

In other instances, the nucleic acid encoding the co-stimulatory polypeptide and the nucleic acid encoding the ACTR polypeptide may be cloned into the same expression vector. Polynucleotides (including vectors in which such polynucleotides are operably linked to at least one regulatory element) for expression of the ACTR and co-stimulatory polypeptide are also within the scope of the present disclosure. Non-limiting examples of useful vectors of the disclosure include viral vectors such as, e.g., retroviral vectors including gamma retroviral vectors, adeno-associated virus vectors (AAV vectors), and lentiviral vectors.

In some instances, the nucleic acid(s) encoding the co-stimulatory polypeptide and/or the ACTR polypeptide may be delivered into host cells via transposons. In some instances, the encoding nucleic acid(s) may be delivered into host cells via gene editing, for example, by CRISPR, TALEN, ZFN, or meganucleases.

In some instances, the nucleic acid described herein may comprise two coding sequences, one encoding an ACTR polypeptide as described herein, and the other encoding a polypeptide capable of modulating a co-stimulatory pathway (i.e., a co- stimulatory polypeptide). The nucleic acid comprising the two coding sequences described herein may be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically separate) polypeptides. To achieve this goal, the nucleic acid described herein may contain a third nucleotide sequence located between the first and second coding sequences. This third nucleotide sequence may, for example, encode a ribosomal skipping site. A ribosomal skipping site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of additional open reading frames from one messenger RNA. This third nucleotide sequence may, for example, encode a P2A, T2A, or F2A peptide (see, for example, Kim et al., PLoS One.2011; 6(4):e18556). As a non-limiting example, an exemplary P2A peptide may have the amino acid sequence of

ATNFSLLKQAGDVEENPGP SEQ ID NO.: 151.

In another embodiment, the third nucleotide sequence may encode an internal ribosome entry site (IRES). An IRES is an RNA element that allows translation initiation in an end-independent manner, also permitting the translation of additional open reading frames from one messenger RNA. Alternatively, the third nucleotide sequence may encode a second promoter controlling the expression of the second polypeptide. The third nucleotide sequence may also encode more than one ribosomal skipping sequence, IRES sequence, additional promoter sequence, or a combination thereof.

The nucleic acid may also include additional coding sequences (including, but not limited to, fourth and fifth coding sequences) and may be configured such that the polypeptides encoded by the additional coding sequences are expressed as further independent and physically separate polypeptides. To this end, the additional coding sequences may be separated from other coding sequences by one or more nucleotide sequences encoding one or more ribosomal skipping sequences, IRES sequences, or additional promoter sequences.

In some examples, the nucleic acid (e.g., an expression vector or an RNA molecule as described herein) may comprise coding sequences for both the co-stimulatory polypeptide (e.g., those described herein) and a suitable ACTR polypeptide, the two coding sequences, in any order, being separated by a third nucleotide sequence coding for a P2A peptide (e.g., ATNFSLLKQAGDVEENPGP; SEQ ID NO: 151). As a result, two separate polypeptides, the co-stimulatory polypeptide and the ACTR, can be produced from such a nucleic acid, wherein the P2A portion ATNFSLLKQAGDVEENPG (SEQ ID NO: 152) is linked to the upstream polypeptide (encoded by the upstream coding sequence) and residue P from the P2A peptide is linked to the downstream polypeptide (encoded by the downstream coding sequence). In some examples, the ACTR polypeptide is the upstream one and the co-stimulatory polypeptide is the downstream one. In other examples, the co-stimulatory polypeptide is the upstream one and the ACTR polypeptide is the downstream one.

In some examples, the nucleic acid described above may further encode a linker (e.g., a GSG linker) between two segments of the encoded sequences, for example, between the upstream polypeptide and the P2A peptide.

In specific examples, the nucleic acid described herein is configured such that it expresses two separate polypeptides in the host cell to which the nucleic acid is transfected: (i) the first polypeptide that contains, from the N-terminus to the C-terminus, a suitable ACTR (e.g., any of SEQ ID NOs:1-80 described herein, for example, SEQ ID NO:1 or SEQ ID NO:57), a peptide linker (e.g., the GSG linker), and the

ATNFSLLKQAGDVEENPG (SEQ ID NO: 152) segment derived from the P2A peptide; and (ii) a second polypeptide that contains, from the N-terminus to the C-terminus, the P residue derived from the P2A peptide and the co-stimulatory polypeptide (e.g., any of SEQ ID NOs: 81-140).

In some examples, the genetically engineered immune cells co-express the ACTR construct of SEQ ID NO:57 in combination with a co-stimulatory polypeptide such as 4- 1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some embodiments, the genetically engineered immune cells co-express an ACTR construct of SEQ ID NO:57 in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In other examples, the genetically engineered immune cells co-express the ACTR construct of SEQ ID NO:58 in combination of a co-stimulatory polypeptide such as 4- 1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some embodiments, the genetically engineered immune cells co-express an ACTR construct of SEQ ID NO:58 in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In yet other embodiments, the genetically engineered immune cells co-express the ACTR of SEQ ID NO:1 in combination with a co-stimulatory polypeptide such as 4-1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, CD70, OX40, OX40L, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some

embodiments, the genetically engineered immune cells co-express an ACTR construct of SEQ ID NO:1 in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

Alternatively, the genetically engineered immune cells may co-express an ACTR comprising a CD28 co-stimulatory domain in combination with a co-stimulatory polypeptide that also comprises a CD28 co-stimulatory domain. In other embodiments, the genetically engineered immune cells may co-express an ACTR comprising a CD28 co- stimulatory domain in combination with a co-stimulatory polypeptide that is not CD28, for example, 4-1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR, or CD27. In some embodiments, the genetically engineered immune cells co-express an ACTR comprising a CD28 co-stimulatory domain in combination with a co- stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In some embodiments, the genetically engineered immune cells may co-express an ACTR comprising a CD28 co-stimulatory domain and a CD28 hinge domain in combination with a co-stimulatory polypeptide that also comprises a CD28 co-stimulatory domain. In other embodiments, the genetically engineered immune cells may co-express an ACTR comprising a CD28 co-stimulatory domain and a CD28 hinge domain in combination with a co-stimulatory polypeptide that is not CD28, for example, 4-1BB, 4- 1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. Alternatively or in addition, the ACTR construct disclosed herein may comprise a transmembrane domain of CD8 or a portion thereof. In some embodiments, the genetically engineered immune cells co-express an ACTR comprising a CD28 co-stimulatory domain and a CD28 hinge domain in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In some embodiments, the genetically engineered immune cells may co-express an ACTR comprising a 4-1BB co-stimulatory domain in combination a co-stimulatory polypeptide such as 4-1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some embodiments, the genetically engineered immune cells co- express an ACTR comprising a 4-1BB co-stimulatory domain in combination with a co- stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In some embodiments, the genetically engineered immune cells may co-express an ACTR comprising a CD28 hinge domain in combination with a co-stimulatory polypeptide such as 4-1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some embodiments, the genetically engineered immune cells co- express an ACTR comprising a CD28 hinge domain in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27. Any of the CD28 hinge domain disclosed herein may consists of about 25-45 amino acid residues (e.g., about 35-40 amino acid residues).

In some embodiments, the genetically engineered immune cells may co-express an ACTR that is free of any hinge domain in combination with a co-stimulatory polypeptide, e.g., 4-1BB, 4-1BBL (e.g., a variant of a native 4-1BBL such as those described herein), ICOS, ICOSL, OX40, OX40L, CD70, LIGHT, CD30L, GITRL, CD40, CD40L, TL1A, BAFFR (e.g., a variant of a native BAFFR such as those described herein), or CD27. In some embodiments, the genetically engineered immune cells co-express an ACTR that is free of any hinge domain in combination with a co-stimulatory polypeptide of ICOSL, BAFFR (e.g., a variant of a native BAFFR such as those described herein), LIGHT, CD30L, or CD27.

In some embodiments, the genetically engineered immune cells may co-express an ACTR (e.g., those described herein) and a co-stimulatory polypeptide, which is 4-1BBL. In some instances, the 4-1BBL can be a functional variant of a naturally occurring 4-1BBL (e.g., human 4-1BBL), for example, any of the variants disclosed herein (e.g., 4-1BBL Q89A, 4-1BBL L115A, 4-1BBL K127A, or 4-1BBL Q227A). In some examples, the 4- 1BBL polypeptide is a truncated variant of a naturally occurring counterpart, wherein the truncated variant lacks the cytoplasmic fragment.

In some instances, additional polypeptides of interest may also be introduced into the host immune cells.

Following introduction into the host cells a vector encoding any of the co- stimulatory polypeptides and/or the ACTR polypeptides provided herein, or the nucleic acid encoding the ACTR and/or co-stimulatory polypeptide (e.g., an RNA molecule), the cells may be cultured under conditions that allow for expression of the co-stimulatory polypeptide and/or the ACTR polypeptide. In examples in which the nucleic acid encoding the co-stimulatory polypeptide and/or the ACTR polypeptide is regulated by a regulatable promoter, the host cells may be cultured in conditions wherein the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or in conditions in which the inducing molecule is produced. Determining whether the co-stimulatory polypeptide and/or the ACTR polypeptide is expressed will be evident to one of skill in the art and may be assessed by any known method, for example, detection of the co- stimulatory polypeptide and/or the ACTR polypeptide-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of the co-stimulatory polypeptide and/or the ACTR polypeptide protein by methods including Western blotting, fluorescence microscopy, and flow cytometry.

