Immunomodulatory fusion proteins and their uses

Abstract

The present disclosure relates to immunomodulatory fusion proteins comprising an extracellular binding domain and an intracellular signaling domain, wherein binding of a target can generate a regulatory signal in a host cell such as a T cell. The present disclosure also relates to the use of immune cells expressing such immunomodulatory fusion proteins in the treatment of certain diseases, such as cancer or infectious diseases.

Description

[0001] Statement regarding the Sequence Listing

[0002] The sequence listing associated with the present application is provided in text format in lieu of a paper copy and is incorporated by reference into the specification herein. The name of the text file containing the sequence listing is 360056_447WO_SEQUENCE_LISTING.txt. The text file is 322 KB and was created on March 5, 2018, and was submitted electronically via EFS-Web. Background Art

[0003] When tumor-reactive T cells were found in tumor-infiltrating lymphocyte (TIL) populations, the development of T cell-based immunotherapies began (Clark et al., Cancer Res. 29:705, 1969). A strategy known as adoptive T cell transfer, in some cases involves the isolation of tumor-infiltrating lymphocytes preselected for tumor reactivity, the clonal expansion of tumor-reactive T cells induced by anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and finally the reinfusion of the expanded cell population into the tumor-bearing patient (along with repeated administration of chemotherapy and IL-2) (Dudley et al., Science 298:850, 2002). This form of adoptive T cell therapy using tumor-infiltrating lymphocytes can be technically cumbersome and leads to complete remission in only a small fraction of patients with melanoma and is rarely effective in other cancers (Besser et al., Clin. Cancer Res. 16:2646, 2010).

[0004] The isolation of tumor-reactive T cell clones led to the development of another immunotherapy approach - the generation of recombinant T cell receptors (TCRs) specific for a particular antigen, which can be introduced into T cells, for example, using a vector delivery system to confer specificity for a desired target, such as a tumor-associated peptide presented by major histocompatibility complex (MHC) molecules expressed on tumor cells (known as human leukocyte antigen (HLA) molecules in humans). Another approach introduces a synthetic receptor, called a chimeric antigen receptor (CAR), which typically contains an antigen-binding domain that can bind, for example, a tumor-specific or associated antigen in the case of anti-tumor therapy, linked to one or more intracellular components containing effector domains, such as a primary signaling domain, such as a TCR signaling domain, or in some contexts a co-stimulatory signaling domain. Different from the administration of TILs, the basic procedure for engineered TCR or CAR T cell immunotherapy is usually to genetically modify human T cells with a transgene encoding a tumor-targeting moiety, expand the recombinant T cells ex vivo, and reinfuse the expanded recombinant T cells into the patient.

[0005] Adoptive T cell therapy using T cells expressing recombinant TCRs has shown promising clinical benefits, especially in certain B cell cancers. However, effective T cell activation often requires or is enhanced by concurrent co-stimulatory signals (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). In the tumor microenvironment, co-stimulatory molecules are often downregulated. As a result, for T cells expressing recombinant TCRs specific for cancer antigens, exogenous stimulation by IL-2 is typically required.

[0006] When the TCR engages a specific peptide presented in MHC on an antigen-presenting cell (APC), activation of the T cell is initiated (Rossy et al., Frontiers in Immunol. 3:1-12, 2012). The point of interaction between the T cell and the APC becomes the immunological synapse, which includes three concentric supramolecular activation clusters (SMACs), comprising a central cSMAC, a peripheral pSMAC, and a distal dSMAC (Rossy et al., Frontiers in Immunol. 3:1-12, 2012). Within the cSMAC, co-stimulatory receptors can recruit signaling molecules to amplify TCR signals. Such co-stimulatory receptors can include CD28 and, in some cases, form microclusters with the TCR to lower the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). Entry of transmembrane proteins expressed by T cells into the cSMAC may be limited by the size of the extracellular domain. For example, CD45 has a large extracellular domain and is typically excluded from the immunological synapse, thereby preventing its ability to inhibit TCR signaling (James and Vale, Nature 487:64-69, 2012).

[0007] There remains a need in the field of immunotherapy for alternative compositions and methods of providing immunomodulatory signals to host cells to treat various diseases (such as cancer or infection). The presently disclosed embodiments address these needs and provide other related advantages. Summary of the Invention

[0008] In certain aspects, the present disclosure relates to a fusion protein comprising an extracellular component containing a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse.

[0009] In some embodiments, the length or spatial distance of the complex formed between the fusion protein and the target, or a portion of such a fusion protein::target complex (typically the extracellular portion of such a complex), is or spans a particular distance, e.g., in some embodiments, is a distance less than or less than about a certain distance. In some aspects, the distance of the fusion protein::target complex (or typically its extracellular portion) is less than or about 50 nm, less than or about 40 nm, less than or about 30 nm, or less than or about 20 nm or equal to or less than or equal to or about 15 nm. In some embodiments, it is or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm, e.g., is or about 14 or 15 nm. In some aspects, the distance is a distance similar to the distance between membranes in an immunological synapse, or is the same, approximately the same, or substantially the same as the distance between the nearest membrane portions relative to the TCR-peptide / MHC complex (e.g., residues of the extracellular domain of the TCR) and the nearest membrane portion (residues of the MHC, e.g., residues of HLA, such as residues of MHC I or MHC II), or the distance spanned by the extracellular portion of such a complex (or the spatial distance spanned by the extracellular portion known to be contained within the synapse, e.g., a complex containing CD8, CD4, CD28, and their respective binding partners or ligands). In some embodiments, the spatial distance of the complex refers to the distance between the membranes of two different cells, where the first cell and the second cell each express a binding partner on their surface, and when the cells approach each other, the binding partners can form a complex between the membranes. In some aspects, the distance is the same, substantially the same, or approximately the same as the distance spanned by the extracellular portion of the complex formed between the TCR and the homologous interaction with the MHC molecule. In some aspects, e.g., when the fusion protein contains a binding domain from a molecule that is typically able to enter the immunological synapse or co-localize with the antigen receptor, the distance is similar to or the same as the distance spanned by the complex formed between the molecule (having the binding domain used in the fusion protein) and its natural binding partner. In some aspects, e.g., when the fusion protein contains a binding domain from a molecule that is typically unable to enter the immunological synapse or typically unable to co-localize with the antigen receptor, the distance is different from, e.g., less than or substantially less than the distance spanned by the complex formed between the molecule (having the binding domain used in the fusion protein or a functional portion thereof) and its natural binding partner.

[0010] In some embodiments, the binding domain within the extracellular component of the fusion protein of the present disclosure comprises a target binding portion of a molecule capable of delivering an inhibitory signal (e.g., an inhibitory molecule, such as an immunosuppressive molecule, e.g., an immunosuppressive receptor or an immune checkpoint molecule). In some aspects, such a molecule is a glycoprotein, a checkpoint family member. In certain embodiments, the fusion protein comprising a glycoprotein, a checkpoint family member is not a B7 or B7-binding molecule or not a CD28-B7-superfamily member (e.g., not CD28, CTLA4, ICOS, or other B7-family binding molecules). Exemplary glycoproteins, checkpoint family members include CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CTLA4 (CD152), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5, or a binding variant of any such molecule. In some embodiments, the binding domain within the extracellular component of the fusion protein of the present disclosure comprises a binding partner of any of the foregoing, or a binding variant of any such molecule. In some aspects of such embodiments, the intracellular portion of the fusion protein includes a signaling domain capable of transmitting a stimulatory signal (e.g., a co-stimulatory signal) to lymphocytes (e.g., T cells, e.g., the co-stimulatory region of CD28, 4-1BB, ICOS, or other co-stimulatory molecules). In some aspects, when the extracellular binding portion is from a checkpoint or immunosuppressive molecule, the intracellular portion of the fusion protein does not include the intracellular signaling domain of an inhibitory molecule such as a checkpoint or immunosuppressive molecule. In some aspects, the fusion protein does not include a primary signaling domain, such as the CD3ζ signaling domain or other domains capable of delivering a primary signal to T cells.

[0011] In some aspects, the extracellular component or its binding portion contains or is a binding domain of a molecule or extracellular domain capable of specifically binding CD200, e.g., the binding portion of CD200R or its variant. In some embodiments, the binding domain is or comprises a binding region of a molecule or extracellular domain capable of specifically binding CD47, e.g., the extracellular domain of SIRP or its CD47-binding region, e.g., the extracellular domain of SIRPα or its CD47-binding region. In some embodiments, the binding domain is capable of binding to a PD-L1 or PD-L2 or LAG3 molecule. Exemplary targets can be one or more proteins whose expression is increased or upregulated in certain cells or tissues, said certain cells or tissues being associated with a disease or disorder to be treated or ameliorated with the fusion proteins and compositions provided herein, e.g., tumor cells or the tumor microenvironment, or binding to receptors that are typically upregulated on immune cells (e.g., lymphocytes infiltrating diseased tissues such as tumors).

[0012] In some embodiments, the extracellular component further comprises one or more additional regions or domains, e.g., from such molecules: molecules different from the derived binding domain or molecules different from those sharing identity with the binding domain. One or more additional extracellular domains can include spacer regions, e.g., spacer regions from immunoglobulin molecules, which can include all or part of the hinge, or constant region domains such as CH2 or CH3 domains, or from another cell surface molecule such as a co-stimulatory receptor, such as CD28. In some aspects, the additional extracellular domain can include a multimerization domain, e.g., a dimerization domain or sequence that can promote homodimerization or heterodimerization with another molecule, e.g., multimerization of two or more fusion proteins. In some embodiments, such domains include a portion of the extracellular domain of the CD28 molecule that includes at least the most membrane-proximal cysteine, and typically the extracellular portion between such cysteine and the membrane, or a modified variant thereof. In some aspects, such domains include the amino acid sequence shown in SEQ ID NO: 32, or a portion thereof, or a variant having, for example, at least 90%, 95% or 99% identity thereto. In some aspects, such domains can be included to assist or promote multimerization. In some embodiments, the fusion protein contains an extracellular component that includes a CD200 binding domain, e.g., the extracellular portion of CD200R (or a portion thereof, e.g., its binding domain), e.g., the extracellular portion of CD200R having the amino acid sequence shown in SEQ ID NO: 25, or the extracellular portion of CD200R encoded by the nucleic acid molecule shown in SEQ ID NO: 2, or its CD200 binding portion or a variant or a binding portion thereof. In some aspects of such embodiments, the extracellular portion of the fusion protein further comprises a portion of the CD28 extracellular domain, e.g., up to about 9 to about 12 amino acids (e.g., 9 amino acids or 12 amino acids), and in some aspects, includes the most membrane-proximal cysteine residue of the CD28 extracellular region. In some such embodiments, the length of the CD200R portion of the extracellular region is reduced by an amount corresponding to the number of additional residues in the CD28-derived portion, e.g., reduced by about 9 to about 12 amino acids (e.g., 9 amino acids or 12 amino acids), or reduced by a sufficient number of amino acids such that the distance spanned by the extracellular portion of the complex between the fusion protein and the CD200 molecule is similar, substantially similar or the same as the distance spanned by the extracellular portion of the complex between human CD200R (e.g., CD200R) and CD200, or the distance spanned by the extracellular portion of the complex between a TCR and an MHC molecule (e.g., MHC I or MHCII) in a homologous interaction with a cognate peptide-MHC complex, or the distance of the immunological synapse.In some aspects, the fusion protein further includes a transmembrane domain, such as CD28 transmembrane, for example, the transmembrane domain encoded by the sequence shown in SEQ ID NO: 4 or a portion thereof, or a modified version thereof such as a variant, which is modified to include additional charged regions or residues or hydrophilic residues to facilitate intermolecular interactions. In some embodiments, the protein further includes a CD28 intracellular signaling domain, such as the co-stimulatory domain of CD28, for example, a domain capable of recruiting one or more adaptor molecules to CD28 in response to ligation. In some aspects, the CD28 intracellular domain includes or is encoded by the nucleotide sequence of SEQ ID NO: 5 or a portion or functional variant thereof.

[0013] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component including a binding domain that specifically binds a target, an intracellular component including an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein:target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD200R, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0014] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component including a binding domain that specifically binds a target, an intracellular component including an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein:target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD200R, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28 and the intracellular signaling domain of CD137 (4-1BB).

[0015] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component including a binding domain that specifically binds a target, an intracellular component including an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD200R, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD137 (4-1BB).

[0016] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein:target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of SIRPα, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0017] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD279 (PD-1), (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0018] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD279 (PD-1), (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0019] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein:target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of TIM3, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0020] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein:target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of LAG3, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0021] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component that links the extracellular and intracellular components, provided that the length of the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse. Wherein, (a) the extracellular component comprises the extracellular portion of CD2, (b) the hydrophobic component comprises the transmembrane domain of CD28, and (c) the intracellular component comprises the intracellular signaling domain of CD28.

[0022] In some embodiments, the present disclosure relates to a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component that links the extracellular and intracellular components, wherein the extracellular portion of the complex formed by specific binding of the fusion protein to the target (fusion protein::target complex) has the following size or spans the following distance: (i) up to the distance between the two cell membranes of an immunological synapse, (ii) up to a distance approximately or substantially the same as the distance spanned by the extracellular portion of the complex, the complex being between a T cell receptor (TCR) and an MHC-peptide complex specifically bound by the TCR, (iii) up to a distance approximately or substantially the same as the distance spanned by the extracellular portion of the complex, the complex being between a native molecule comprising the binding domain and its cognate binding partner, (iii) less than or up to about 40 nm, 25 nm, 20 nm, 15 nm, or 14 nm; or (iv) any combination thereof; wherein the extracellular component is or comprises the extracellular domain of CD95 (Fas) or a functional fragment thereof, and the intracellular component is or comprises the intracellular signaling domain of CD137 (4-1BB) or a functional portion thereof.

[0023] In some embodiments, the present disclosure relates to a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component that links the extracellular and intracellular components, wherein the binding domain has or has at least 95% identity with an inhibitory molecule binding domain, and the intracellular signaling domain has or comprises at least 95% identity with a co-stimulatory or stimulatory molecule binding domain, and wherein the inhibitory molecule is or comprises the extracellular domain of CD95 (Fas) or a functional fragment thereof, and the co-stimulatory or stimulatory molecule is or comprises the intracellular signaling domain of CD137 (4-1BB) or a functional portion thereof.

[0024] In some embodiments, the present disclosure relates to a fusion protein comprising: (a) an extracellular component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 71, (b) a hydrophobic component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 197, and (c) an intracellular component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 13.

[0025] In some embodiments, the present disclosure relates to a fusion protein comprising: (a) an extracellular component comprising a binding domain having the amino acid sequence shown in SEQ ID NO.: 72, (b) a hydrophobic component comprising the amino acid sequence shown in SEQ ID NO: 198, and (c) an intracellular component comprising the amino acid sequence shown in SEQ ID NO.: 36.

[0026] In certain aspects, the present disclosure relates to a nucleic acid molecule encoding a fusion protein as described herein.

[0027] In certain aspects, the present disclosure relates to a vector comprising a nucleic acid molecule encoding a fusion protein as described herein.

[0028] In certain other aspects, the present disclosure relates to a host cell comprising a nucleic acid molecule, vector, or fusion protein encoding a fusion protein as described herein.

[0029] In certain other aspects, provided is a method of increasing the activity of immune cells, which comprises administering to a subject in need thereof an effective amount of a host cell as described herein.

[0030] In other aspects, the present disclosure relates to a method of enhancing or prolonging an immune response, which comprises administering to a subject in need thereof an effective amount of a host cell as described herein.

[0031] In other aspects, the present disclosure provides methods of stimulating antigen-specific T cell responses, the methods comprising administering to a subject in need of enhanced immune cell activity an effective amount of the host cells described herein.

[0032] In other aspects, the present disclosure relates to methods of inhibiting immunosuppressive signaling pathways, comprising administering to a subject in need thereof an effective amount of the host cells described herein.

[0033] In other aspects, the present disclosure relates to methods of treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of the host cells described herein.

[0034] In other aspects, the present disclosure relates to methods of inhibiting the immune resistance of cancer cells, comprising administering to a subject in need thereof an effective amount of the host cells described herein.

[0035] In other aspects, the present disclosure relates to methods of treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of the host cells described herein, wherein the administered host cells are capable of proliferating in an immunosuppressive tumor microenvironment.

[0036] The present disclosure also provides methods of treating an infection, the methods comprising administering to a subject having the infection a therapeutically effective amount of the host cells described herein.

[0037] These and other aspects of the invention will become apparent with reference to the following detailed description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A and 1B shows in primary murine CD8 +CD200R-CD28 constructs highly expressed on T cells. (A) Schematic diagrams of exemplary CD200R-CD28 constructs. Construct "I" contains the extracellular ("EC") and transmembrane ("TM") domains of CD200R and the intracellular ("IC") signaling domain of CD28 (CD200Rtm-CD28). Construct "II" contains the extracellular domain of CD200R and the transmembrane and intracellular domains of CD28 (CD200R-CD28tm). Constructs "III-V" also incorporate a portion of the extracellular domain of CD28 to the juxtamembrane cysteine to promote multimerization and enhance CD28 signaling. To illustrate any additional extracellular amino acids (e.g., from one to about 50 amino acids; for example, the exemplary murine constructs disclosed herein contain an additional nine (9) amino acids, and the exemplary human constructs disclosed herein contain twelve (12) amino acids), some constructs have truncated portions of the extracellular or intracellular domains (e.g., CD200R that retains the N-linked glycosylation site). For example, construct IV has a truncated portion of CD200R with 3 amino acids truncated. For example, construct V has a truncated portion of CD200R with 9 amino acids truncated. Constructs "I", "II", and "V" maintain a shorter spatial distance between cells (e.g., between T cells and antigen-presenting cells), and can colocalize with the TCR in the cSMAC and transmit strong costimulatory signals. (B) Transgenic expression of murine CD200R-CD28 constructs on TCR gag T cells detected by anti-CD200R antibody. The control vector contains green fluorescent protein (GFP).

[0039] Figures 2A to 2G showed that CD200R-CD28 constructs promoted proliferation, accumulation, and effector functions in response to CD200 + tumor target cells in vitro and accumulated in the immunological synapse. Splenocytes from naive TCR gag mice were stimulated in vitro with anti-CD3, anti-CD28, and recombinant human IL-2 (100 U / ml) and transduced with retroviral supernatant for 2 days. Cells were restimulated every 7 days with irradiated FBL and splenocytes and cultured with rhIL-2 (50 U / mL) for up to 3 stimulations. Five to 7 days after the last stimulation, T cells were used for assays. (A) Proliferation of CD200R-CD28 and GFP control TCR gag T cells measured by CellTrace Violet dilution. T cells were stimulated with CD200 - FBL (upper panel) or CD200 + FBL (lower panel) for 3 days. (B) Weekly with irradiated CD200 +During the cycle of FBL and splenocyte stimulation, compared with untransduced TCR gag T cells, during co - culture of transduced TCR gag T cells, preferential expansion / survival of transduced T cells. (C) Enrichment of transduced T cells. Compared with wild - type T cells transduced with an empty GFP control vector, irradiated CD200 + Tumor cells repeatedly restimulated enriched cells transduced with CD200R - 9aas - CD28Cys. (D) Increased CD200R and CD200 signal intensities at the T cell:FBL synapse. Lipid rafts are increased at the immunological synapse (I). The CD200R - 9aas - CD28Cys fusion protein co - localizes with lipid rafts, indicating that the fusion protein is concentrated within the immunological synapse (III, IV). (E) CD200R - CD28 + CD8 + T cells show higher in vitro ability to lyse CD200 + FBL cells. As indicated, target tumor cells were labeled with different dilutions of the fluorescent dye 5,6 - carboxyfluorescein diacetate succinimidyl ester (CFSE). Effector TCR gag T cells transduced with the indicated CD200R - CD28 fusion protein or empty vector control were incubated with CD200 + FBL (CFSE hi ) and a 1:1 mixture of non - specific EL4 (CFSElo) controls at the indicated effector - to - target ratios for 5 hours. The percentage of FBL in the sum of FBL and control tumor cells was determined by flow cytometry. The lysis percentage was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells. (F) Target tumor cells used for the CFSE assay in (G). Target tumor cells were labeled with different dilutions of the fluorescent dye CellTrace Violet (CTV) or CFSE. A 1:1:1 mixture of EL4 cells (CTV + ), CD200 + FBL (CFSEhi) and non - specific EL4 (CFSElo) controls was generated. (G) CFSE cytotoxicity assay. TCR gag T cells were transduced with the CD200R - CD28 receptor or GFP control vector. Effector TCR gag T cells were incubated with CD200 - FBL or CD200 +A 1:1 mixture of FBL and non-specific EL4 control targets was incubated for 4 hours at the indicated effector-to-target ratios. The percentage of FBL in the total of FBL and control tumor cells was determined by flow cytometry. The lysis percentage was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells.

[0040] Figures 3A to 3D It was shown that T cells transduced with CD200R-9aas-CD28Cys preferentially accumulated in response to tumor challenge in vivo and expressed surface proteins consistent with an effector phenotype after injection into cyclophosphamide-treated mice bearing FBL. Transduced TCRs were generated as described in Example 2. gag T cells. (A) Schematic of the experiment. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, CD200R-9aas-CD28Cys (Thy1.1 homozygous) and eGFP control (Thy1.1 heterozygous) TCR gag T cells were co-injected into cyclophosphamide-treated B6 mice bearing FBL at a concentration of 4×10 6 cells per mouse. IL-2 (2×10 4 U / dose) was administered every 2 days. Mice were euthanized on day 8 after T cell transfer, and spleens and inguinal lymph nodes were collected. (B) CD200R-9aas-CD28Cys TCR gag T cells accumulated in the spleen in response to FBL. (LN = lymph node; Spl = spleen). (C) Comparison of surface proteins of T cells transduced to express CD200R-9aas-CD28Cys, T cells transduced with empty vector, and endogenous T cells 3 days after transfer. Compared to control TCR gag T cells, CD200R-9aas-CD28Cys TCR gag T cells expressed reduced CD62L, indicating an effector T cell phenotype. (D) Comparison of surface proteins of T cells transduced to express CD200R-9aas-CD28Cys + T cells, T cells transduced with empty vector, and endogenous T cells 15 days after transfer. Compared to control TCR gag T cells, CD200R-9aas-CD28Cys TCR gag T cells expressed similar levels of cell surface proteins.

[0041] Figures 4A to 4DAdoptive immunotherapy with CD200R-CD28-transduced T cells can eradicate disseminated leukemia. (A) Schematic experimental diagram. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, CD200R-CD28tm, CD200R-CD28Cys, CD200R-9aas-CD28Cys or eGFPTCR 5 T cells were intraperitoneally injected into Cy-treated mice bearing FBL at a dose of 10 gag cells / mouse. As shown, IL-2 (2×10 4 U / dose) was given every 2 days in one group of mice. (B) Representative examples of the expression of cell surface proteins in CD200R-CD28tm-transduced T cells and untransduced T cells on the day of IL-2 injection, as determined by injection flow cytometry. (C) Survival rate of mice treated in the presence of IL-2 injection. (C) Survival rate of mice treated without IL-2 injection. In the absence of IL-2 injection, the transfer of CD200R-9aas-CD28Cys TCR gag T cells significantly improved the survival rate (P < 0.05, log-rank Mantel-Cox test).

[0042] Figures 5A to 5C T cells expressing CD200R-9aas-CD28Cys do not induce detectable autoimmune liver injury or infiltrate normal tissues. (A) Schematic experimental diagram. Cyclophosphamide-treated Alb / Gag mice were injected with 4×10 6 CD200 + FBL cells. Five days later, CD200R-9aas-CD28Cys and eGFP TCR 5 T cells were intraperitoneally injected into cyclophosphamide-treated mice bearing FBL at a dose of 10 gag cells / mouse. As shown, IL-2 (2×10 4 U / dose) was given every 2 days in one group of mice. Three and seven days after transfer, liver injury was evaluated by quantifying the serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). (B) For mice that did not receive T cells, received control T cells expressing GFP, or received T cells expressing CD200R-9aas-CD28Cys, the AST and ALT levels measured 3 and 7 days after transfer did not change due to the treatment. (C) Evaluation of T cell infiltration of normal tissues. Using an antibody specific for the T cell marker CD3, limited presence of T cells in liver tissue was observed (left panel), while CD200R-9aas-CD28Cys TCRgag or control TCR gag There was no significant difference among the recipients of T cells (right panel).

[0043] Figures 6A to 6D Show that the 4-1BB costimulatory signaling domain promotes the accumulation and effector functions of transduced T cells in vitro and promotes the tumor-bearing recipients of transduced T cells to respond to CD200 + Survival of tumor target cells. (A) Schematic diagrams of CD200R-CD28 (“V”), -4-1BB (“VI”), and –CD28-4-1BB (“VII”) constructs. (B) After weekly stimulation with irradiated CD200 + FBL and splenocytes, the transduced TCR gag T cells were expanded relative to untransduced TCR gag T cells. CD200R-4-1BB and CD200R-CD28-4-1BB also promoted the accumulation of transduced T cells in vitro. (C) Using a standard CFSE-based cytotoxicity assay, CD200R-9aas-4-1BB + CD8 + T cells showed higher in vitro lysis of CD200 + FBL cells compared to the control. The percentage of FBL in the sum of FBL and control tumor cells was determined by flow cytometry. The lysis percentage was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL without T cell incubation. (D) CD200R-41BB-transduced T cells also promoted survival relative to the control. C57BL / 6 mice were injected with 4 × 10 6 CD200 + FBL cells. Five days later, CD200R-9aas-CD28, CD200R-9aas-4-1BB, CD200R-9aas-CD28-4-1BB, or eGFP TCR 5 T cells were injected intraperitoneally at a dose of 10 gag cells / mouse into cyclophosphamide-treated mice bearing FBL.