Alternatively, expression of the ACTR polypeptide may take place in vivo after the immune cells are administered to a subject. As used herein, the term“subject” refers to any mammal such as a human, monkey, mouse, rabbit, or domestic mammal. For example, the subject may be a primate. In a preferred embodiment, the subject is human.

Alternatively, expression of a co-stimulatory polypeptide and/or an ACTR polypeptide in any of the immune cells disclosed herein can be achieved by introducing RNA molecules encoding the co-stimulatory polypeptides and/or the ACTR polypeptides. Such RNA molecules can be prepared by in vitro transcription or by chemical synthesis. The RNA molecules can then be introduced into suitable host cells such as immune cells (e.g., T cells, NK cells, or both T cells and NK cells) by, e.g., electroporation. For example, RNA molecules can be synthesized and introduced into host immune cells following the methods described in Rabinovich et al., Human Gene Therapy, 17:1027- 1035 and WO WO2013/040557.

In certain embodiments, a vector(s) or RNA molecule(s) comprising the co- stimulatory polypeptide and/or the ACTR polypeptide may be introduced to the host cells or immune cells in vivo. As a non-limiting example, this may be accomplished by administering a vector or RNA molecule encoding one or more co-stimulatory polypeptides and/or one or more ACTR polypeptides described herein directly to the subject (e.g., through intravenous administration), producing host cells comprising co- stimulatory polypeptides and/or ACTR polypeptides in vivo.

Methods for preparing host cells expressing any of the co-stimulatory polypeptides and/or the ACTR polypeptides described herein may also comprise activating the host cells ex vivo. Activating a host cell means stimulating a host cell into an activated state in which the cell may be able to perform effector functions (e.g., ADCC). Methods of activating a host cell will depend on the type of host cell used for expression of the co- stimulatory polypeptides and/or ACTR polypeptides. For example, T cells may be activated ex vivo in the presence of one or more molecules including, but not limited to: an anti-CD3 antibody, an anti-CD28 antibody, IL-2, phytohemagglutinin, engineered artificial stimulatory cells or particles, or a combination thereof. The engineered artificial stimulatory cells may be artificial antigen-presenting cells as known in the art. See, e.g., Neal et al., J. Immunol. Res. Ther.2017, 2(1):68-79 and Turtle et al., Cancer J.2010, 16(4):374-381, the relevant disclosures of each of which are hereby incorporated by reference for the purpose and subject matter referenced herein.

In other examples, NK cells may be activated ex vivo in the presence of one or more molecules such as a 4-1BB ligand, an anti-4-1BB antibody, IL-15, an anti-IL-15 receptor antibody, IL-2, IL12, IL-21, K562 cells, and/or engineered artificial stimulatory cells or particles. In some embodiments, the host cells expressing any of the co- stimulatory polypeptides and/or the ACTR polypeptides (ACTR- and/or co-stimulatory polypeptide-expressing cells) described herein are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be evident to one of skill in the art and may include assessing expression of one or more cell surface markers associated with cell activation, expression or secretion of cytokines, and cell morphology.

Methods for preparing host cells expressing any of the co-stimulatory polypeptides and/or the ACTR polypeptides described herein may comprise expanding the host cells ex vivo. Expanding host cells may involve any method that results in an increase in the number of cells expressing co-stimulatory polypeptides and/or ACTR polypeptides, for example, allowing the host cells to proliferate or stimulating the host cells to proliferate. Methods for stimulating expansion of host cells will depend on the type of host cell used for expression of the co-stimulatory polypeptides and/or the ACTR polypeptides and will be evident to one of skill in the art. In some embodiments, the host cells expressing any of the co-stimulatory polypeptides and/or the ACTR polypeptides described herein are expanded ex vivo prior to administration to a subject.

In some embodiments, the host cells expressing the co-stimulatory polypeptides and/or the ACTR polypeptides are expanded and activated ex vivo prior to administration of the cells to the subject. Host cell activation and expansion may be used to allow integration of a viral vector into the genome and expression of the gene encoding a co- stimulatory polypeptide and/or an ACTR polypeptide as described herein. If mRNA electroporation is used, no activation and/or expansion may be required, although electroporation may be more effective when performed on activated cells. In some instances, a co-stimulatory polypeptide and/or an ACTR polypeptide is transiently expressed in a suitable host cell (e.g., for 3-5 days). Transient expression may be advantageous if there is a potential toxicity and should be helpful in initial phases of clinical testing for possible side effects.

Any of the host cells expressing the co-stimulatory polypeptides and/or the ACTR polypeptides may be mixed with a pharmaceutically acceptable carrier to form a

pharmaceutical composition, which is also within the scope of the present disclosure.

The phrase“pharmaceutically acceptable”, as used in connection with compositions of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.“Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and/or non- ionic surfactants. See, e.g. Remington: The Science and Practice of Pharmacy 20^th Ed.

(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions of the disclosure may also contain one or more additional active compounds as necessary for the particular indication being treated and/or for the enhancement of ADCC, preferably those with complementary activities that do not adversely affect each other. Non-limiting examples of possible additional active

compounds include, e.g., IL-2 as well as various agents known in the field and listed in the discussion of combination treatments, below. IV. Combined Immunotherapy of Genetically Engineered Hematopoietic Cells

Described Herein and Fc-Containing Therapeutic Agents

The exemplary ACTR polypeptides of the present disclosure confer antibody- dependent cell cytotoxicity (ADCC) capacity to T lymphocytes and enhance ADCC in NK cells. When the receptor is engaged by an antibody bound to cells, it triggers T-cell activation, sustained proliferation and specific cytotoxicity against the bound cells.

The degree of affinity of CD16 for the Fc portion of Ig is a critical determinant of ADCC and thus to clinical responses to antibody immunotherapy. The CD16 with the V158 polymorphism which has a high binding affinity for Ig and mediates superior ADCC was selected as an example. Although the F158 receptor has lower potency than the V158 receptor in induction of T cell proliferation and ADCC, the F158 receptor may have lower in vivo toxicity than the V158 receptor making it useful in some clinical contexts.

The co-stimulatory polypeptides to be co-expressed with ACTR polypeptides in immune cells would facilitate cell-based immune therapy such as T-cell therapy or NK-cell therapy by inducing the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. Antibody- directed cytotoxicity could be stopped whenever required by simple withdrawal of antibody administration. Clinical safety can be further enhanced by using mRNA electroporation to express the co-stimulatory polypeptides and/or the ACTR polypeptides transiently, to limit any potential autoimmune reactivity.

Thus, in one embodiment, the disclosure provides a method for enhancing efficacy of an antibody-based immunotherapy of a cancer in a subject in need thereof, which subject is being treated with an Fc-containing therapeutic agent such as a therapeutic antibody, which can bind to antigen-expressing cells. The Fc-containing therapeutic agent contains an Fc portion, for example, a human or humanized Fc portion, which can be recognized and bound by the Fc-binding portion (e.g., the extracellular domain of human CD16A) of the ACTR expressed on the engineered immune cells.

The methods described herein may comprise introducing into the subject a therapeutically effective amount an antibody and a therapeutically effective amount of the genetically engineered host cells such as hematopoietic cells, for example, immune cells (e.g., T lymphocytes or NK cells), which co-express a co-stimulatory polypeptide and an ACTR polypeptide of the disclosure. The subject (e.g., a human patient such as a human cancer patient) has been treated or is being treating with an Fc-containing therapeutic agent specific to a target antigen. A target antigen may be any molecule that is associated with a disease or condition, including, but are not limited to, tumor antigens, pathogenic antigens (e.g., bacterial or viral), or antigens present on diseased cells, such as those described herein.

In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms“treat”,“treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. For example, in connection with cancer the term“treat” may mean eliminate or reduce a patient's tumor burden, or prevent, delay or inhibit metastasis, etc.

As used herein the term“therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered (e.g., a first pharmaceutical composition comprising an antibody, and a second pharmaceutical composition comprising a population of T lymphocytes or NK cells that express a co-stimulatory polypeptide and/or an antibody- coupled T-cell receptor (ACTR) construct), the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. Within the context of the present disclosure, the term“therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. A. Enhancing Immune Therapy Efficacy

Host cells (e.g., immune cells such as T cells and NK cells) expressing co- stimulatory polypeptides and ACTR polypeptides described herein are useful for enhancing ADCC in a subject and/or for enhancing the efficacy of an antibody-based immunotherapy and/or for enhancing growth and/or proliferation of immune cells. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has been treated or is being treated with any of the therapeutic antibodies described herein. To practice the method described herein, an effective amount of the immune cells (NK cells and/or T lymphocytes) expressing any of the co-stimulatory polypeptides and the ACTR polypeptides described herein and an effective amount of an antibody, or compositions thereof may be administered to a subject in need of the treatment via a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of the respective agent (e.g., the NK cells and/or T lymphocytes expressing co-stimulatory polypeptides, ACTR polypeptides, antibodies, or compositions thereof) that upon administration confers a therapeutic effect on the subject.

Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender, sex, and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient. In some embodiments, the subject in need of treatment suffers from one or more pathogenic infections (e.g., viral, bacterial, and/or fungal infections).

The methods of the disclosure may be used for treatment of any cancer or any pathogen. Specific non-limiting examples of cancers which can be treated by the methods of the disclosure include, for example, lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, thyroid cancer, hepatocellular cancer, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor.

The methods of this disclosure may also be used for treating infectious diseases, which may be caused by bacterial infection, viral infection, or fungus infection. In such instances, the genetically engineered immune cells can be co-used with an Fc-containing therapeutic agent (e.g., an antibody) that targets a pathogenic antigen (e.g., an antigen associated with the bacterium, virus, or fungus that causes the infection). Specific non- limiting examples of pathogenic antigens include, but are not limited to, bacterial, viral, and/or fungal antigens. Some examples are provided below: influenza virus neuraminidase, hemagglutinin, or M2 protein, human respiratory syncytial virus (RSV) F glycoprotein or G glycoprotein, herpes simplex virus glycoprotein gB, gC, gD, or gE, Chlamydia MOMP or PorB protein, Dengue virus core protein, matrix protein, or glycoprotein E, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV 1, hepatitis B core antigen or surface antigen, diptheria toxin,

Streptococcus 24M epitope, Gonococcal pilin, pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus III (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, or human hepatitis C virus glycoprotein E1 or E2.

In some embodiments, the immune cells are administered to a subject in an amount effective in enhancing ADCC activity by least 20% and/or by at least 2-fold, e.g., enhancing ADCC by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more.

The immune cells are co-administered with an Fc-containing therapeutic agent such as a therapeutic antibody in order to target cells expressing the antigen to which the Fc-containing therapeutic agent binds. In some embodiments, more than one Fc- containing therapeutic agents, such as more than one antibodies can be co-used with the immune cells. Antibody-based immunotherapy may be used to treat, alleviate, or reduce the symptoms of any disease or disorder for which the immunotherapy is considered useful in a subject.

An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term“antibody”

encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof which comprise an Fc region, mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric

antibodies, diabodies, single domain antibodies (e.g., nanobodies), linear antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and an Fc region, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The antibody for use in the present disclosure contains an Fc region recognizable by the co-used ACTR- and/or co- stimulatory polypeptide-expressing immune cells. The Fc region may be a human or humanized Fc region.

Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a“polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human

immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human

immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO

99/58572. The antibodies used herein may be glycosylated (e.g., fucosylated) or

afucoslylated. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as a human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

The immune cells (e.g., T lymphocytes and/or NK cells) expressing any of the co- stimulatory polypeptides and/or the ACTR polypeptides disclosed herein may be

administered to a subject who has been treated or is being treated with an Fc-containing antibody. For example, the immune cells may be administered to a human subject simultaneously with an antibody. Alternatively, the immune cells may be administered to a human subject during the course of an antibody-based immunotherapy. In some examples, the immune cells and an antibody can be administered to a human subject at least 4 hours apart, e.g., at least 12 hours apart, at least 1 day apart, at least 3 days apart, at least one week apart, at least two weeks apart, or at least one month apart.

In some embodiments, the antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof. An antibody that“specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody“specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such,“specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that“specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen.

In some embodiments, an antibody as described herein has a suitable binding affinity for the target antigen (e.g., any one of the targets described herein) or antigenic epitopes thereof. As used herein,“binding affinity” refers to the apparent association constant or K_A. The K_A is the reciprocal of the dissociation constant (K_D). The antibody for use in the methods described herein may have a binding affinity (K_D) of at least 10^-5, 10^-6, 10^-7, 10^-8, 10- 9, 10^-10 M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased K_D. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher K_A (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. In some embodiments, any of the antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005 % (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free]/(Kd+[Free]) It is not always necessary to make an exact determination of K_A, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_A, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The antibodies for use in the immune therapy methods described herein may bind to (e.g., specifically bind to) a target antigen of interest, or a specific region or an antigenic epitope therein. Table 4 below lists exemplary target antigens of interest and exemplary antibodies specific to such. Table 4. Exemplary Target Antigens and Binding Antibodies Thereof

The efficacy of an antibody-based immunotherapy may be assessed by any method known in the art and would be evident to a skilled medical professional. For example, the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or tumor or cancer burden in the subject or tissue or sample thereof. In some embodiments, the immune cells are administered to a subject in need of the treatment in an amount effective in enhancing the efficacy of an antibody-based immunotherapy by at least 20% and/or by at least 2-fold, e.g., enhancing the efficacy of an antibody-based immunotherapy by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more, as compared to the efficacy in the absence of the immune cells expressing the co- stimulatory polypeptide and/or the ACTR polypeptide and/or the antibody.

In any of the compositions or methods described herein, the immune cells (e.g., NK and/or T cells) may be autologous to the subject, i.e., the immune cells may be obtained from the subject in need of the treatment, genetically engineered for expression of the co- stimulatory polypeptides and/or the ACTR polypeptides, and then administered to the same subject. In one specific embodiment, prior to re-introduction into the subject, the autologous immune cells (e.g., T lymphocytes or NK cells) are activated and/or expanded ex vivo.

Administration of autologous cells to a subject may result in reduced rejection of the host cells as compared to administration of non-autologous cells.

Alternatively, the host cells are allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of the co-stimulatory polypeptide and/or the ACTR polypeptide, and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor. In a specific embodiment, the T lymphocytes are allogeneic T lymphocytes in which the expression of the endogenous T cell receptor has been inhibited or eliminated. In one specific embodiment, prior to introduction into the subject, the allogeneic T lymphocytes are activated and/or expanded ex vivo. T lymphocytes can be activated by any method known in the art, e.g., in the presence of anti-CD3/CD28, IL-2, phytohemagglutinin, engineered artificial stimulatory cells or particles, or a combination thereof.

NK cells can be activated by any method known in the art, e.g., in the presence of one or more agents selected from the group consisting of CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, K562 cell line, and/or engineered artificial stimulatory cells or particles. See, e.g., U.S. Patents Nos. 7,435,596 and 8,026,097 for the description of useful methods for expanding NK cells. For example, NK cells used in the compositions or methods of the disclosure may be

preferentially expanded by exposure to cells that lack or poorly express major histocompatibility complex I and/or II molecules and which have been genetically modified to express membrane bound IL-15 and 4-1BB ligand (CDI37L). Such cell lines include, but are not necessarily limited to, K562 [ATCC, CCL 243; Lozzio et al., Blood 45(3): 321-334 (1975); Klein et al., Int. J. Cancer 18: 421-431 (1976)], and the Wilms tumor cell line HFWT (Fehniger et al., Int Rev Immunol 20(3-4):503-534 (2001); Harada H, et al., Exp Hematol 32(7):614-621 (2004)), the uterine endometrium tumor cell line HHUA, the melanoma cell line HMV-II, the hepatoblastoma cell line HuH-6, the lung small cell carcinoma cell lines Lu- 130 and Lu-134-A, the neuroblastoma cell lines NB 19 and N1369, the embryonal carcinoma cell line from testis NEC 14, the cervix carcinoma cell line TCO-2, and the bone marrow- metastasized neuroblastoma cell line TNB 1 [Harada, et al., Jpn. J. Cancer Res 93: 313-319 (2002)]. Preferably the cell line used lacks or poorly expresses both MHC I and II molecules, such as the K562 and HFWT cell lines. A solid support may be used instead of a cell line. Such support should preferably have attached on its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and/or a proliferative response or capable of binding a molecule having such an affect thereby acting as a scaffold. The support may have attached to its surface the CD137 ligand protein, a CD137 antibody, the IL-15 protein or an IL-15 receptor antibody. Preferably, the support will have IL-15 receptor antibody and CD137 antibody bound on its surface.

In one embodiment of the described compositions or methods, introduction (or re- introduction) of T lymphocytes, NK cells, or T lymphocytes and NK cells to the subject is followed by administering to the subject a therapeutically effective amount of IL-2.

In accordance with the present disclosure, patients can be treated by infusing therapeutically effective doses of immune cells such as T lymphocytes or NK cells comprising a co-stimulatory polypeptide and/or an ACTR polypeptide of the disclosure in the range of about 10⁵ to 10¹⁰ or more cells per kilogram of body weight (cells/Kg). The infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient, but can be determined by the treating physician for a particular patient. Typically, initial doses of approximately 10⁶ cells/Kg will be infused, escalating to 10⁸ or more cells/Kg. IL-2 can be co-administered to expand infused cells. The amount of IL-2 can about 1-5 x 10⁶ international units per square meter of body surface.