[0044] Figures 7A to 7D Show that human primary T cells transduced to express a WT1-specific TCR and a CD200Rtm-CD28 fusion protein showed enhanced proliferation against target cells expressing CD200 and showed increased cytokine production in response to tumor cells expressing CD200. (A) WT1 126Expression of specific TCR, C4, and CD200Rtm-CD28. (B) Expression of CD200 in T2 and K562 cells. T2 cells exhibit low levels of endogenous CD200 expression. (C) Proliferation of T cells as shown by CFSE. Cells that proliferate in response to antigen show decreased CFSE fluorescence intensity. T cells transduced with both C4 and IFP show enhanced proliferation against target cells expressing low levels of CD200 compared to T cells transduced with C4 alone. (D) Cytokine production measured by flow cytometry in response to exposure to CD200dim tumor cells. T cells transduced with both C4 and IFP CD200Rtm-CD28 show increased cytokine production relative to control T cells transduced with TCR C4 alone.

[0045] Figures 8A to 8E It is shown that a fusion protein comprising the extracellular component of SIRPα and a CD28 costimulatory signaling domain promotes the accumulation and proliferation of transduced T cells in vitro. (A) Schematic of exemplary SIRPα-CD28 constructs. Construct “I” comprises the extracellular (“EC”) and transmembrane (“TM”) domains of SIRPα and the intracellular (“IC”) signaling domain of CD28 (SIRPαtm-CD28). Construct “II” comprises the extracellular domain of SIRPα and the transmembrane and intracellular domains of CD28 (SIRPα-CD28tm). Constructs “III-VI” also incorporate a portion of the extracellular domain of CD28 to the juxtamembrane cysteine to promote multimerization and enhance CD28 signaling. To illustrate additional extracellular amino acids (e.g., nine (12) additional amino acids for murine constructs, or twelve (12) amino acids for human constructs), some constructs have truncated portions of the extracellular or intracellular domains (e.g., SIRPα that retains the N-linked glycosylation site). Construct IV has a truncated portion of SIRPα that is truncated by six amino acids to retain the N-linked glycosylation site. For example, construct V has a truncated portion of SIRPα that is truncated by nine amino acids. For example, construct VI has a truncated portion of SIRPα that is truncated by twenty-three amino acids. Constructs “I”, “II”, and “V” maintain a shorter spatial distance between cells (e.g., between T cells and antigen-presenting cells), and can colocalize with the TCR in the cSMAC and deliver a potent costimulatory signal. (B) After weekly stimulation with irradiated SIRPα + FBL and splenocytes, the transduced TCR gag T cells relative to untransduced TCR gagExpansion of T cells. The SIRPα-CD28 construct promotes the accumulation of transduced T cells in vitro, with SIRPα-9aas-CD28Cys showing enhanced accumulation. (C) Proliferation of T cells transduced with the SIRPα-CD28 construct in the CellTrace Violet (CTV) dilution proliferation assay. T cells expressing the SIRPα-CD28 construct, engineered to maintain the distance between T cells and tumor cells, showed enhanced proliferation relative to untransduced T cells. (D) Killing of CD47 + tumor cells after co-culture with SIRPα-CD28 + T cells. In contrast, tumor cells were not eradicated when co-cultured with T cells that received an empty vector or truncated SIRPα lacking its intracellular domain. (E) Assay for quantifying the killing of CD47 + tumor cells Results of the assay. CD47 + FBL tumor cells were transduced with mCherry. Loss of the red signal indicates killing of the tumor cells. Killing of the tumor cells was tested at effector:target ratios of 10:1, 2:1, and 0.4:1. Even at the lowest effector-to-target ratio, SIRPα-CD28 + T cells killed CD47 + tumor cells.

[0046] Figure 9A and 9BIt is shown that a fusion protein comprising the extracellular component of PD-1 and the CD28 costimulatory signaling domain promotes cytokine production in vitro. (A) Schematic diagrams of exemplary PD-1-CD28 constructs. Construct “I” comprises the extracellular (“EC”) and transmembrane (“TM”) domains of PD-1 and the intracellular (“IC”) signaling domain of CD28 (PD1tm-CD28). Construct “II” comprises the extracellular domain of PD-1 and the transmembrane and intracellular domains of CD28 (PD1-CD28tm). Constructs “III-VII” also incorporate a portion of the extracellular domain of CD28 that is adjacent to the cysteine near the transmembrane end to promote multimerization and enhance CD28 signaling. To illustrate the additional extracellular amino acids (e.g., nine (12) additional amino acids for the murine construct, or twelve (12) amino acids for the human construct), constructs IV-VII have truncated portions of PD-1. Construct IV has a truncated portion of PD-1 with 9 amino acids truncated. Construct V has a truncated portion of PD-1 with 12 amino acids truncated. Construct VI has a truncated portion of PD-1 with 15 amino acids truncated. Construct VII has a truncated portion of PD-1 with 21 amino acids truncated. Constructs “I”, “II”, and “V” maintain a shorter spatial distance between cells (e.g., between T cells and antigen-presenting cells), and can colocalize with the TCR in the cSMAC and deliver a potent costimulatory signal. (B) In response to stimulation with FBL cells that endogenously express the PD-1 ligands, PD-L1 and PD-L2, for 5 hours in the presence of brefeldin A, PD1-CD28 + T cells showed increased cytokine production. Intracellular expression of the effector cytokines IFNγ and TNFα in the stimulated T cells was evaluated by flow cytometry.

[0047] Figure 10 Co-expression of TCR C4 and PD-1IFP (PD1-12aas-CD28Cys, PD1-15aas-CD28Cys, or PD1-21aas-CD28Cys) is shown. T cells transduced with C4 and PD1-12aas-CD28Cys or PD1-15aas-CD28Cys exhibited high transduction efficiency and expression of both proteins.

[0048] Figures 11A to 11CIt is shown that, after stimulation with irradiated FBL cells, a fusion protein comprising the extracellular portion of Fas and the CD28 co-stimulatory signaling domain accumulates in vitro. (A) Schematic representation of exemplary Fas-CD28 constructs. Construct “I” comprises the extracellular (“EC”) and transmembrane (“TM”) domains of Fas and the intracellular (“IC”) signaling domain of CD28 (Fastm-CD28). Construct “II” comprises the extracellular domain of Fas and the transmembrane and intracellular domains of CD28 (Fas-CD28tm). Constructs “III” and “IV” also incorporate a portion of the extracellular domain of CD28 (the portion adjacent to the cysteine near the transmembrane end) to promote multimerization and enhance CD28 signaling. To illustrate the additional extracellular amino acids (e.g., nine (12) additional amino acids for murine constructs, or twelve (12) amino acids for human constructs), construct IV has a truncated portion of Fas, where the extracellular domain of Fas is truncated by nine amino acids. Constructs “I”, “II” and “V” maintain a shorter spatial distance between cells (e.g., between T cells and antigen presenting cells), and can co-localize with the TCR in the cSMAC and deliver a potent co-stimulatory signal. (B) TCR transduced with Fas constructs gag Accumulation of T cells upon multiple stimulations with irradiated FBL cells. All constructs promoted T cell accumulation relative to control T cells. (C) Expression of Fas-CD28 constructs rather than full-length (FL) Fas promoted T cell survival or expansion after multiple stimulations in vitro.

[0049] Figure 12A and 12BShows the structure and expression of a fusion protein comprising the extracellular component of LAG3 and a CD28 co-stimulatory signaling domain. (A) Schematic of exemplary LAG3-CD28 constructs. Construct “I” contains the extracellular (“EC”) and transmembrane (“TM”) domains of LAG3 and the intracellular (“IC”) signaling domain of CD28 (LAG3tm-CD28). Construct “II” contains the extracellular domain of LAG3 and the transmembrane and intracellular domains of CD28 (LAG3-CD28tm). Constructs “III” and “IV” also incorporate a portion of the extracellular domain of CD28 (the portion adjacent to the transmembrane proximal cysteine) to promote multimerization and enhance CD28 signaling. To account for additional extracellular amino acids (e.g., nine (12) additional amino acids for murine constructs or twelve (12) additional amino acids for human constructs), construct IV has a truncated portion of LAG3 where the LAG3 extracellular domain is truncated by nine amino acids. Constructs “I”, “II”, and “V” maintain a shorter spatial distance between cells (e.g., between T cells and antigen-presenting cells) and can co-localize with the TCR in the cSMAC and deliver potent co-stimulatory signals. (B) Murine CD8 + T cells express the LAG3-CD28 constructs. In contrast to control T cells receiving the empty vector, T cells transduced to express the LAG3-CD28 constructs (LAG3tm-CD28, LAG3-CD28tm, LAG3-CD28Cys, LAG3-9aas-CD28Cys) showed expression of the construct.

[0050] Figure 13A and 13BShows the structure and expression of a fusion protein comprising the extracellular component of TIM3 and the CD28 co-stimulatory signaling domain. (A) Schematic diagram of exemplary TIM3-CD28 constructs. Construct "I" contains the extracellular ("EC") and transmembrane ("TM") domains of TIM3 and the intracellular ("IC") signaling domain of CD28 (TIM3tm-CD28). Construct "II" contains the extracellular domain of TIM3 and the transmembrane and intracellular domains of CD28 (TIM3-CD28tm). Constructs "III" and "IV" also incorporate a portion of the extracellular domain of CD28 (the portion adjacent to the transmembrane proximal cysteine) to promote multimerization and enhance CD28 signaling. To account for additional extracellular amino acids (e.g., nine (12) additional amino acids for the murine construct, or twelve (12) amino acids for the human construct), construct IV has a truncated portion of TIM3 where the TIM3 extracellular domain is truncated by nine amino acids. Constructs "I", "II", and "V" maintain a shorter spatial distance between cells (e.g., between T cells and antigen-presenting cells) and can co-localize with the TCR in the cSMAC and deliver a potent co-stimulatory signal. (B) Murine CD8 + T cells expressing the TIM3-CD28 constructs. In contrast to control T cells receiving the empty vector, T cells transduced to express the TIM3-CD28 constructs (TIM3tm-CD28; TIM3-CD28tm; TIM3-CD28Cys; TIM3-9aas-CD28Cys) generally showed expression of the construct.

[0051] Figure 14A and 14B Shows in primary murine CD8 +CD200R-CD28 constructs highly expressed on T cells. (A) Schematic diagrams of representative CD200R-CD28 constructs. Construct "I" contains the extracellular ("EC") and transmembrane ("TM") domains of CD200R and the intracellular ("IC") signaling domain of CD28 (CD200Rtm-CD28). Construct "II" contains the extracellular domain of CD200R and the transmembrane and intracellular domains of CD28 (CD200R-CD28tm). Constructs "III-V" also incorporate a portion of the extracellular domain of CD28 to the juxtamembrane cysteine to promote multimerization and enhance CD28 signaling. To illustrate any additional extracellular amino acids resulting from the incorporation of a portion of the extracellular domain of CD28 (e.g., from one to about 50 amino acids; e.g., the exemplary murine constructs disclosed herein contain an additional three (3) or nine (9) amino acids, and the exemplary human constructs disclosed herein contain an additional nine (9) or twelve (12) amino acids), some constructs have truncated portions of the extracellular or intracellular domains (e.g., CD200R that retains the N-linked glycosylation site). For example, construct IV has a truncated portion of CD200R with 3 amino acids truncated. For example, construct V has a truncated portion of CD200R with 9 amino acids truncated. Constructs "I", "II", and "V" maintain the spatial distance between cells (e.g., between T cells and antigen-presenting cells), and as indicated by the dashed lines, can colocalize with the TCR in the cSMAC and transmit potent co-stimulatory signals. (B) Transgenic expression of murine CD200R-CD28 constructs on TCR gag T cells detected by anti-CD200R antibody.

[0052] Figures 15A to 15N showed that CD200R-CD28 constructs promoted the proliferation, accumulation, and effector functions of T cells stimulated by CD200 + tumor target cells in vitro and accumulated in the immunological synapse. Unless otherwise indicated, all results are representative of at least 2 experiments with similar results. Splenocytes from naive TCR gag mice were stimulated in vitro with anti-CD3, anti-CD28, and recombinant human IL-2 (rhIL-2, 100 U / ml) and transduced with retroviral supernatant for 2 days. Cells were restimulated every 7 days with irradiated FBL and splenocytes and cultured with rhIL-2 (50 U / mL) for up to 3 stimulations. Five to seven days after the last stimulation, T cells were used for assays. (A) Proliferation of CD200R-CD28 and GFP control TCR gag T cells measured by CellTrace Violet (CTV) dilution, relative to unstimulated cells (shaded). With CD200 -FBL (upper panel) or CD200 + FBL (lower panel) stimulated T cells for 3 days. (B) Schematic of truncated CD200R (trCD200R). (C) Costimulation requires the CD28 signaling domain. Transgenic expression of the trCD200R construct on TCR gag T cells detected by anti-CD200R antibody. (D) Proliferation of trCD200R (blue line) and GFP control (red line) TCR gag T cells measured by CellTrace Violet dilution. T cells were stimulated with CD200 + FBL for 3 days. (E) Restimulation with CD200 + FBL enriched CD200R-IFP-transduced T cells. In a cycle of restimulation with irradiated CD200 - (left panel) or CD200 + (right panel) FBL and splenocytes, the transduced TCR gag T cells were enriched in a mixed population including untransduced TCR gag T cells. TCR gag T cells were transduced with CD200R-CD28 (upper panel) or GFP control (lower panel). (F, G) In a cycle of weekly stimulation with irradiated CD200 + FBL and splenocytes, the transduced TCR gag T cells were enriched in a mixed population including untransduced TCR gag T cells. *P < 0.05, **P < 0.01 (t test). (H) CD200R-9aas-CD28Cys + CD8 + T cells showed higher in vitro ability to lyse CD200 + FBL cells. Target tumor cells were labeled with the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). TCR gag T cells were transduced with CD200R-9aas-CD28Cys or mock-transduced cells (black symbols). Effector TCR gag T cells were incubated with a 1:1 mixture of CD200 + FBL and non-specific EL4 control targets at the indicated effector-to-target ratios for 4 hours. The percentage of FBL in the sum of FBL and control tumor cells was determined by flow cytometry. The lysis percentage was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells. (I) Pie chart depicting the response to FBL stimulation at a 1:1 ratio, TCR gagPatterns of cytokine production in T cells. Each slice in the pie chart represents a combination of cytokine staining, including IFNγ, TNFα, and IL-2. (J) Histogram of cytokine production as shown in (I) measured by flow cytometry. The shaded histogram represents GFP control-transduced cells. (K–M) The CD200R-9aas-CD28Cys fusion protein co-localizes with lipid rafts, indicating that the fusion protein is concentrated in the T cell:target contact area, suggesting that the size of the fusion protein can be accommodated in the immunological synapse. Effector T cells expanded in vitro expressing TCR gag were combined with FBL at an E:T ratio of 10:1 for 20 minutes at 37 °C. The conjugates were loaded onto μ-Slide VI.4 chambers (Ibidi) and left for a further 15 minutes. The fixed cells were stained and visualized by microscopy. In Figure 15K , the upper panel shows the contacting cells, while the lower panel shows the non-contacting cells. (N) Expression of LCKY394 in TCR gag T cells transduced with CD200R-9aas-CD28Cys (red line), CD200R-CD28Cys (blue line), and GFP control (black line) stimulated as indicated for 10 minutes.

[0053] Figures 16A to 16E showed that in vivo in response to tumor challenge, T cells transduced with CD200R-9aas-CD28Cys preferentially accumulated and enhanced adoptive immunotherapy of disseminated leukemia. Transduced TCR gag T cells were generated as described in Example 15. C57BL / 6 mice were injected with 4 × 10 6 CD200 + FBL cells. Five days later, CD200R-9aas-CD28Cys (Thy1.1 homozygous) and eGFP (Thy1.1 heterozygous) TCR gag T cells were co-injected into Cy-treated B6 mice bearing FBL at a concentration of 4 × 10 6 cells per mouse. IL-2 (2 × 10 4 U / dose) was administered every 2 days. Mice were euthanized on day 8 after T cell transfer, and spleens and inguinal lymph nodes were collected. (A) Relative to empty vector control TCR gag T cells, CD200R-9aas-CD28Cys TCR gag T cells accumulated in the spleen in response to FBL. (B) The phenotype of IFP-transduced T cells was similar to that of the control. Five days after in vitro stimulation, TCR gagExpression of surface markers on T cells. (C) CD200R-9aas-CD28Cy and empty vector control TCRs in response to FBL gag T cells accumulate in lymph nodes (LN) and spleen (Spl). Calculated by dividing by the empty vector control (Thy1.1 + Thy1.2 + ) TCR gag The percentage of T cells to calculate the fold increase of CD200R-9aas-CD28Cys TCR gag T cells (Thy1.1 homozygous). (D, E) On day 8 (D) and day 15 (E), CD200R-9aas-CD28Cys TCR gag T cells (blue line), control TCR gag T cells (red line), and surface marker expression on endogenous T cells (shaded). Fifteen days after transfer, compared with control TCR gag T cells, CD200R-9aas-CD28Cys TCR gag T cells express similar levels of cell surface proteins.

[0054] Figure 17A and 17B Show the survival rates of mice treated in the presence (A) or absence (B) of IL-2 injection. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, CD200R-9aas-CD28Cys and eGFP TCR 5 T cells were intraperitoneally injected into Cy-treated FBL-bearing mice at a dose of 10 gag cells / mouse (as indicated by the arrow). In one group of mice, IL-2 was given every 2 days for a total of 10 days (2×10 4 U / dose). In the absence of IL-2 injection, the transfer of CD200R-9aas-CD28Cys TCR gag T cells significantly improved the survival rate (P < 0.05, log-rank Mantel-Cox test) (B). In (A), the data are from experiment 1 (n = 3 - 4 mice / group). In (B), the data are from a total of 3 independent experiments (n = 6 - 10 mice / group).

[0055] Figures 18A to 18EShown are human primary T cells transduced to express a WT1-specific TCR and a CD200Rtm-CD28 fusion protein that showed enhanced proliferation against target cells expressing CD200 and showed increased cytokine production in response to CD200-expressing tumor cells. (A) Expression of CD200 on CD34 + cells from healthy donor leukapheresis (upper panel) or leukemic blasts (lower panel). (B) Expression of WT1 126 -specific TCR, TCR C4 and CD200Rtm-CD28 in primary human T cells. The figure shows constructs that incorporate IFP, TCRα, and TCRβ chains. (C,D) Proliferation of T cells as shown by CFSE. Cells that proliferate in response to antigen show reduced CFSE fluorescence intensity. T2 cells pulsed with WT1 126 were used to stimulate T cells transduced with TCR C4 or with TCR C4 and CD200Rtm-CD28. (E) Cytokine production in response to exposure to T2 cells measured by flow cytometry. As shown, titration of T2 cells pulsed with WT1 126 was used to stimulate T cells transduced with TCR C4 alone (upper panel) or T cells transduced with both TCR C4 and an IFP targeting CD200 (lower panel). T cells transduced with both TCR C4 and IFP CD200Rtm-CD28 showed increased cytokine production relative to control T cells transduced with TCR C4 alone.

[0056] Figures 19A to 19C Shows the CD200R-CD28 construct co-expressed with a WT1-specific TCR in primary human T cells. (A) Schematic of a representative CD200R-CD28 construct. (B) Figure showing constructs incorporating IFP, TCRα, and TCRβ chains. (C) Expression of WT1 126 -specific TCR, TCR C4 and CD200R-CD28 fusion protein in primary human T cells.

[0057] Figures 20A to 20D Shows the results of an assay for enrichment of T cells expressing the CD200R-CD28 construct.

[0058] Figures 21A to 21K Shows the effector function assays (cytokine production, cytotoxicity) of T cells expressing the CD200R-CD28 construct.

[0059] Figures 22A to 22DShows the in vivo study results of immunotherapy with T cells expressing the Fas IFP construct. (A) Study design. (B - C) After intraperitoneal inoculation of 4×10 6 tumor cells (day 0) and after cyclophosphamide treatment, in vivo bioluminescence imaging of firefly luciferase + FBL tumors was performed in C57BL / 6 mice at the indicated time points, followed by no further treatment or adoptive transfer of 10 6 GFP - transduced or Fas - CD28 - transduced TCR gag transgenic CD8 + T cells (day 5). Figure 22C The two mice shown represent n = 4 mice. (D) Biodistribution of FBL tumor cells quantified by IVIS imaging. After intraperitoneal inoculation of 4×10 6 tumor cells (day 0) and after cyclophosphamide treatment, FBL tumors in C57BL / 6 mice at the indicated time points, followed by no other treatment (white circles) or adoptive transfer of 10 6 GFP - transduced (black circles) or Fas - CD28 - transduced (red circles) TCR gag transgenic CD8 + T cells (day 5).

[0060] Figures 23A to 23D Shows that a fusion protein containing the extracellular component of Fas and the 4 - 1BB costimulatory signaling domain accumulates and proliferates in vitro after stimulation with tumor cells and also reduces Fas - induced cell death. (A) Schematic of an exemplary Fas - 4 - 1BB construct. The construct contains the Fas extracellular ("EC") domain and the 4 - 1BB transmembrane ("TM") and intracellular ("IC") signaling domains ("Fas - 4 - 1BBtm"). (B) Co - expression of transgenic TCR and Fas - 4 - 1BBIFP (Fas - 4 - 1BBtm) in murine T cells. Retroviral supernatants were generated by transfecting Plat - E cells with DNA constructs encoding only TCR gag or encoding TCR gag and Fas - 4 - 1BBtm (SEQ ID NO.: 187). Naive P14 T cells were stimulated with anti - CD3 and anti - CD28 and then transduced with the retroviral supernatants for 2 days. Five days after stimulation, the transduced T cells were stained with specific antibodies against TCR and Fas and analyzed by flow cytometry. (C) As measured by the CellTrace Violet (CTV) dilution method, TCR gag alone or TCR gagProliferation of Fas-4-1BBtm-transduced T cells. Transduced P14 T cells were stained with CellTrace Violet (CTV) proliferation dye and left unstimulated (left) or stimulated with FBL tumor cells at an effector-to-target ratio of 8:1 for 6 days (right). T cells were then harvested and analyzed by flow cytometry. (D) Cell death Activity of the Fas signaling pathway in: (i) T cells expressing the transgenic TCR gag but lacking Fas expression; (ii) wild-type T cells expressing the transgenic TCR gag ; and (iii) T cells expressing the transgenic TCR gag and Fas-4-1BBtm. P14 T cells were stimulated and transduced with TCR gag or TCR gag +Fas-4-1BBtm IFP. After 7 days, T cells were stained for active caspase-8 expression using the FLICA method to measure cell death via the Fas pathway.

[0061] Figures 24A to 24B Shown is that Fas-4-1BB T cells control tumor growth and promote survival in the ID8 model of ovarian cancer. (A) Results of the assay used to quantify killing of ID8 ovarian tumor cells. Murine transduced T cells (anti-mesothelin TCR or anti-mesothelin TCR + Fas-4-1BBtm) were co-incubated with red fluorescent ID8 ovarian tumor cells for 2 days, and growth of ID8 cells was quantified by analysis. Loss of the red signal indicates killing of tumor cells. (B) Survival of ID8 mice treated with (i) anti-mesothelin TCR cells or (ii) anti-mesothelin TCR + Fas-4-1BBtm cells. In the ID8 murine ovarian cancer model, 5 × 10 6 ID8 tumor cells were implanted and disseminated for 6 weeks. After cyclophosphamide treatment, mice received 10 7 T cells and 5.0 × 10 8 mesothelin-pulsed splenocytes, then IL-2 was injected for a total of 10 days. Mice were treated every two weeks until euthanasia.

[0062] Figures 25A to 25DFas-4-1BB T cells are shown to display greater persistence and promote survival in a KPC mouse model of pancreatic cancer. (A) Ultrasonography images of a healthy mouse with a normal pancreas (left) and a pancreatic tumor in a "breeder" mouse (KPC genetically engineered mouse) (right). (B) Schematic of the experiment. KPC mice were screened by ultrasonography to determine when tumors appeared and were enrolled in the study at approximately 8 weeks of age when tumors were detected. Mice were randomly assigned to treatment groups, treated with cyclophosphamide, and at 14 days after cyclophosphamide, mesothelin-specific T cells and mesothelin peptide-pulsed splenocytes were injected into the mice receiving TCR-T cells, 10 7 each. Starting 14 days after enrollment, T cell / APC infusions (without cyclophosphamide) were repeated every 2 weeks for a total of 3 infusions, and IL-2 was not injected. At the end of the study, the survival of the mice was evaluated. (C) Mice that survived 28 days after the last T cell infusion were bled, and the persistence of transferred T cells was evaluated by detecting T cells with innate markers by flow cytometry. (D) Survival of KPC mice treated with (i) anti-mesothelin TCR cells or (ii) anti-mesothelin TCR + Fas-4-1BBtm cells.

[0063] Figure 26 Survival of mice in an AML model (injected with FBL) treated with (i) TCR gag T cells or (ii) TCR gag + Fas-4-1BBtm T cells is shown. Mice were injected with FBL cells. Five days later, the mice were treated with cyclophosphamide with or without 10 6 T cells. DETAILED DESCRIPTION

[0064] The present disclosure provides fusion proteins that regulate signal transduction in host cells such as immune cells. For example, the fusion proteins of the present disclosure can provide activation or co-stimulatory signals in human T cells, where the T cells can optionally be engineered to have a preferred antigen-specific TCR. These immunomodulatory fusion proteins (IFPs) can interact with ubiquitously expressed targets or targets that are typically upregulated or overexpressed in abnormal cells (such as cancer cells). Such IFPs have an extracellular binding domain and an intracellular signal transduction domain. By transducing T cells with an engineered TCR (e.g., a high-affinity TCR) and a fusion protein of the present disclosure that generates an activation signal, certain embodiments of T cells may no longer require exogenous co-stimulation when interacting with, for example, tumor cells.