In some embodiments, the antibody is administered to the subject in one or more doses of about 100-500 mg, 500-1000 mg, 1000-1500 mg or 1500-2000 mg. In some embodiments, the antibody is administered to the subject in one or more doses of about 500 mg, about 600 mg, about 700 mg, about 800 mg, or about 900 mg. In some embodiments, the antibody is administered to the subject in one or more doses of about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, or about 1800 mg. In some embodiments, the antibody is administered to the subject in one or more doses of about 1600 mg.

The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example,“about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively,“about” can mean a range of up to ± 20%, preferably up to ± 10%, more preferably up to ± 5%, and more preferably still up to ± 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term“about” is implicit and in this context means within an acceptable error range for the particular value.

The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history. The appropriate dosage of the antibody used will depend on the type of cancer to be treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody can be administered to the patient at one time or over a series of treatments. The progress of the therapy of the disclosure can be easily monitored by conventional techniques and assays.

The administration of the antibody can be performed by any suitable route, including systemic administration as well as administration directly to the site of the disease (e.g., to a tumor).

In some embodiments, the method involves administering the antibody to the subject in one dose. In some embodiments, the method involves administering the antibody to the subject in multiple dose (e.g., at least 2, 3, 4, 5, 6, 7, or 8 doses). In some embodiments, the antibody is administered to the subject in multiple doses, with the first dose of the antibody administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the immune cells expressing the co-stimulatory polypeptide and/or the ACTR polypeptide. In some embodiments, the first dose of the antibody is administered to the subject between about 24-48 hours prior to the administration of the immune cells expressing the co- stimulatory polypeptide and/or the ACTR polypeptide.

In some embodiments, the antibody is administered to the subject prior to

administration of the immune cells expressing the co-stimulatory polypeptide and/or the ACTR polypeptide and then subsequently about every two weeks. In some embodiments, the first two doses of the antibody are administered about one week (e.g., about 6, 7, 8, or 9 days) apart. In certain embodiments, the third and following doses are administered about every two weeks.

In any of the embodiments described herein, the timing of the administration of the antibody is approximate and includes three days prior to and three days following the indicated day (e.g., administration every three weeks encompasses administration on day 18, day 19, day 20, day 21, day 22, day 23, or day 24).

The efficacy of the compositions or methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional. For example, the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or cancer burden in the subject or tissue or sample thereof. In some embodiments, the antibody-based immunotherapy is assessed based on the safety or toxicity of the therapy (e.g., administration of the antibody and the immune cells expressing the co-stimulatory polypeptides and/or the ACTR polypeptides) in the subject, for example by the overall health of the subject and/or the presence of adverse events or severe adverse events.

B. Combination Treatments

The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth, or anti-infection therapy. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure.

When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

The treatments of the disclosure can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 41BB, OX40, etc.).

Non-limiting examples of other therapeutic agents useful for combination with the immunotherapy of the disclosure include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeutic compounds such as, e.g., pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (etoposide and teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan,

dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L- asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors

(letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (brefeldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); AKT inhibitors (such as MK-22062HCl, Perifosine (KRX-0401), GSK690693, Ipatasertib (GDC-0068), AZD5363, uprosertib, afuresertib, or triciribine); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors;

mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

For examples of additional useful agents see also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

The efficacy of the methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional. For example, the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or cancer burden in the subject or tissue or sample thereof. In some embodiments, the antibody-based immunotherapy is assessed based on the safety or toxicity of the therapy in the subject, for example by the overall health of the subject and/or the presence of adverse events or severe adverse events. V. Kits for Therapeutic Use

The present disclosure also provides kits for use of the compositions described herein. For example, the present disclosure also provides kits for use of an antibody and a population of immune cells (e.g., T lymphocytes or NK cells, constructed in vitro or in vivo) that express a co-stimulatory polypeptide and/or an antibody-coupled T-cell receptor (ACTR) construct in enhancing antibody-dependent cell-mediated cytotoxicity, enhancing an antibody-based immunotherapy, and/or enhancing immune cell growth and/or proliferation. Such kits may include one or more containers comprising a first pharmaceutical composition that comprises a population of T lymphocytes and/or NK cells (immune cells) that express a co-stimulatory polypeptide and/or an antibody-coupled T-cell receptor (ACTR) construct such as those described herein, and a second pharmaceutical composition that comprises an antibody and a pharmaceutically acceptable carrier.

In some embodiments, the kit described herein comprises a co-stimulatory polypeptide-expressing and/or ACTR-expressing immune cells which are expanded in vitro, and an antibody specific to a cell surface antibody that is present on activated T cells, for example, an anti-CD5 antibody, an anti-CD38 antibody or an anti-CD7 antibody. The co-stimulatory polypeptide-expressing and/or ACTR-expressing immune cells may express any of the ACTR constructs known in the art or disclosed herein.

Alternatively, the kit disclosed herein may comprise a nucleic acid or a nucleic acid set as described herein, which collectively encodes any of the ACTR polypeptides and any of the co-stimulatory polypeptides as also described herein.

In some embodiments, the kit can additionally comprise instructions for use in any of the methods described herein. The included instructions may comprise a description of administration of the first and second pharmaceutical compositions to a subject to achieve the intended activity, e.g., enhancing ADCC activity, and/or enhancing the efficacy of an antibody-based immunotherapy in a subject and/or enhancing the growth and/or proliferation of immune cells. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. In some embodiments, the instructions comprise a description of administering the first and second pharmaceutical compositions to a subject who is in need of the treatment. The instructions relating to the use of the first and second pharmaceutical compositions described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also

contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. At least one active agent in the second pharmaceutical composition is an antibody as described herein. At least one active agent in the first pharmaceutical composition is a population of immune cells (e.g., T lymphocytes or NK cells) that express an antibody-coupled T-cell receptor (ACTR) construct and/or a co-stimulatory polypeptide as described herein.

Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed.1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.

1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed.1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (lRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES

Example 1: Impact of expressing a ligand from the tumor necrosis factor (TNF)

superfamily in combination with an ACTR polypeptide on T cell function. T cells were transduced with a virus encoding an ACTR polypeptide and a TNF superfamily ligand separated by a P2A ribosomal skip sequence. In a first experiment, ACTR 1 (SEQ ID NO: 58) was co-expressed in T cells with 4-1BBL (SEQ ID NO: 92), CD70 (SEQ ID NO: 103), or LIGHT (SEQ ID NO: 112). T cells were incubated at a 2:1 E:T ratio with OAW42 target cells, which express FOLRa, and an anti-FOLRa antibody (23 nM) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 40-48 hours. Supernatants were subsequently removed for cytokine analysis.

In a separate experiment, ACTR 2 (SEQ ID NO: 57) was co-expressed in T cells with 4-1BBL (SEQ ID NO: 92). T cells were incubated at a 2:1 E:T ratio with IGROV-1 target cells, which express FOLRa, and an anti-FOLRa antibody (20 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 40-48 hours. Supernatants were subsequently removed for cytokine analysis.

Supernatants were analyzed for IL-2 using a homogenous time resolved fluorescence (HTRF) assay (Cisbio). Briefly, the cytokine standards and conjugates were prepared according to the manufacturer’s protocol. In a low volume 384-well plate, conjugate and cell supernatant were mixed in a final volume of 20 µL and co-incubated for 24 hours. The fluorescence signal was measured using an EnVision Multi-label plate reader and data was analyzed according to the manufacturer’s recommendations.

Measured IL-2 was plotted as a function of the TNF receptor family protein co- expressed with ACTR in the T cell for ACTR 1 co-expressed in T cells with 4-1BBL, CD70, or LIGHT (Figure 1, panel A) and ACTR 2 co-expressed in T cells with 4-1BBL (Figure 1, panel B). These results demonstrated that 4-1BBL, CD70, and LIGHT enhanced T cell function, as measured by IL-2 release, relative to T cells that expressed ACTR alone in the presence of target cells and a cognate targeting antibody. Example 2: Impact of expressing a variant of TNF superfamily ligand 4-1BBL in

combination with an ACTR polypeptide on T cell function.

T cells were transduced with virus encoding an ACTR 1 and 4-1BBL (SEQ ID NO: 92) or 4-1BBL lacking the cytoplasmic domain (SEQ ID NO: 93) separated by a P2A ribosomal skip sequence. T cells were incubated at a 2:1 E:T ratio with OAW42 target cells, which express FOLRa, and an anti-FOLRa antibody (0 - 70 nM) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 40-48 hours. Supernatants were subsequently removed for cytokine analysis.

Measured IL-2 was plotted as a function of antibody concentration for each T cell variant (Figure 2, panel A). These results demonstrated that T cells co-expressing ACTR and 4-1BBL lacking the cytoplasmic domain enhanced T cell function, as measured by IL-2 release, relative to T cells that expressed ACTR and full-length 4-1BBL in the presence of target cells and a cognate targeting antibody.

In a separate set of experiments, T cells were incubated at a 2:1 E:T ratio with IGROV-1 cells and 20 µg/mL anti-FOLRa antibody or at a 4:1 E:T ratio with fixed OVCAR8 cells and 10 µg/mL anti-FOLRa antibody in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 8 days. For experiments with IGROV-1 cells, co-cultures were harvested and stained with an anti- CD3 antibody and a live-dead cell stain. The number of live, CD3-positive cells was evaluated by flow cytometry as a measure of T cell proliferation. For experiments with fixed OVCAR8 cells, cells were harvested and the ATP content, a measure of live cells, was evaluated using an ATPlite 1step Luminescence Assay System (Perkin Elmer) according to the manufacturer’s instructions. The ATPlite luminescence signal was used as a measure of T cell proliferation.