[0065] In some aspects, the present disclosure provides host cells (e.g., immune cells such as T cells, dendritic cells, NK cells) comprising an IFP and a vector encoding the IFP, and methods of activating T cells comprising the IFP for various therapeutic applications, including treating a disease (e.g., cancer, infectious disease) in a subject.

[0066] Before elaborating on the invention in more detail, providing definitions of certain terms used herein may be helpful in understanding the invention. Other definitions are set forth throughout the present invention.

[0067] In this specification, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the stated range and, when appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer), unless otherwise stated. In addition, unless otherwise stated, any numerical range associated with any physical feature described herein, such as polymer subunits, dimensions or thickness, should be understood to include any integer within the stated range. As used herein, the term "about" means ±20% of the stated range, value or structure. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the terms "comprising", "having" and "including" are used synonymously, and their terms and variants are intended to be interpreted as non-limiting.

[0068] The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, or those materials or steps that do not materially affect the basic characteristics of the claimed invention. For example, a protein domain, region, module (e.g., a binding domain, hinge region, linker module) or protein (which may have one or more domains, regions, modules) consists essentially of a specific amino acid sequence (e.g., amino or carboxyl termini or amino acids between domains) when the amino acid sequence of the domain, region, module or protein includes extensions, deletions, mutations or combinations thereof that, in combination, contribute at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) to the length of the domain, region, module or protein and do not materially affect (i.e., do not reduce the activity by more than 50%, e.g., not more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) the activity (e.g., the target binding affinity of a binding protein) of the domain, region, module or protein.

[0069] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, molecule, or activity that is not native to the host cell or subject, or any gene, protein, compound, molecule, or activity that is native to the host or host cell but has been altered or mutated such that the structure, activity, or both are different between the native and mutated molecule. In certain embodiments, a heterologous, non-endogenous, or exogenous molecule (e.g., a receptor, ligand) may not be endogenous to the host cell or subject, but the nucleic acid encoding such a molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may integrate into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The terms "homologous" or "homologue" refer to a molecule or activity that is found in, or is derived from, the host cell, species, or strain. For example, a heterologous or exogenous molecule or gene encoding the molecule may be homologous to the native host or host cell molecule or gene encoding the molecule, respectively, but may have an altered structure, sequence, expression level, or a combination thereof. A non-endogenous molecule may be from the same species, a different species, or a combination thereof.

[0070] As used herein, the terms "endogenous" or "native" refer to a gene, protein, compound, molecule, or activity that is normally present in the host or host cell and has not been engineered to be altered.

[0071] As used herein, a "binding domain" (also referred to as a "binding region" or "binding moiety") refers to a molecule, such as a peptide, oligopeptide, polypeptide, or protein, that has the ability to specifically and non-covalently associate, combine, or interact with a target molecule (e.g., CD200, CD47, CD19, CD20, CD22, ROR1, mesothelin, PD-L1, PD-L2, PSMA, WT-1, cyclin-A1). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner of a biomolecule or other target of interest or its binding protein. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or a T cell receptor (TCR), or a functional binding domain or an antigen-binding fragment thereof. Exemplary binding domains include extracellular domains of receptors (e.g., those of CD200R, PD-1, CTLA4, BTLA, CD2, Fas) or their binding portions, ligands (e.g., cytokines such as IL35, chemokines) or their binding portions, single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab) or their binding portions, antigen-binding regions of T cell receptors (TCRs), such as single-chain TCRs (scTCRs) or synthetic polypeptides, selected for their ability to specifically bind to a biomolecule.

[0072] As used herein, "specifically binds" refers to the association or binding of a binding domain or its fusion protein with a target molecule with an affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction, units 1 / M), where the affinity or Ka is equal to or greater than 10 5 M-1, or binds to such a target molecule without significantly associating or binding with any other molecule or component in the sample. Binding domains (or their fusion proteins) can be classified as "high affinity" binding domains (or their fusion proteins) or "low affinity" binding domains (or their fusion proteins). A "high affinity" binding domain refers to one having at least 10 7 M -1 、at least 10 8 M -1 、at least 10 9 M -1 、at least 10 10 M -1 、at least 10 11 M -1 、at least 10 12 M -1 、or at least 10 13 M -1 Ka. A "low affinity" binding domain refers to those having up to 10 7 M -1 、up to 10 6 M -1 、up to 10 5 M -1 Ka. Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a specific binding interaction in units of M (e.g., 10 -5 M to 10 -13 M). In certain embodiments, a binding domain can have "enhanced affinity", which refers to a selected or engineered binding domain that binds more strongly to a target antigen than the wild-type (or parental) binding domain. For example, enhanced affinity may be due to a higher Ka (equilibrium association constant) of the target antigen than that of the wild-type binding domain, or due to a lower Kd (dissociation constant) of the target antigen than the Kd of the wild-type binding domain, or due to a lower dissociation rate (Koff) of the target antigen than the dissociation rate (Koff) of the wild-type binding domain. A variety of assays are known for identifying the binding domains of the present invention that specifically bind a particular target, and for determining the affinity of a binding domain or fusion protein, such as Western blot, ELISA, and analysis (see also, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents thereof).

[0073] As used herein, a "fusion protein" refers to a polypeptide having at least two different domains in a single chain, where the domains are not naturally found together in a protein. Nucleic acid molecules encoding fusion proteins can be constructed using PCR, recombinant engineering, etc., or such fusion proteins can be prepared using protein synthesis methods. The fusion protein can further comprise other components (e.g., covalently attached), such as tags or bioactive molecules. In certain embodiments, a fusion protein expressed or produced by a host cell (e.g., a T cell) is located on the cell surface, where the fusion protein is anchored to the cell membrane, with a portion of the fusion protein outside the cell (e.g., containing a binding domain) and a portion of the fusion protein inside the cell (e.g., containing a signaling domain).

[0074] As used herein, a "hydrophobic moiety" refers to any amino acid sequence having a three-dimensional structure that is thermodynamically stable in a cell membrane and is typically in the range of about 15 amino acids to about 30 amino acids in length. The structure of the hydrophobic moiety can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof. In certain embodiments, the hydrophobic moiety comprises a "transmembrane domain" from a known transmembrane protein, which is part of a transmembrane protein that can insert or span the cell membrane. In further embodiments, the hydrophobic moiety or transmembrane domain can be placed between and linked to the extracellular and intracellular portions of the fusion protein. Additionally, the hydrophobic moiety can be modified to contain charged regions or hydrophilic residues to facilitate intermolecular interactions.

[0075] As used herein, an "intracellular signaling domain" is the intracellular portion of a molecule, such as one of the molecules in a fusion protein of the invention, that can directly or indirectly promote a response, such as a co-stimulatory, positive, or activation of a biological or physiological response in a cell, when an appropriate signal is received. In certain embodiments, the intracellular signaling domain is part of a protein or protein complex that receives a signal upon binding, or that can itself directly bind to a target molecule to transmit a signal to other components in the cell. When the intracellular signaling domain contains one or more signaling domains or motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs), kinase domains, co-stimulatory domains, etc., it can directly promote a cellular response. In other embodiments, the intracellular signaling domain will indirectly promote a cellular response by associating with one or more other proteins, which in turn directly promote the cellular response. In some embodiments, the intracellular signaling domain or a functional fragment thereof can be from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the intracellular signaling domain or a functional fragment thereof does not contain CD3ζ.

[0076] As used herein, a "multimerization domain" refers to a polypeptide molecule or region that directly or indirectly preferentially interacts or associates with another polypeptide molecule or region, where the interaction of the multimerization domain substantially contributes to or effectively promotes multimerization (i.e., formation of a dimer, trimer, tetramer, or higher-order multimer, which can be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer, etc.). For example, multimerization may be attributed to one or more types of molecular forces, including covalent bonds (e.g., disulfide bonds or bridges), ionic bonds, metal bonds, electrostatic interactions, salt bridges, dipole-dipole forces, hydrogen bonds, van der Waals forces, hydrophobic interactions, or any combination thereof. The multimer is stable under appropriate conditions (e.g., physiological conditions, in an aqueous solution suitable for expressing, purifying, or storing a recombinant or engineered protein, or under non-denaturing or non-reducing electrophoresis conditions). Exemplary multimerization domains can contain one or more disulfide bonds, zinc finger motifs, leucine zipper motifs, helix-turn-helix, helix-loop-helix, etc.

[0077] In certain embodiments, the fusion protein may comprise a "linker" that can provide a spacer function to facilitate the interaction of two single-chain fusion proteins or the positioning of one or more binding domains such that the resulting polypeptide structure maintains specific binding affinity for a target molecule or retains signaling activity (e.g., effector domain activity) or both. Exemplary linkers include repeats of Gly x Ser y from 1 to about ten, where x and y are independently integers from 1 to 5.

[0078] A "linking amino acid" or "linking amino acid residue" refers to one or more (e.g., about 2 - 20) amino acid residues between two adjacent motifs, regions, or domains of a fusion protein, such as between a binding domain and an adjacent hydrophobic moiety, or at one or both ends of a hydrophobic moiety. Linking amino acids can be generated by the design of the fusion protein construct (e.g., amino acid residues generated using restriction enzyme sites during the construction of the nucleic acid molecule encoding the fusion protein). In certain embodiments, the linking amino acids form a linker, such as those having repeats of Gly x Ser y from 1 to about ten, where x and y are independently integers from 1 to 5.

[0079] As used herein, an "immune system cell" refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow, which gives rise to two main lineages, namely myeloid progenitors (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitors (which give rise to lymphocytes such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - double negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells may be referred to as "antigen - presenting cells" or "APCs", which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with the TCR on the surface of the T cell.

[0080] A "T cell" is an immune system cell that matures in the thymus and produces a T cell receptor (TCR). T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA compared to T CM ; decreased expression of CD45RO), memory T cells (T M )(cells that have experienced antigen and survive long - term), and effector cells (cells that have experienced antigen and are cytotoxic). T MIt can be further divided into central memory T cells (with increased expression of TCD62L, CCR7, CD28, CD127, CD45RO, and CD95 and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (T EM , compared to naive T cells or T CM , with decreased expression of CD62L, CCR7, CD28, and CD45RA and increased expression of CD127). Effector T cells (T E ) refer to CD8 + cytotoxic T lymphocytes that have experienced antigen, with decreased expression of CD62L, CCR7, and CD28 compared to T CM and being positive for granzyme and perforin. Other exemplary T cells include regulatory T cells such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8 + CD28- and Qa-1 restricted T cells.

[0081] The "T cell receptor" (TCR) refers to a molecule found on the surface of T cells (or T lymphocytes) that associates with CD3 and is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The TCR is a disulfide-linked heterodimer of highly variable α and β chains (also referred to as TCRα and TCRβ, respectively) in most T cells. In a small subset of T cells, the TCR consists of a heterodimer of variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus (see Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). As used in the present invention, the TCR can be from various animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. The TCR can be cell-bound (i.e., having a transmembrane region or domain) or in a soluble form.

[0082] "Major histocompatibility complex molecules" (MHC molecules), which can be used interchangeably and are also understood to refer to the corresponding human leukocyte antigen (HLA molecules) in humans, are glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a transmembrane α chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC (HLA) class I molecules deliver peptides derived from the cytosol to the cell surface, where the peptide:MHC complex (or peptide:human HLA) is recognized by CD8 + T cells. MHC (HLA) class II molecules deliver peptides originating from the vesicular system to the cell surface, where they are recognized by CD4 + T cells. MHC molecules can be from various animal species, including humans, mice, rats, or other mammals.

[0083] A "nucleic acid molecule" or "polynucleotide" can be in the form of RNA or DNA, which includes cDNA, genomic DNA, and synthetic DNA. The nucleic acid molecule can be double-stranded or single-stranded, and if single-stranded, it can be the coding strand or the non-coding strand (antisense strand). The coding molecule can have the same coding sequence as a coding sequence known in the art, or it can have a different coding sequence. Due to the redundancy or degeneracy of the genetic code or through splicing, the coding molecule can encode the same polypeptide.

[0084] Variants of the nucleic acid molecules or polynucleotides of the present disclosure are also contemplated. The variant polynucleotide has at least 80%, preferably at least 85%, 90%, 95%, 99%, or 99.9% identity with one of the polynucleotides of the defined sequences described herein, or hybridizes with one of those polynucleotides of the defined sequences under stringent hybridization conditions (0.015 M sodium chloride, 0.0015 M sodium citrate at about 65 - 68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at about 42 °C). The polynucleotide variant retains the ability to encode a binding domain or a fusion protein thereof having the function described herein.

[0085] The term "stringent" is used to refer to conditions that are generally understood in the art as being stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturants such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65 - 68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at about 42°C (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

[0086] More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide or other denaturants) can also be used; however, the hybridization rate will be affected. In the case of deoxyoligonucleotide hybridization, other exemplary stringent hybridization conditions include washing in 6x SSC, 0.05% sodium pyrophosphate at 37°C (for 14 - base oligonucleotides), 48°C (for 17 - base oligonucleotides), 55°C (for 20 - base oligonucleotides), and 60°C (for 23 - base oligonucleotides).

[0087] "Vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, cosmid, virus, or phage. An "expression vector" is a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector.

[0088] "Retrovirus" is a virus having an RNA genome. "Gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0089] "Lentivirus" refers to a genus of retroviruses capable of infecting both dividing and non - dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0090] In the context of two or more polypeptide or nucleic acid molecule sequences, the terms "identical" or "percent identity" refer to the situation where, when compared and aligned for maximum correspondence over a comparison window or specified region, such as using methods known in the art (e.g., sequence comparison algorithms), by manual alignment, or by visual inspection, two or more sequences or subsequences are identical or have the same specified percentage of amino acid residues or nucleotides in the specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity). For example, preferred algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0091] "Treatment" or "treating" or "ameliorating" refers to the medical management of a disease, disorder or condition in a subject (e.g., a human or non-human mammal, such as a primate, horse, dog, mouse, rat). Generally, a host cell expressing a fusion protein of the present disclosure, along with an appropriate dose or treatment regimen of an optional adjuvant or adjunctive therapy, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic benefits include improving clinical outcomes, alleviating or relieving symptoms associated with the disease, reducing the incidence of symptoms, improving quality of life, longer disease-free status, reducing the severity of the disease, stabilizing the disease state, delaying disease progression, remission, survival, extended survival period, or any combination thereof.

[0092] A "therapeutically effective amount" or "effective amount" of a cell expressing a fusion protein of the present disclosure or of the fusion protein (e.g., CD200R-CD28, SIRPα-CD28, CD200R-41BB, SIRPα-41BB, CD200R-CD28-41BB, SIRPα-CD28-4-1BB or other such fusion proteins) in the context of the disease or condition being treated refers to the amount of the fusion protein or the number of cells that is sufficient to alleviate one or more symptoms of the disease being treated in a statistically significant manner (e.g., reducing infection, reducing tumor size, inhibiting cancer growth, etc.).

[0093] Immunomodulatory fusion protein (IFP)

[0094] In some aspects, the present disclosure provides fusion proteins that include an extracellular component, a hydrophobic component, and an intracellular component. In some embodiments, the extracellular component includes a binding domain, such as a binding domain that specifically binds a target. In some embodiments, the binding domain is from a molecule that is capable of delivering a negative or inhibitory signal when bound to its binding partner or ligand or receptor (such as an immunosuppressive receptor or checkpoint molecule), for example, in its natural environment, or the target is an inhibitory receptor or ligand or checkpoint molecule or other inhibitory ligand. In some embodiments, the intracellular component includes a signaling domain, such as a co-stimulatory signaling domain or a signaling domain of a molecule that is generally capable of delivering a co-stimulatory signal or a positive signal to an immune cell. Thus, in some aspects, the fusion protein is capable of delivering a positive or co-stimulatory signal in response to a binding event that would result in an inhibitory signal in the natural environment.

[0095] In some embodiments, the fusion protein enables a specific distance to be achieved. For example, in some embodiments, the fusion protein::target complex (such as a complex that includes the extracellular portion of the complex formed by specific binding between the fusion protein and the target) has a specific length or spans a specific distance, such as a distance that reaches the distance between membranes in an immunological synapse, or the distance spanned by the extracellular portion of a homologous complex between a TCR and an MHC molecule, for example, after specific recognition by the TCR, or the distance spanned by the extracellular portion of a complex formed between a native molecule and its native binding partner. In some embodiments, when expressed in an immune cell, such as a T cell, or when entering an immunological synapse, the distance or length is sufficient to promote co-localization of the fusion protein with an antigen receptor or other signaling molecule.

[0096] As background, an immunological synapse is an interface between cells, which can form between a variety of cells, such as between immune cells (Rossy et al., Frontiers in Immunol. 3:1-12, 2012; Hatherley et al., Structure 21:820, 2013). For example, in the case of contact between a T cell and an antigen-presenting cell (APC), an immunological synapse can form by binding the TCR (located on the surface of the T cell) to an HLA peptide (MHC peptide bound to a non-human peptide) complex (e.g., found on the surface of the APC; class I HLA molecules can be found on the surface of all nucleated cells, while class II HLA molecules can be conditionally expressed on all cell types but are typically found on APCs). Additionally, immunological synapses can be organized into supramolecular activation clusters (SMACs), which can influence lymphocyte activation, direct presentation of antigen-HLA (or antigen-MHC) complexes to lymphocytes, and direct secretion of cytokines or lytic granules between cells. A SMAC can include three structures arranged in concentric circles: a central region (cSMAC) containing a large number of TCRs as well as co-stimulatory and inhibitory molecules, a peripheral region (pSMAC) where LFA-1 and talin aggregate, and a distal region (dSMAC) rich in CD43 and CD45 molecules. In certain embodiments, the immunological synapse will span from about 10 nm to about 15 nm. For example, protein interactions found in immunological synapses, such as TCR::HLA peptide interactions or fusion protein-target interactions, typically span about 14 nm between membranes. In certain embodiments, the width of the SMAC in an immunological synapse does not exceed 15 nm.

[0097] In some embodiments, the extracellular span of the fusion protein::target complex enables it to localize to specific compartments of the immunological synapse. Complexes that are thought to be located in the various compartments of the immunological synapse are well characterized in terms of the length of their extracellular spans. For example, the MHC-TCR complex is thought to have an extracellular span of about 10-15 nm, while integrin-based complexes are thought to have an extracellular span on the order of about 40 nm (Alakoskela et al., Biophys J 100:2865, 2011). Additional exemplary complexes include the CD2-CD48 complex, which is thought to have an extracellular span of about 12.8 nm (Milstein et al., J Biol Chem 283:34414, 2008). Additionally, exemplary ligand-binding molecules that are thought to localize to the cSMAC include TCR and MHC complexes, CD2, CD4, CD8, CD28, and their ligands (Dustin et al., CSH Perspectives in Biology 2:a002311, 2010); thus, it is expected that these molecules complexed with their native ligands will have an appropriate size to localize to the cSMAC.

[0098] In some aspects, the length or distance or approximate length or distance of a particular construct or its engineered extracellular portion (e.g., the extracellular portion of a fusion protein, or a complex of any of the foregoing (e.g., complexed with its binding partner)) can be determined or modeled by known methods. In certain exemplary models, the input amino acid or nucleic acid sequence can be used to estimate the tertiary structure, binding domains, and other features of the protein. The tertiary structure of the protein can be used to estimate the size, flexibility, and other features that can be used to determine the approximate length of the extracellular portion of the protein or its complex. Generally, methods for modeling or estimating the length of the extracellular portion of a protein are known. For example, molbiol-tools.ca and Swiss-Model contain multiple tools that can be used to predict protein structure (see also Schwede, T., Structure 21:1531, 2013).

[0099] In certain embodiments, the fusion proteins of the present disclosure that complex, associate, or interact with a target are capable of being located within the immunological synapse. In some embodiments, the extracellular portion of the fusion protein::target complex spans the immunological synapse. In other embodiments, the fusion protein::target complex is located in the supramolecular activation cluster (SMAC), such as the cSMAC. In further embodiments, the extracellular portion of the fusion protein::target complex spans the immunological synapse defined by the extracellular portion of the TCR::HLA-peptide interaction. In other embodiments, the length of the extracellular portion of the fusion protein::target complex is from about 12 nm to about 15 nm, or is about 14 nm.

[0100] The distance between the cell membranes of cells interacting in an immunological synapse can be measured by any method known in the art. For example, in certain embodiments, the distance can be measured by sub-diffraction resolution methods or electron microscopy (James and Vale, Nature 487:64-69, 2012).

[0101] In certain embodiments, the fusion proteins disclosed herein comprise an extracellular portion that extends less than 40 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein comprise an extracellular portion that extends less than 30 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein comprise an extracellular portion that extends less than 20 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein comprise an extracellular portion that extends less than 15 nm from the cell membrane.

[0102] In some embodiments, the provided fusion proteins offer the advantage of having an extracellular length or spatial distance compared to the distance between cell membranes that permits entry into the synapse or co-localization with an antigen receptor or mimics the length or distance present in a native protein. In some embodiments, when the extracellular portion of the fusion protein comprises a domain from an additional molecule, the additional molecule being from a different molecule from which the binding domain was obtained, the length of the extracellular component comprising the binding domain is reduced, e.g., truncated compared to the extracellular region of the native molecule, to provide a similar length or distance. In some embodiments, the fusion proteins described herein comprise an extracellular component that comprises the extracellular domain of a cell surface receptor and a second domain (e.g., a linker or extracellular domain of a second cell surface receptor). In some such embodiments, one or more domains of the extracellular component can be truncated in order to maintain an extracellular component that can remain within or span the immunological synapse when complexed with a target molecule.

[0103] In certain diseases (e.g., cancer), due to the imbalance between co-stimulatory signals and inhibitory signals, the amplitude and quality of the T cell response caused by antigen recognition by the T cell receptor (TCR) may be dysregulated (e.g., reduced), which can lead to immune resistance. One advantage of certain fusion proteins of the present disclosure is that the first signal can be converted into a second signal of a different nature. For example, in some embodiments, the fusion protein enables a negative or inhibitory signal to be effectively converted into a positive or co-stimulatory signal, thereby alleviating or minimizing immune resistance associated with diseases such as cancer. For example, in the binding to a target, if bound by its natural binding partner, it will result in inhibition or delivery of a negative signal. In some embodiments, the fusion proteins provided herein are capable of alternatively delivering a positive, e.g., co-stimulatory signal, to the cells that express it, such as T cells. In certain embodiments, the fusion proteins of the present invention comprise an extracellular component associated with a negative signal and an intracellular component associated with a positive signal. An exemplary receptor found on the surface of T cells, cytotoxic T lymphocyte-associated antigen 4 (CTLA4 or CD152), can receive an inhibitory signal when bound to one of the ligands CD80 or CD86 found on APCs. CTLA4 regulates the amplitude of early T cell activation by counteracting the T cell co-stimulatory receptor CD28 (see Rudd et al., Immunol. Rev. 229:12, 2009). Another exemplary receptor found on the surface of T cells, programmed cell death protein 1 (PD-1 or CD279), can receive an inhibitory signal when bound to one of its ligands PD-L1 (B7-H1, CD274) or PD-L2 (B7-DC, CD73) found on APCs. PD-1 restricts the activity of T cells in peripheral tissues during inflammation and minimizes autoimmunity (see Keir et al., Annu. Rev. Immunol. 26:677, 2008). Representative fusion proteins of the present disclosure comprising an extracellular component associated with a negative signal (e.g., CTLA4 or PD-1) and an intracellular component associated with a positive signal (e.g., CD28, CD137) include CTLA4-CD28 fusion proteins, CTLA4-CD137 fusion proteins, CTLA4-CD28-CD137 fusion proteins, PD1-CD28 fusion proteins, PD1-CD137 fusion proteins or PD1-CD28-CD137 fusion proteins.

[0104] The fusion proteins of the present disclosure can block or reduce the number of inhibitory signals received by immune cells. For example, in some embodiments, the fusion proteins disclosed herein convert inhibitory signals into positive signals, thereby reducing the total number of inhibitory signals received by immune cells or converting normal negative or inhibitory signals into positive signals. In other embodiments, the fusion proteins disclosed herein block the signal transduction of wild-type receptors. For example, dominant-negative fusion proteins are included within the scope of the present disclosure. In some embodiments, the fusion proteins disclosed herein bind to wild-type receptors and block the signal transduction of wild-type receptors by forming oligomers with wild-type receptors.

[0105] Another advantage of certain fusion proteins of the present disclosure is that cells can express more than one such fusion protein, thereby providing multiple stimulatory signals. It has been observed that recombinant TCRs with multiple co-stimulatory domains may not generate sufficient co-stimulatory signals. Co-expressing multiple immunomodulatory fusion proteins, especially those that can remain in the immunological synapse, can provide the co-stimulatory signal transduction necessary for T cells to avoid anergy and proliferation.

[0106] In some embodiments, the fusion proteins of the present disclosure act in trans relative to a TCR or a chimeric antigen receptor (CAR) or other antigen receptor. In some embodiments, the fusion proteins disclosed herein act outside the immunological synapse.

[0107] On the other hand, the fusion proteins of the present disclosure allow for the enrichment of transduced T cells by restimulation with tumor cells expressing a ligand that binds to the fusion protein without sorting.

[0108] In an exemplary embodiment, a fusion protein is provided that comprises (a) an extracellular portion of CD200R, (b) a transmembrane domain of CD28, and (c) an intracellular signaling domain of CD28. In some embodiments, the extracellular portion further comprises an extracellular portion of CD28 extending from the CD28 transmembrane domain. In further embodiments, the extracellular portion of CD200R comprises at least about 231 amino acids from the N-terminus of CD200R. In other embodiments, the fusion protein further comprises an intracellular signaling domain of CD137 (4-1BB).