Proliferation, relative to ACTR co-expressed with full-length 4-1BBL, was plotted as a function of T cell and target cell (Figure 2, panel B). These results demonstrated that T cells co-expressing ACTR and 4-1BBL lacking the cytoplasmic domain enhanced T cell function, as measured by proliferation, relative to T cells that expressed ACTR and full- length 4-1BBL in the presence of target cells and a cognate targeting antibody. Example 3: Impact of expressing TNF superfamily ligand BAFFR in combination with an ACTR polypeptide on T cell function.

T cells were transduced with virus encoding an ACTR 2 alone or ACTR 2 (SEQ ID NO: 57) and BAFFR (SEQ ID NO: 101) separated by a P2A ribosomal skip sequence. T cells were incubated at a 2:1 E:T ratio with IGROV-1 target cells, which express FOLRa, and an anti-FOLRa antibody (0– 20 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 8 days. Co-cultures were harvested and stained with an anti-CD3 antibody and a live-dead cell stain. The number of live, CD3-positive cells was evaluated by flow cytometry as a measure of T cell proliferation.

Proliferation was plotted as a function of antibody concentration for T cells expressing ACTR alone or co-expressing ACTR and BAFFR (Figure 3). These results demonstrated that T cells co-expressing ACTR and BAFFR enhanced T cell function, as measured by proliferation, relative to T cells that expressed ACTR alone in the presence of target cells and a cognate targeting antibody. Example 4: Impact of expressing B7/CD28 and TNF superfamily receptors in

combination with an ACTR polypeptide on T cell function.

T cells were transduced with virus encoding ACTR 2 (SEQ ID NO: 57) alone or in combination with CD40 (SEQ ID NO: 106), OX40 (SEQ ID NO: 115), ICOS (SEQ ID NO: 84), and 4-1BB (SEQ ID NO: 91) separated by a P2A ribosomal skip sequence. T cells were incubated at a 2:1 E:T ratio with IGROV-1 target cells, which express FOLRa, and an anti- FOLRa antibody (1 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 8 days. Co- cultures were harvested and stained with an anti-CD3 antibody and a live-dead cell stain. The number of live, CD3-positive cells was evaluated by flow cytometry as a measure of T cell proliferation.

Proliferation was plotted as a function of T cell variant (Figure 4). These results demonstrated that T cells co-expressing ACTR and CD40, OX40, ICOS, or 4-1BB enhanced T cell function, as measured by proliferation, relative to T cells that expressed ACTR alone in the presence of target cells and a cognate targeting antibody. Example 5. T Cells Co-Expressing ACTR and Co-stimulatory Molecule Showed

Enhanced Activity

This example demonstrates that co-expressing tumor necrosis factor (TNF) or B7/CD28 superfamily members or ligands thereof and an ACTR construct in T cells enhances the activity of the T cell relative to the ACTR alone in the presence of target cells and a targeting antibody.

In these experiments, T cells were transduced with virus encoding ACTR 2 (SEQ ID NO: 57) and a TNF or B7/CD28 superfamily protein separated by a P2A ribosomal skip sequence. In these experiments ACTR 2 was co-expressed in T cells with ICOSL (SEQ ID NO: 85), CD30L (SEQ ID NO: 105); BAFFR (SEQ ID NO: 101), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115). ACTR 2 was used as a control in this experiment. T cells were incubated at a 2:1 E:T ratio with IGROV-1 target cells, which express FOLRa, and an anti- FOLRa antibody (10 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 40-48 hours. Supernatants were removed for cytokine analysis.

Measured IL-2 is plotted as a function of T cell variant (Figure 5). These results demonstrate that T cells co-expressing ACTR and ICOSL, CD30L, BAFFR, CD27, or OX40 enhance T cell function, as measured by IL-2 release, relative to T cells that express ACTR alone in the presence of target cells and a cognate targeting antibody. Example 6. T Cells Co-Expressing ACTR and Co-Stimulatory Molecules Showed

Enhanced Proliferation in the Presence of Solid Tumor-related Inhibitory Agents.

This example demonstrates that expressing tumor necrosis factor (TNF) superfamily costimulatory polypeptide 4-1BB in T cells in combination with ACTR enhances the activity of the T cell relative to ACTR alone in the presence of target cells, a targeting antibody, and inhibitors that are present in solid tumor microenvironments. In these experiments, T cells were transduced with virus encoding an ACTR polypeptide (SEQ ID NO: 57) alone or an ACTR polypeptide and 4-1BB (SEQ ID NO: 91) separated by a P2A ribosomal skip sequence. Transduced T cells were incubated at a 2:1 E:T ratio with live IGROV-1 target cells, which express FOLRa, an anti-FOLRa antibody (1 µg/mL), and varying concentrations of PGE₂ (0– 16 µM), TGF-beta (0– 10 ng/mL), or adenosine (0– 2 mM) in RPMI 1640 media supplemented with 10 % fetal bovine serum. For experiments with adenosine, erythro- 9-(2-Hydroxy-3-nonyl)adenine hydrochloride (EHNA; 1 µM) was added to reactions after 48 hours. In a separate experiment, T cells were incubated at a 4:1 E:T ratio with fixed IGROV- 1 target cells, which express FOLRa, an anti-FOLRa antibody (1 µg/mL), and varying concentrations of kynurenine (0– 1 mM) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. For experiments with live target cells, T cell proliferation was evaluated using flow cytometry by measuring live, CD3-positive cells. For experiments with fixed target cells, T cell proliferation was evaluated by measuring ATP content in the reaction using an ATPlite 1 step kit (Perkin Elmer) according to the manufacturer’s recommendation.

Proliferation (live CD3+ cell count or ATP content) is plotted as a function of inhibitor concentration for T cells expressing ACTR alone (SEQ ID NO: 57) and ACTR in combination with 4-1BB (SEQ ID NO: 91) (Figure 6). These experiments demonstrate that T cells expressing ACTR in combination with 4-1BB show superior proliferation relative to T cells expressing ACTR alone in the presence of the inhibitors PGE₂ (Figure 6, panel A), TGF- beta (Figure 6, panel B), and adenosine (Figure 6, panel C); in the presence of the inhibitor kynurenine (Figure 6, panel D), the T cells show similar proliferative capacity. These experiments demonstrate that co-expressing TNF family member polypeptides like 4-1BB in T cells that also express ACTR can enhance T cell activity in the presence of inhibitors that are known to be present in solid tumor microenvironments. Example 7. T Cells Co-Expressing ACTR and Co-Stimulatory Molecules Showed

Enhanced Proliferation Under Chronic Stimulation Conditions.

This example demonstrates that expressing tumor necrosis factor (TNF) superfamily costimulatory polypeptides 4-1BB, CD27, or OX40 in T cells in combination with ACTR enhances the activity of the T cell relative to ACTR alone in chronic stimulation assays. In these experiments, T cells were transduced with virus encoding an ACTR polypeptide (SEQ ID NO: 57) alone or an ACTR polypeptide and 4-1BB (SEQ ID NO: 91), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115) separated by a P2A ribosomal skip sequence.

Transduced T cells were incubated at a 2.7:1 E:T ratio with fixed IGROV-1 target cells, which express FOLRa, and anti-FOLRa antibody (1 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum in a 250-µL reaction volume. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. Cells were mixed and 50 µL was removed for analysis. T cell proliferation was evaluated by measuring ATP content in the reaction using an ATPlite 1 step kit (Perkin Elmer) according to the manufacturer’s recommendation. Another 150-µL aliquot was removed, cells were pelleted, resuspended in media, and restimulated with 30,000 fixed IGROV-1 target cells in the presence of anti- FOLRa antibody (1 µg/mL). Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. Cells were mixed and 50 µL was removed for analysis. T cell proliferation was evaluated by measuring ATP content in the reaction using an ATPlite 1 step kit (Perkin Elmer) according to the manufacturer’s recommendation.

Fold-change in proliferation relative to ACTR alone (ACTR parent) was plotted as a function of co-expressed TNF family member polypeptide for the first stimulation and second stimulation rounds of the experiment (Figure 7, panel A). These results demonstrate that T cells co-expressing ACTR and 4-1BB, CD27, or OX40 show similar proliferation to T cells expressing ACTR alone (ACTR parent) after the first stimulation. T cells co-expressing ACTR and 4-1BB or OX40 show superior proliferation to T cells expressing ACTR alone (ACTR parent) after the second stimulation; T cells co-expressing ACTR and CD27 show similar proliferation to T cells expressing ACTR alone (ACTR parent) after the second stimulation.

In a similar experiment, chronic stimulation was carried out in the absence or presence of inhibitors PGE₂ and TGF-beta. T cells were incubated at a 4:1 E:T ratio with fixed IGROV-1 target cells, which express FOLRa, and anti-FOLRa antibody (1 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum in a 250-µL reaction volume. Some reactions also contained either PGE₂ (16 nM) or TGF-beta (0.1 ng/mL). Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. Cells were mixed and 75 µL was removed for analysis. T cell proliferation was evaluated by measuring ATP content in the reaction using an ATPlite 1 step kit (Perkin Elmer) according to the manufacturer’s recommendation. A 100-µL aliquot was removed and restimulated with 20,000 fixed IGROV-1 target cells in the presence of anti-FOLRa antibody (1 µg/mL); some reactions also contained either PGE₂ (16 nM) or TGF-beta (0.1 ng/mL). Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. Cells were mixed and 100 µL was removed for analysis. T cell proliferation was evaluated by measuring ATP content in the reaction using an ATPlite 1 step kit (Perkin Elmer) according to the manufacturer’s recommendation.

Fold-change in proliferation relative to ACTR alone (ACTR parent) was plotted as a function of co-expressed TNF family member polypeptide for the second stimulation round of the experiment (Figure 7, panel B). These results demonstrate that T cells co-expressing ACTR and CD27, or OX40 show superior proliferation to T cells expressing ACTR alone (ACTR parent) in the presence of PGE2. T cells co-expressing ACTR and OX40 also show superior proliferation to T cells expressing ACTR alone (ACTR parent) in the presence of TGF-beta. These experiments demonstrate that co-expressing TNF family member polypeptides like 4-1BB, CD27, or OX40 in T cells that also express ACTR can enhance T cell activity under chronic stimulation conditions in the absence and presence of inhibitors that are known to be present in solid tumor microenvironments. Example 8. Activity of T cells Co-Expressing ACTR and Co-stimulatory Molecules in the Presence of Regulatory T Cells

This example demonstrates that expressing tumor necrosis factor (TNF) superfamily costimulatory polypeptides 4-1BB, 4-1BBL, CD27, or OX40 in T cells in combination with ACTR enhances the activity of the T cell relative to ACTR alone in the presence of suppressive regulatory T cells. Inducible regulatory T cells (Tregs) were isolated and expanded from PBMCs. Briefly, CD4+ CD127-/dim cells were isolated from PBMCs using a Regulatory T Cell Isolation Kit II (Miltenyi) according to the manufacturer’s protocol.

Isolated cells were expanded by stimulating with Human Treg Expander Dynabeads (Gibco) every 4– 6 days and culturing in RPMI 1640 media supplemented with 10 % fetal bovine serum in the presence of human IL-2 (1000 U/mL), human TGF-beta (10 ng/mL), and rapamycin (100 nM). Cells were maintained at approximately 0.5 x 10⁶ cells per mL throughout the expansion. Cell phenotype was monitored by flow cytometry using anti-CD4, anti-CD25, anti-CD127, and anti-FoxP3 antibodies. Activated inducible Tregs were defined as a cell population with >10 µm diameter and a phenotype of >95 %

CD4+/CD25high/FoxP3+/CD127dim. Activated inducible Tregs were used in subsequent experiments.

In these experiments, T cells were transduced with virus encoding an ACTR polypeptide (SEQ ID NO: 57) alone or an ACTR polypeptide and 4-1BB (SEQ ID NO: 91), CD27 (SEQ ID NO: 102), or OX40 (SEQ ID NO: 115) separated by a P2A ribosomal skip sequence. Transduced T cells were incubated at a 1:1 E:T ratio with live IGROV-1 target cells, which express FOLRa, an anti-FOLRa antibody (1 µg/mL) in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were carried out in the absence or presence of donor-matched inducible Tregs at varying ratios relative to T cells (T cell:Treg = 1:0, 4:1, 2:1, or 1:1). Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. Supernatants were removed from each reaction after 4 days and IFN-gamma levels were measured using a Human IFN-gamma Kit (Cisbio). The amount of IFN-gamma is plotted as a function of condition for ACTR T cells (SEQ ID NO: 57) made from two different donors (Figure 8, panel A). These data demonstrate a dose-dependent inhibition of IFN-gamma in the presence of increasing amounts of inducible Tregs. The percentage of maximum IFN-gamma, determined by the amount of IFN-gamma produced relative to a matched reaction in the absence of inducible Tregs, is plotted as a function of TNF superfamily polypeptide co-expressed with ACTR in T cells (Figure 8, panel B). Experiments in Donor 1 were carried out with a 4:1 T cell:Treg ratio; experiments in Donor 2 were carried out with a 1:1 T cell:Treg ratio. IFN-gamma production from T cells expressing ACTR alone (parent) is suppressed in the presence of Tregs to a level that is 30– 40 % that of cells in the absence of Tregs in both donors. Similar results are observed with T cells co-expressing both ACTR and 4-1BB in both donors and with T cells co-expressing ACTR and OX40 and T cells co-expressing ACTR and CD27 in Donor 1. T cells co-expressing ACTR and 4-1BBL are more resistant to Treg suppression than T cells expressing ACTR alone as evidenced by higher relative IFN-gamma production; T cells co-expressing ACTR and OX40 and T cells co-expressing ACTR and CD27 also show more resistance to Treg suppression than T cells expressing ACTR alone in Donor 2.

These experiments demonstrate that co-expressing TNF family member polypeptides like 4-1BBL, CD27, or OX40 in T cells that also express ACTR can enhance T cell activity by making them more resistant to suppression by Tregs, which are known to be present in solid tumor microenvironments. Example 9. Effect of Myeloid-Derived Suppressor Cells on T Cells Co-expressing

ACTR and Co-stimulatory Molecule This example demonstrates that expressing tumor necrosis factor (TNF) superfamily costimulatory polypeptides 4-1BBL, CD27, OX40, and CD40 in T cells in combination with ACTR enhances the activity of the T cell relative to ACTR alone in the presence of suppressive myeloid-derived suppressor cells (MDSCs). MDSCs were isolated and differentiated from PBMCs. Briefly, CD14-positive cells were isolated using the EasySep Human CD14 Positive Selection Kit II (Gibco) according to the manufacturer’s protocol. CD14+ cells were cultured in RPMI 1640 media supplemented with 10 % fetal bovine serum in the presence of GMCSF (10 ng/mL) and PGE₂ (1 ng/mL). Cells were incubated in a CO₂ (5 %) incubator at 37 degrees C for 6 days. Cultures were supplemented with GMCSF (10 ng/mL) and PGE₂ (1 ng/mL) on day 2; on day 4, media was removed and replenished with fresh RPMI 1640 supplemented with 10 % fetal bovine serum and GMCSF (10 ng/mL) and PGE₂ (1 ng/mL). Cells were harvested for use in assays as MDSCs at day 6. Cells were characterized by flow cytometry to confirm that they were CD14_low/HLA- DRlow/CD33high/PDL1high.

In these experiments, T cells were transduced with virus encoding an ACTR polypeptide (SEQ ID NO: 57) alone or an ACTR polypeptide and 4-1BBL (SEQ ID NO: 92), CD27 (SEQ ID NO: 102), OX40 (SEQ ID NO: 115), or CD40 (SEQ ID NO: 106) separated by a P2A ribosomal skip sequence. Transduced T cells were incubated at a 2:1 E:T ratio with live IGROV-1 target cells, which express FOLRa, an anti-FOLRa antibody (1 µg/mL), and recombinant human annexin V (1 µg/mL) to block phagocytosis of activated T cells by MDSCs in RPMI 1640 media supplemented with 10 % fetal bovine serum. Reactions were carried out in the absence or presence of donor-matched MDSCs at a 1:3 ratio with T cells. The total reaction volume was 215 µL. Reactions were incubated in a CO₂ (5 %) incubator at 37 degrees C for 7 days. At day 4, reactions were supplemented with 50 µL of RPMI 1640 media supplemented with 10 % fetal bovine serum. Cells were analyzed by flow cytometry after staining with an anti-CD3 antibody, and a viability dye. The number of live CD3+ cells were measured and the percentage of maximum proliferation was determined by dividing the number of cells observed in reactions in the presence of MDSCs by that in the absence of MDSCs for each T cell variant.

The percentage of maximal proliferation was plotted as a function of TNF

superfamily polypeptide co-expressed with ACTR in T cells (Figure 9). The presence of MDSCs suppresses the proliferation of T cells expressing ACTR alone (parent) to approximate 30 % of that observed without MDSCs. Proliferation of T cells co-expressing ACTR and 4-1BBL, OX40, CD40, and CD27 are either minimally or not suppressed by MDSCs, demonstrating superior proliferative ability relative to T cells expressing ACTR alone in the presence of MDSCs.

These experiments demonstrate that co-expressing TNF family member polypeptides like 4-1BBL, CD27, OX40, or CD40 in T cells that also express ACTR can enhance T cell activity by making them more resistant to suppression by MDSCs, which are known to be present in solid tumor microenvironments. OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an

alternative feature serving the same, equivalent, or similar purpose. Thus, unless

expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one of skill in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.” The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of” or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.,“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

WHAT IS CLAIMED IS: 1. A genetically engineered hematopoietic cell, wherein the hematopoietic cell co- expresses:

(i) an antibody-coupled T cell receptor (ACTR) polypeptide; wherein the ACTR polypeptide comprises:

(a) an extracellular Fc binding domain;

(b) a transmembrane domain; and

(c) a cytoplasmic signaling domain; and

(ii) a co-stimulatory polypeptide, wherein the co-stimulatory polypeptide is a member of the B7/CD28 superfamily, a member of the tumor necrosis factor (TNF) superfamily, or a ligand thereof, and wherein the co-stimulatory polypeptide is encoded by an exogenous nucleic acid.