[0109] In another exemplary embodiment, a fusion protein is provided that comprises (a) the extracellular portion of SIRPα, (b) the transmembrane domain of CD28, and (c) the intracellular signaling domain of CD28. In some embodiments, the fusion protein further comprises the extracellular portion of CD28 extending from the CD28 transmembrane domain. In further embodiments, the extracellular portion of SIRPα comprises at least about 361 amino acids from the N-terminus of SIRPα. In other embodiments, the fusion protein further comprises the intracellular signaling domain of CD137 (4-1BB).

[0110] In another exemplary embodiment, a fusion protein is provided that comprises (a) the extracellular portion of CD95 (Fas), (b) the transmembrane domain of CD137 (4-1BB), and (c) the intracellular signaling domain of CD137 (4-1BB).

[0111] In another exemplary embodiment, a fusion protein is provided that comprises (a) the extracellular portion of CD95 (Fas), (b) the transmembrane domain of CD28, and (c) the intracellular signaling domain of CD137 (4-1BB). In some embodiments, the fusion protein further comprises the extracellular portion of CD28 extending from the CD28 transmembrane domain.

[0112] In another exemplary embodiment, the present disclosure provides a fusion protein that comprises (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component that links the extracellular and intracellular components, wherein the extracellular portion of the complex formed by specific binding of the fusion protein to the target (fusion protein::target complex) has the following size or spans the following distance: (i) up to the distance between the two cell membranes of an immunological synapse, (ii) up to a distance that is about or substantially the same as the distance spanned by the extracellular portion of the complex between a T cell receptor (TCR) and an MHC-peptide complex specifically bound by the TCR, (iii) up to a distance that is about or substantially the same as the distance spanned by the extracellular portion of the complex between a native molecule comprising the binding domain and its cognate binding partner, (iii) less than or up to about 40 nm, 25 nm, 20 nm, 15 nm, or 14 nm; or (iv) any combination thereof; wherein the extracellular component is or comprises the CD95 (Fas) extracellular domain or a functional fragment thereof, and the intracellular component is or comprises the CD137 (4-1BB) intracellular signaling domain or a functional portion thereof.

[0113] In another exemplary embodiment, the present disclosure provides a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component that links the extracellular and intracellular components, wherein the binding domain has or has at least 95% identity with an inhibitory molecule binding domain, and the intracellular signaling domain has or comprises at least 95% identity with a co-stimulatory or stimulatory molecule binding domain, and wherein the inhibitory molecule is or comprises the extracellular domain of CD95 (Fas) or a functional fragment thereof, and the co-stimulatory or stimulatory molecule is or comprises the intracellular signaling domain of CD137 (4-1BB) or a functional portion thereof.

[0114] In another exemplary embodiment, the present disclosure provides a fusion protein comprising: (a) an extracellular component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 71, (b) a hydrophobic component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 197, and (c) an intracellular component comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 13.

[0115] In another exemplary embodiment, the present disclosure provides a fusion protein comprising: (a) an extracellular component comprising a binding domain having the amino acid sequence shown in SEQ ID NO.: 72, (b) a hydrophobic component comprising the amino acid sequence shown in SEQ ID NO: 198, and (c) an intracellular component comprising the amino acid sequence shown in SEQ ID NO.: 36.

[0116] The components of the fusion proteins of the present disclosure are described in further detail herein.

[0117] Extracellular component

[0118] As described herein, the fusion proteins of the present invention generally comprise an extracellular component that comprises a binding domain that specifically binds a target. The binding of the fusion protein binding domain to the target can (1) block the interaction of the target with another molecule (e.g., block or interfere with receptor-ligand interactions), (2) interfere with, reduce, or eliminate certain functions of the target (e.g., inhibit signal transduction) (3) induce certain biological pathways that are not normally induced upon binding to the target (e.g., convert an inhibitory or negative signal to a stimulatory or positive signal) such as in the cell in which the fusion protein is expressed, or any combination thereof. In some embodiments, the fusion proteins described herein comprise an extracellular portion, wherein the extracellular portion comprises the extracellular portion of a protein associated with a negative signal.

[0119] Exemplary binding domains of the present disclosure can be extracellular domains or binding portions thereof of cell surface receptors, extracellular domains of cell surface ligands, cytokines (such as IL35), chemokines, antibody-based binding domains, TCR-based binding domains, unconventional binding domains, or any combination thereof. For example, binding domains comprising extracellular domains of CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CTLA4 (CD152), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5 are within the scope of the present disclosure. As used herein, an "extracellular domain" from a cell surface receptor or ligand includes the full extracellular domain or a functional (binding) fragment thereof. In certain embodiments, the extracellular domain comprises a mutated extracellular domain or a functional (binding) fragment thereof that has a higher affinity for a target compared to a wild-type or reference protein. In certain embodiments, the extracellular domain comprises a variable-like domain or CDRs of a variable-like domain.

[0120] In some embodiments, the fusion protein comprises an extracellular component containing a CD200 binding domain, such as the extracellular domain of CD200R or its CD200 binding portion. As background, CD200R is a receptor that binds to CD200, a type I membrane protein of the immunoglobulin superfamily (Tonks et al., Leukemia 21:566-568, 2007). CD200 has been reported to be upregulated in various malignancies, including leukemia, multiple myeloma, and various solid tumors (e.g., melanoma, breast cancer, and squamous cell carcinoma). Indeed, high levels of CD200 expression are associated with poor prognosis in acute myeloid leukemia (AML), and CD200R signaling has been shown to have an inhibitory effect on T cells (Coles et al., Leukemia 26:2148-2151, 2012). In certain embodiments, the extracellular domain of CD200R includes the full-length extracellular portion of the CD200R protein, the full-length mature extracellular portion of the CD200R protein, a binding fragment of the extracellular portion of the CD200R protein, or a binding fragment of the extracellular portion of the CD200R protein with a portion of the CD200R transmembrane domain, or any combination thereof.

[0121] In a further embodiment, CD200R is encoded by the nucleic acid molecule shown in SEQ ID NO.: 2. In certain other embodiments, the extracellular domain of CD200R comprises at least 200 amino acids from the N-terminus of CD200R. In some other embodiments, CD200R is encoded by the nucleic acid molecule shown in SEQ ID NO.: 11. In some additional embodiments, the extracellular portion of CD200R comprises at least about 180, 190, 200, 210, 220, 230, 231, 234 or 243 amino acids from the N-terminus of CD200R. For example, CD200R is encoded by the nucleic acid molecule shown in SEQ ID NO.: 8. In any of the foregoing embodiments, CD200R, the extracellular domain of CD200R, or any CD200R fragment used in the fusion proteins of the present disclosure is human CD200R. In a further embodiment, an extracellular domain of CD200R is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 2.

[0122] In some embodiments, CD200R comprises the amino acid sequence shown in SEQ ID NO.: 25. In some embodiments, CD200R comprises the amino acid sequence shown in SEQ ID NO.: 34. In certain embodiments, CD200R comprises the amino acid sequence shown in SEQIDNO.: 31. In any of the foregoing embodiments, CD200R, the extracellular domain of CD200R, or any CD200R fragment used in the fusion proteins of the present disclosure is human CD200R. In a further embodiment, an extracellular domain of CD200R is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID NO.: 25.

[0123] In some embodiments, the fusion protein comprises an extracellular component containing a CD47 binding domain, such as the extracellular domain of SIRPα or a binding portion thereof. As background, CD47 is a widely expressed transmembrane protein that plays a role in protecting cells from phagocytosis (Willingham et al., PNAS 109:6662–6667, 2012). The binding of CD47 to SIRPα initiates SIRPα signaling, thereby inhibiting macrophage phagocytosis. Thus, downregulation of SIRPα will result in increased macrophage phagocytosis. SIRPα is expressed in a variety of human tumor types, including AML, chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), lung, bladder, and other solid tumors. In certain embodiments, the extracellular domain of SIRPα comprises the full-length extracellular portion of the SIRPα protein, the full-length mature extracellular portion of the SIRPα protein, a binding fragment of the extracellular portion of the SIRPα protein, and a binding fragment of the extracellular portion of the SIRPα protein with a portion of the SIRPα transmembrane domain, or any combination thereof.

[0124] In further embodiments, the extracellular domain of SIRPα or a binding portion thereof is encoded by the nucleic acid molecule shown in SEQ ID NO.: 17. In certain embodiments, the extracellular domain of SIRPα comprises at least about 300, 310, 320, 330, 340, 350, 360, 361, 370, 373 or more amino acids from the N-terminus of SIRPα. In some other embodiments, SIRPα is encoded by the nucleic acid molecule shown in SEQID NO.: 21. In any of the foregoing embodiments, the SIRPα, the extracellular domain of SIRPα, or any SIRPα fragment used in the fusion protein of the present disclosure is human SIRPα. In further embodiments, an extracellular domain of SIRPα is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 17.

[0125] In a further embodiment, the extracellular domain of SIRPα comprises the amino acid sequence shown in SEQ ID NO.: 40. In some embodiments, SIRPα comprises the amino acid sequence shown in SEQ ID NO.: 44. In any of the foregoing embodiments, the SIRPα, the extracellular domain of SIRPα, or any SIRPα fragment thereof used in the fusion proteins of the present disclosure is human SIRPα. In a further embodiment, an extracellular domain of SIRPα is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 40.

[0126] In some embodiments, the fusion protein comprises an extracellular component that comprises a binding domain that binds to PD-L1, PD-L2, or both. In some embodiments, the fusion protein comprises an extracellular component that comprises the extracellular domain of PD1 or a ligand-binding portion thereof. In certain embodiments, the extracellular domain of PD-1 includes the full-length extracellular portion of the PD-1 protein, the full-length mature extracellular portion of the PD-1 protein, a binding fragment of the extracellular portion of the PD-1 protein, or a binding fragment of the extracellular portion of the PD-1 protein with a portion of the PD-1 transmembrane domain, or any combination thereof. In certain embodiments, the extracellular domain of PD-1 comprises at least 80, 90, 100, 110, 120, 125, 130, 132, 135, 137, 140, 149, 150, 155, 158, 160, or 170 amino acids from the N-terminus of PD-1. For example, in certain embodiments, the extracellular domain of PD-1 is encoded by a nucleic acid molecule shown in SEQ ID NO.: 91, 93, or 95. In further embodiments, the extracellular domain of PD-1 comprises at least from about 90 amino acids to at least about 130 amino acids, as shown in SEQ ID NO.: 60. In still further embodiments, the extracellular domain of PD-1 comprises a 170 amino acid sequence from the N-terminus of the PD-1 extracellular domain, as shown in SEQ ID NO.: 90. In some embodiments, PD-1 is encoded by a nucleic acid molecule shown in SEQ ID NO.: 89. In any of the foregoing embodiments, the PD-1, the extracellular domain of PD-1, or any PD-1 fragment used in the fusion proteins of the present disclosure is human PD-1. In further embodiments, an extracellular domain of PD-1 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 60. In further embodiments, an extracellular domain of PD-1 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 90. In further embodiments, an extracellular domain of PD-1 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 89.

[0127] In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence shown in SEQ ID NO.: 92, 94, or 96. In further embodiments, a PD-1 extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 92, 94, or 96. In any of the foregoing embodiments, the PD-1, PD-1 extracellular domain, or any PD-1 fragment thereof used in the fusion proteins of the present disclosure is human PD-1.

[0128] In some embodiments, the fusion protein comprises an extracellular component comprising the CD2 extracellular domain. In certain embodiments, CD2 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 61. In certain embodiments, the CD2 extracellular domain comprises the full-length extracellular portion of the CD2 protein, the full-length mature extracellular portion of the CD2 protein, a binding fragment of the extracellular portion of the CD2 protein, or a binding fragment of the extracellular portion of the CD2 protein with a portion of the CD2 transmembrane domain, or any combination thereof. In any of the foregoing embodiments, the CD2, CD2 extracellular domain, or any CD2 fragment thereof used in the fusion proteins of the present disclosure is human CD2. In further embodiments, a CD2 extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in GenBank Accession No. NM_001767.3. In further embodiments, a GenBank AccessionNo. NM_001767.3 extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 61.

[0129] In some embodiments, the CD2 extracellular domain comprises the amino acid sequence shown in SEQ ID NO.: 62. In further embodiments, a CD2 extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 62. In any of the foregoing embodiments, the CD2, CD2 extracellular domain, or any CD2 fragment thereof used in the fusion proteins of the present disclosure is human CD2.