2. The hematopoietic cell of claim 1, wherein the co-stimulatory polypeptide is a member of the B7/CD28 superfamily or a ligand thereof, which is selected from the group consisting of CD28, CD80, CD86, ICOS, ICOSL, B7-H3, B7-H4, VISTA, TMIGD2, B7-H6, and B7-H7.

3. The hematopoietic cell of claim 2, wherein the member of the B7/CD28 superfamily or the ligand thereof is selected from the group consisting of CD80, CD86, ICOS, and ICOSL.

4. The hematopoietic cell of claim 2 or claim 3, wherein the B7/CD28 superfamily member or the ligand thereof lacks a cytoplasmic domain.

5. The hematopoietic cell of claim 1, wherein the co-stimulatory polypeptide is a member of the TNF superfamily or a ligand thereof, which is selected from the group consisting of 4-1BB, 4-1BBL, BAFF, BAFFR, CD27, CD70, CD30, CD30L, CD40, CD40L, DR3, GITR, GITRL, HVEM, LIGHT, TNF-beta, OX40, OX40L, RELT, TACI, TL1A, TNF- alpha, TNFRII, BCMA, EDAR2, TROY, LTBR, EDAR, NGFR, OPG, RANK, DCR3, TNFR1, FN14 (TweakR), APRIL, EDA-A2, TWEAK, LTb (TNF-C), NGF, EDA-A1, APP amyloid precursor protein (APP), and TRAIL.

6. The hematopoietic cell of claim 5, wherein the member of the TNF superfamily or the ligand thereof is selected from the group consisting of 4-1BB, 4-1BBL, BAFF, BAFFR, CD27, CD70, CD30, CD30L, CD40, CD40L, GITR, GITRL, HVEM, LIGHT, OX40, OX40L, and TL1A.

7. The hematopoietic cell of claim 6, wherein the 4-1BBL is 4-1BBL Q89A, 4-1BBL L115A, 4-1BBL K127A, or 4-1BBL Q227A.

8. The hematopoietic cell of claim 6 or claim 7, wherein the member of the TNF superfamily or the ligand thereof lacks a cytoplasmic domain.

9. The hematopoietic cell of claim 8, wherein the co-stimulatory polypeptide is 4-1BBL, and wherein the 4-1BBL lacks a cytoplasmic domain.

10. The hematopoietic cell of any one of the preceding claims, wherein the co-stimulatory polypeptide is free of a FK506 binding protein (FKBP).

11. The hematopoietic cell of any one of claims 1-10, wherein the ACTR polypeptide further comprises at least one co-stimulatory signaling domain.

12. The hematopoietic cell of any one of claims 1-10, wherein the ACTR polypeptide is free of any co-stimulatory signaling domain.

13. The hematopoietic cell of any of claims 1-12, wherein the cytoplasmic signaling domain (c) in the ACTR polypeptide comprises an immunoreceptor tyrosine-based activation motif (ITAM).

14. The hematopoietic cell of any one of claims 1-13, wherein (c) is located at the C- terminus of the ACTR polypeptide.

15. The hematopoietic cell of any one of claims 1-14, wherein the ACTR polypeptide further comprises a hinge domain, which is located at the C-terminus of (a) and the N- terminus of (b).

16. The hematopoietic cell of any one of claims 1-15, wherein the ACTR polypeptide further comprises a signal peptide at its N-terminus.

17. The hematopoietic cell of any one of claims 1-16, wherein the Fc binding domain of (a) is selected from the group consisting of:

(A) an extracellular ligand-binding domain of an Fc-receptor,

(B) an antibody fragment that binds the Fc portion of an immunoglobulin, (C) a naturally-occurring protein that binds the Fc portion of an immunoglobulin or an Fc-binding fragment thereof, and

(D) a synthetic polypeptide that binds the Fc portion of an immunoglobulin.

18. The hematopoietic cell of claim 17, wherein the Fc binding domain is (A), which is an extracellular ligand-binding domain of an Fc-gamma receptor, an Fc-alpha receptor, or an Fc- epsilon receptor.

19. The hematopoietic cell of claim 18, wherein the Fc binding domain is an extracellular ligand-binding domain of CD16A, CD32A, or CD64A.

20. The hematopoietic cell of claim 19, wherein the Fc binding domain is an extracellular ligand-binding domain of F158 CD16A or V158 CD16A.

21. The hematopoietic cell of claim 17, wherein the Fc binding domain is (B), which is a single chain variable fragment (scFv), or a single domain antibody.

22. The hematopoietic cell of claim 17, wherein the Fc binding domain is (C), which is Protein A or Protein G, or an Fc-binding fragment thereof.

23. The hematopoietic cell of claim 17, wherein the Fc binding domain is (D), which is a Kunitz peptide, a SMIP, an avimer, an affibody, a DARPin, or an anticalin.

24. The hematopoietic cell of any one of claims 1-23, wherein the transmembrane domain of (b) is of a single-pass membrane protein.

25. The hematopoietic cell of claim 24, wherein the transmembrane domain is of a membrane protein selected from the group consisting of CD8a, CD8b, 4-1BB, CD28, CD34, CD4, FceRIg, CD16A, OX40, CD3z, CD3e, CD3g, CD3d, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B.

26. The hematopoietic cell of any one of claims 1-23, wherein the transmembrane domain of (b) is a non-naturally occurring hydrophobic protein segment.

27. The hematopoietic cell of any one of claims 11 and 13-26, wherein the at least one co-stimulatory signaling domain is of a co-stimulatory molecule selected from the group consisting of 4-1BB, CD28, CD28_LL^GG variant, OX40, ICOS, CD27, GITR, ICOS, HVEM, TIM1, LFA1, and CD2.

28. The hematopoietic cell of claim 27, wherein the at least one co-stimulatory signaling domain is a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain.

29. The hematopoietic cell of any one of claims 11 and 13-28, wherein the ACTR polypeptide comprises two co-stimulatory signaling domains.

30. The hematopoietic cell of claim 29, wherein the two co-stimulatory domains are:

(i) CD28 and 4-1BB; or

(ii) CD28LL_^GG variant and 4-1BB.

31. The hematopoietic cell of claim 29, wherein one of the co-stimulatory signaling domains is a CD28 co-stimulatory signaling domain; and wherein the other co-stimulatory domain is selected from the group consisting of a 4-1BB co-stimulatory signaling domain, an OX40 co-stimulatory signaling domain, a CD27 co-stimulatory signaling domain, and an ICOS co-stimulatory signaling domain.

32. The hematopoietic cell of any one of claims 1-31, wherein the cytoplasmic signaling domain of (c) is a cytoplasmic domain of CD3z or FceR1g.

33. The hematopoietic cell of any one of claims 15-32, wherein the hinge domain is 1 to 60 amino acids in length.

34. The hematopoietic cell of any one of claims 15-33, wherein the hinge domain is of CD28, CD16A, CD8a, or IgG.

35. The hematopoietic cell of any one of claims 15-33, wherein the hinge domain is a non-naturally occurring peptide.

36. The hematopoietic cell of claim 35, wherein the hinge domain is an extended recombinant polypeptide (XTEN) or a (Gly₄Ser)_n polypeptide, in which n is an integer of 3-12, inclusive.

37. The hematopoietic cell of any one of claims 1-14 and 16-32, wherein the ACTR polypeptide is free of any hinge domain.

38. The hematopoietic cell of any one of claims 1-32, wherein the ACTR polypeptide is free of a hinge domain from any non-CD16A receptor.

39. The hematopoietic cell of claim 1, wherein the ACTR polypeptide comprises components (a)-(e) as shown in Table 3.

40. The hematopoietic cell of any one of claims 1-39, wherein the ACTR polypeptide comprises the amino acid sequence selected from SEQ ID NOs: 1-80.

41. The hematopoietic cell of claim 28, wherein the ACTR polypeptide comprises a CD28 co-stimulatory domain; and wherein the co-stimulatory polypeptide is ICOS, ICOSL, 4-1BB, BAFFR, CD27, CD40, OX40, 4-1BBL, CD70, CD30L, or LIGHT.

42. The hematopoietic cell of claim 41, wherein the ACTR polypeptide further comprises a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof, optionally wherein the CD28 transmembrane domain consists of about 30-45 amino acid residues, preferably wherein the CD28 transmembrane domain consists of about 35-40 amino acid residues.

43. The hematopoietic cell of claim 41, wherein the ACTR polypeptide lacks a hinge domain.

44. The hematopoietic cell of any one of claims 41-43, wherein the co-stimulatory polypeptide is 4-1BBL.

45. The hematopoietic cell of claim 44, wherein the 4-1BBL is 4-1BBL Q89A, 4-1BBL L115A, 4-1BBL K127A, or 4-1BBL Q227A.

46. The hematopoietic cell of claim 44 or claim 45, wherein the 4-1BBL lacks a cytoplasmic domain.

47. The hematopoietic cell of any one of claims 41-46, wherein the co-stimulatory polypeptide is ICOSL, BAFFR, LIGHT, CD30L, or CD27.

48. The hematopoietic cell of any one of claims 44-47, wherein the ACTR polypeptide comprises SEQ ID NO: 57 or SEQ ID NO: 58.

49. The hematopoietic cell of any one of claims 1-48, wherein the hematopoietic cell is a hematopoietic stem cell or an immune cell, optionally wherein the immune cell is a natural killer cell, macrophage, neutrophil, eosinophil, or T cell.

50. The hematopoietic cell of claim 49, wherein the immune cell is a T cell, in which the expression of an endogenous T cell receptor, an endogenous major histocompatibility complex, an endogenous beta-2-microglobulin, or a combination thereof has been inhibited or eliminated.

51. The hematopoietic cell of any one of claims 1-50, wherein the hematopoietic cell is derived from peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSCs), or inducible pluripotent stem cells (iPSCs).

52. The hematopoietic cell of any one of claims 1-51, wherein the hematopoietic cell comprises a nucleic acid or nucleic acid set, which collectively comprises:

(A) a first nucleotide sequence encoding the co-stimulatory polypeptide; and (B) a second nucleotide sequence encoding the antibody-coupled T cell receptor (ACTR) polypeptide.

53. The hematopoietic cell of claim 52, wherein the nucleic acid or the nucleic acid set is an RNA molecule or a set of RNA molecules.

54. The hematopoietic cell of claim 52 or 53, wherein the hematopoietic cell comprises the nucleic acid, which comprises both the first nucleotide sequence and the second nucleotide sequence.

55. The hematopoietic cell of claim 54, wherein the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosomal skipping site, an internal ribosome entry site (IRES), or a second promoter.

56. The hematopoietic cell of claim 55, wherein the third nucleotide sequence encodes a ribosomal skipping site, which is a P2A peptide.

57. The hematopoietic cell of any one of claims 53-56, wherein the nucleic acid or the nucleic acid set is comprised within a vector or a set of vectors.

58. The hematopoietic cell of claim 57, wherein the vector or set of vectors is an expression vector or a set of expression vectors.

59. The hematopoietic cell of claim 57 or 58, wherein the vector or set of vectors comprises one or more viral vectors.

60. The hematopoietic cell of claim 59, wherein the one or more viral vectors is a retroviral vector, which optionally is a lentiviral vector or a gammaretroviral vector.

61. A pharmaceutical composition, comprising a hematopoietic cell of any one of claims 1-60, and a pharmaceutically acceptable carrier.

62. The pharmaceutical composition of claim 61, wherein the composition further comprises an Fc-containing therapeutic agent.

63. The pharmaceutical composition of claim 62, wherein the Fc-containing therapeutic agent is a therapeutic antibody or an Fc fusion protein.

64. The pharmaceutical composition of claim 62 or 63, wherein the Fc-containing therapeutic agent binds to a target antigen, which optionally is a tumor antigen or a pathogenic antigen, or the Fc-containing therapeutic agent binds an immune cell specific to an autoantigen.

65. The pharmaceutical composition of claim 64, wherein the tumor antigen is associated with a hematologic tumor, and optionally wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, Kappa-chain, CD30, CD123, CD33, LeY, CD138, CD5, BCMA, CD7, CD40, and IL-1RAP.

66. The pharmaceutical composition of claim 64, wherein the tumor antigen is associated with a solid tumor, and optionally wherein the tumor antigen is selected from the group consisting of GD2, GPC3, FOLR, HER2, EphA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, Mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD70, MUC16, L1-CAM, and CAIX.

67. The pharmaceutical composition of claim 64, wherein the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen.

68. The pharmaceutical composition of claim 63, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of Adalimumab, Ado- Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab,

Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab, Gemtuzumab, Golimumab, hu14.18K322A, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Trastuzumab,

Tositumomab, Ustekinumab, and Vedolizumab.

69. A kit, comprising:

a first pharmaceutical composition that comprises a hematopoietic cell of any one of claims 1-60, and a pharmaceutically acceptable carrier; and

a second pharmaceutical composition that comprises an Fc-containing therapeutic agent and a pharmaceutically acceptable carrier.

70. The kit of claim 69, wherein the Fc-containing therapeutic agent is an Fc fusion protein or a therapeutic antibody.

71. The kit of claim 69 or claim 70, wherein the Fc-containing therapeutic agent binds to a target antigen, which optionally is a tumor antigen or a pathogenic antigen, and optionally wherein the tumor antigen or pathogenic antigen is set forth in any one of claims 65-67.

72. The kit of claim 69 or claim 70, wherein the Fc-containing therapeutic agent binds an immune cell specific to an autoantigen.

73. The kit of claim 70, wherein the therapeutic antibody is selected from the group consisting of Adalimumab, Ado-Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab, Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab,

Gemtuzumab, Golimumab, hu14.18K322A, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Trastuzumab, Tositumomab, Ustekinumab, and Vedolizumab.

74. A method for inhibiting cells expressing a target antigen in a subject, the method comprising administering to a subject in need thereof a population of the hematopoietic cells set forth in any one of claims 1-60, wherein the subject has been treated or is being treating with an Fc-containing therapeutic agent specific to the target antigen.

75. The method of claim 74, wherein the target antigen is a tumor antigen, which optionally is set forth in claim 65 or claim 66.

76. The method of claim 74, wherein the target antigen is a pathogenic antigen, which optioanlly is a bacterial antigen, a viral antigen, or a fungal antigen.

77. The method of claim 74, wherein the target antigen is an antigen of an immune cell specific to an autoantigen.

78. The method of any one of claims 74-77, wherein the hematopoietic cells are autologous.

79. The method of any one of claims 74-77, wherein the hematopoietic cells are allogeneic.

80. The method of any one of claims 74-79, wherein the hematopoietic cells are activated, expanded, or both ex vivo.

81. The method of any one of claims 74-80, wherein the Fc-containing therapeutic agent is a therapeutic antibody or an Fc fusion protein.

82. The method of claim 81, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of Adalimumab, Ado- Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab,

Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab, Gemtuzumab, Golimumab, hu14.18K322A, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Obinutuzumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Tositumomab, Trastuzumab, Ustekinumab, and Vedolizumab.

83. The method of any one of claims 71-82, wherein the subject is a human patient suffering from a cancer and the target antigen is a tumor antigen.

84. The method of claim 83, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia.

85. The method of claim 83 or 84, wherein the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell

carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer.

86. The method of claim 85, wherein the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin’s lymphoma.

87. The method of claim 80, wherein the hematopoietic cells are immune cells comprising T cells, which are activated in the presence of one or more of anti-CD3 antibody, anti-CD28 antibody, IL-2, phytohemoagglutinin, and an engineered artificial stimulatory cell or particle.

88. The method of claim 80, wherein the hematopoietic cells are immune cells comprising natural killer cells, which are activated in the presence of one or more of 4- 1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21 and K562 cells, and an engineered artificial stimulatory cell or particle.

89. A nucleic acid or nucleic acid set, which collectively comprises:

(A) a first nucleotide sequence encoding an antibody-coupled T cell receptor (ACTR) polypeptide set forth in any one of claims 1 and 11-40; and

(B) a second nucleotide sequence encoding a co-stimulatory polypeptide.

90. The nucleic acid or nucleic acid set of claim 89, wherein the co-stimulatory polypeptide is set forth in any one of claims 2-9.

91. The nucleic acid or nucleic acid set of claim 89 or 90, wherein the nucleic acid or the nucleic acid set is an RNA molecule or a set of RNA molecules.

92. The nucleic acid or nucleic acid set of any one of claims 89-91, wherein the nucleic acid comprises both the first nucleotide sequence and the second nucleotide sequence, and wherein the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, the third nucleotide sequence encoding a ribosomal skipping site, an internal ribosome entry site (IRES), or a second promoter.

93. The nucleic acid or nucleic acid set of claim 92, wherein the ribosomal skipping site is a P2A peptide.

94. The nucleic acid or nucleic acid set of any one of claims 89-93, wherein the nucleic acid or the nucleic acid set is comprised within a vector or a set of vectors.

95. The nucleic acid or nucleic acid set of claim 94, wherein the vector or set of vectors is an expression vector or a set of expression vectors.

96. The nucleic acid or nucleic acid set of claim 94 or 95, wherein the vector or set of vectors comprises one or more viral vectors.

97. The nucleic acid or nucleic acid set of claim 96, wherein the one or more viral vectors is a lentiviral vector or gammaretroviral vector.

98. A method for generating modified hematopoietic cells in vivo, the method comprising administering to a subject in need thereof the nucleic acid or nucleic acid set of any one of claims 89-97.

99. The method of claim 98, further comprising administering to the subject an Fc- containing therapeutic agent specific to the target antigen.