[0130] In some embodiments, the fusion protein comprises an extracellular component, and the extracellular component comprises a binding domain that binds to FasL. In some embodiments, the fusion protein comprises an extracellular component containing the extracellular domain of Fas (CD95). Fas is expressed on tumor-associated blood vessels and prevents CD8 cell infiltration by inducing cell death. FasL is expressed in AML, pancreatic cancer, ovarian cancer, and other cancers (Kornmann et al., Annals of Surgery 231:368-379, 2000; Contin et al., Leukemia 21:253–260, 2007; Motz et al., Nature Medicine 20:607–615, 2014). Additionally, it has been reported that many chemotherapies cause upregulation of FasL on tumors, and FasL can also be upregulated on T cells in response to engagement of CD44. In certain embodiments, the extracellular domain of Fas comprises the full-length extracellular portion of the Fas protein, the full-length mature extracellular portion of the Fas protein, a binding fragment of the extracellular portion of the Fas protein, or a binding fragment of the extracellular portion of the Fas protein with a portion of the Fas transmembrane domain, or any combination thereof. In some embodiments, the extracellular domain of Fas is encoded by the nucleic acid molecule shown in SEQ ID NO.: 71. In some other embodiments, the extracellular domain of Fas comprises at least 150, 160, 161, 166, 170, or 173 amino acids from the N-terminus of Fas. For example, in certain embodiments, Fas is encoded by the nucleic acid molecule shown in SEQ ID NO.: 73. In certain other embodiments, Fas is encoded by the nucleic acid molecule shown in SEQ ID NO.: 75. In any of the foregoing embodiments, the Fas, the extracellular domain of Fas, or any Fas fragment used in the fusion protein of the present disclosure is human Fas. In a further embodiment, there is provided an extracellular domain of Fas that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in GenBank Accession No. NM_000043.4. In a further embodiment, there is provided an extracellular domain of Fas that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 71.

[0131] In some embodiments, the Fas extracellular domain comprises the amino acid sequence shown in SEQ ID NO.: 72. In some embodiments, the Fas extracellular domain comprises the amino acid sequence shown in SEQ ID NO.: 74. In certain embodiments, the Fas extracellular domain comprises the amino acid sequence shown in SEQ ID NO.: 76. In any of the foregoing embodiments, the Fas, Fas extracellular domain, or any Fas fragment thereof used in the fusion proteins of the present disclosure is human Fas. In further embodiments, a Fas extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 72.

[0132] In some embodiments, the fusion protein comprises an extracellular component comprising the extracellular domain of LAG3 (CD223). In certain embodiments, the LAG3 extracellular domain comprises the full-length extracellular portion of the LAG3 protein, the full-length mature extracellular portion of the LAG3 protein, a binding fragment of the extracellular portion of the LAG3 protein, or a binding fragment of the extracellular portion of the LAG3 protein with a portion of the LAG3 transmembrane domain, or any combination thereof. For example, in some embodiments, the LAG3 extracellular domain comprises from about 420, 416, 415, 413, or 410 amino acids from the N-terminus of LAG3. In any of the foregoing embodiments, the LAG3, LAG3 extracellular domain, or any LAG3 fragment thereof used in the fusion proteins of the present disclosure is human LAG3. In further embodiments, a LAG3 extracellular domain is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in GenBank Accession No. NM_002286.5.

[0133] In a further embodiment, LAG3 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 153. In certain other embodiments, the extracellular domain of LAG3 comprises at least 430, 435, 438, 440, 445 or 450 amino acids from the N-terminus of LAG3. For example, in certain embodiments, LAG3 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 161. In any of the foregoing embodiments, LAG3, the extracellular domain of LAG3, or any LAG3 fragment thereof used in the fusion proteins of the present disclosure is human LAG3. In a further embodiment, an extracellular domain of LAG3 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 153.

[0134] In some embodiments, LAG3 comprises the amino acid sequence shown in SEQ ID NO.: 154. In some embodiments, LAG3 comprises the amino acid sequence shown in SEQ ID NO.: 162. In any of the foregoing embodiments, LAG3, the extracellular domain of LAG3, or any LAG3 fragment thereof used in the fusion proteins of the present disclosure is human LAG3. In a further embodiment, an extracellular domain of LAG3 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID NO.: 154.

[0135] In some embodiments, the fusion protein comprises an extracellular component containing the extracellular domain of TIM3. In certain embodiments, the extracellular domain of TIM3 comprises the full-length extracellular portion of the TIM3 protein, the full-length mature extracellular portion of the TIM3 protein, a binding fragment of the extracellular portion of the TIM3 protein, or a binding fragment of the extracellular portion of the TIM3 protein with a part of the TIM3 transmembrane domain, or any combination thereof. In any of the foregoing embodiments, the TIM3, extracellular domain of TIM3, or any TIM3 fragment thereof used in the fusion protein of the present disclosure is human TIM3. In a further embodiment, there is provided an extracellular domain of TIM3 that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in GenBank Accession No. NM_032782.4.

[0136] In a further embodiment, TIM3 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 167. In certain other embodiments, the extracellular domain of TIM3 comprises at least 180, 185, 190, 195, or 200 amino acids from the N-terminus of TIM3. For example, in certain embodiments, TIM3 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 177. In any of the foregoing embodiments, the TIM3, extracellular domain of TIM3, or any TIM3 fragment thereof used in the fusion protein of the present disclosure is human TIM3. In a further embodiment, there is provided an extracellular domain of TIM3 that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 167.

[0137] In some embodiments, TIM3 comprises the amino acid sequence shown in SEQ ID NO.: 168. In some embodiments, TIM3 comprises the amino acid sequence shown in SEQ ID NO.: 178. In any of the foregoing embodiments, TIM3, the extracellular domain of TIM3, or any TIM3 fragment thereof used in the fusion proteins of the present disclosure is human TIM3. In further embodiments, an extracellular domain of TIM3 is provided that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the extracellular domain of a molecule having the amino acid sequence shown in SEQ ID No.: 168.

[0138] The binding domain can be any peptide that specifically binds to a target of interest. Sources of the binding domain include the variable regions of antibodies from different species (which can be in the form of antibodies, sFvs, scFvs, Fabs, scFv-based grababodies, or soluble VH domains or domain antibodies), including human, rodent, avian, or ovine. Other sources of the binding domain include the variable regions of antibodies from other species, such as camelids (from camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett. 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature 363:446, 1993, and Nguyen et al., J. Mol. Biol. 275:413, 1998), nurse sharks (Roux et al., Proc. Nat′l. Acad. Sci. (USA) 95:11804, 1998), channel catfish (Nguyen et al., Immunogen. 54:39, 2002), or lampreys (Herrin et al., Proc. Nat′l. Acad. Sci. (USA) 105:2040, 2008, and Alder et al., Nat. Immunol. 9:319, 2008). These antibodies can form antigen-binding regions using only the variable regions of the heavy chains, i.e., these functional antibodies are only homodimers of the heavy chains (referred to as "heavy-chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006, and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).

[0139] Alternative sources of the unconventional binding domains of the present invention include sequences encoding random peptide libraries or sequences encoding engineered diverse amino acids in loop regions of alternative non-antibody scaffolds, such as scTCR (see, e.g., Lake et al., Int. Immunol. 11:745, 1999; Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Patent No. 8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388, 1985), Kunitz domains (see, e.g., U.S. Patent No. 6,423,498), designed ankyrin repeat proteins (DARPins) (Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin-binding domains (adnectins or monobodies) (Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381:1238-1252), cysteine knot miniproteins (Vita et al. (1995) Proc. Nat′l. Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nat. Biotechnol. 21:71, 2002 and Huang et al. (2005) Structure 13:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471, 2003), lipocalin domains (see, e.g., WO 2006 / 095164, Beste et al., Proc. Nat′l. Acad. Sci. (USA) 96:1898, 1999 and et al., Proc. Nat′l. Acad. Sci. (USA) 106:8198, 2009), V-like domains (see, e.g., U.S. Patent Application Publication No. 2007 / 0065431), C-type lectin domains (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Nat′l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat′l. Acad. Sci. (USA) 100:7779, 2003), mAb 2 or Fcab TM (see, e.g., PCT Patent Application Publication No. WO 2007 / 098934; WO2006 / 072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21:1015, 2012; PCT Patent Application Publication No. WO2009 / 040338), affilin (Ebersbach et al., J. Mol. Biol. 372:172, 2007), affibody, avimers, knottins, fynomer, atrimers, cytotoxic T lymphocyte-associated protein-4 (Weidlee et al., Cancer Gen. Proteo. 10:155, 2013), etc. (Nord et al., Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Plückthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0140] In some embodiments, the binding domain is a single-chain T cell receptor (scTCR) that comprises V α / β and C α / β chains (e.g., V α -C α , V β -C β , V α -V β ) or comprises a V α -C α that is specific for a target of interest, Vβ -C β 、V α -V β pair (e.g., a peptide-MHC complex or a peptide-HLA complex).

[0141] In certain embodiments, the binding domain comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the extracellular domain of a molecule having a TCR Vα, Vβ, Cα or Cβ amino acid sequence, wherein each CDR contains zero or at most one, two or three changes compared to a TCR or a fragment or derivative thereof that specifically binds a target of interest.

[0142] In certain embodiments, when compared to the V α 、V β 、C α or C β of a known TCR, the V α 、V β 、C α or C β regions of the binding domains of the present disclosure may be derived from or based on the V α 、V β 、C α or C β of a known TCR (e.g., a high-affinity TCR), and comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the changes exemplified above. The insertions, deletions or substitutions may be at any position in the V α 、V β 、C α or C β regions, including at the amino or carboxyl terminus or both ends of these regions, provided that each CDR contains zero changes or at most one, two or three changes, and provided that the binding domain comprising the modified V α 、V β 、C α or C β regions can still specifically bind its target with an affinity similar to that of the wild type. In certain embodiments, the TCR has an affinity for the peptide-HLA complex in the range of about 10 μM to about 500 μM. In certain embodiments, the TCR has a high affinity for the peptide-HLA complex in the range of about 10 nM to about 200 pM.

[0143] In some aspects, the fusion proteins according to the present invention have an extracellular component comprising a binding domain that specifically binds a target (e.g., a ligand or a receptor), wherein the extracellular component optionally comprises one or more other functional sub-components or domains, such as a multimerization domain, a linker, a linking amino acid, or any combination thereof.

[0144] In certain embodiments, the fusion proteins disclosed herein further comprise an additional extracellular region, such as a spacer or a multimerization domain, in addition to the binding domain or a portion of the molecule from which the binding domain is derived. For example, in some aspects, the multimerization domain is included in or is part of the extracellular component of the fusion protein. For example, the multimerization domain can be generated by altering (e.g., mutating) the extracellular component, or can be generated by adding 1 to about 50 amino acid residues to the extracellular component. The multimerization domain can be located between the binding domain and the hydrophobic component of the extracellular component of the fusion proteins disclosed herein. In certain embodiments, the fusion protein expressed on the cell surface comprises a multimerization domain within the extracellular component and is close to the cell membrane, within 1 to 50 amino acids from the hydrophobic component. For example, the fusion protein multimerization domain can comprise one or more cysteine residues located within 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids from the hydrophobic component of the fusion protein, wherein one or more such cysteine residues from one fusion protein can form one or more disulfide bonds with one or more other fusion proteins. In some embodiments, the additional extracellular portion is derived from the same molecule from which the transmembrane or stimulatory region of the fusion protein is derived.

[0145] In further embodiments, the interaction between the multimerization domains of two or more fusion proteins substantially contributes to or effectively promotes signal transduction (e.g., immune cell stimulation or activation) as compared to the fusion protein monomer. In certain embodiments, the multimerization of the fusion protein promotes signal transduction in host cells in a statistically significant manner as compared to the fusion protein monomer. In further embodiments, the multimerization of the fusion protein that promotes or enhances signal transduction in host cells is via disulfide bridges.

[0146] An exemplary multimer is a "dimer", which refers to a biological entity comprising two molecules (such as two fusion proteins) associated with each other. When two related fusion proteins have substantially similar or identical amino acid sequences, such a dimer is considered a "homodimer". Similarly, a multimer of three substantially or completely identical fusion proteins is called a " homotrimer". In some embodiments, the multimerization domain comprises at least one cysteine residue, wherein the multimerization domain cysteine residue from the first fusion protein can form a disulfide bridge with the multimerization domain cysteine residue from the second fusion protein. In certain embodiments, a fusion protein dimer is formed by a disulfide bridge. In other embodiments, a fusion protein trimer is formed by two or more disulfide bridges. Alternatively, a dimer, homodimer, trimer or homotrimer can multimerize via a zinc finger motif or a leucine zipper motif. In additional embodiments, the fusion protein comprises multiple multimerization domains, which can be extracellular, intracellular or both.

[0147] In some embodiments, the multimerization domain contained in the extracellular component of the fusion protein comprises an extracellular portion extending from a hydrophobic component. For example, in some embodiments, the multimerization domain contained in the extracellular component of the fusion protein comprises the extracellular portion of CD28 extending from the CD28 transmembrane domain. In some embodiments, the extracellular portion of CD28 comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or up to about 25 amino acids adjacent to the transmembrane domain. In some embodiments, the extracellular portion of CD28 comprises 9 amino acids or 12 amino acids adjacent to the transmembrane domain. In some embodiments, the extracellular portion of CD28 comprises the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 9. In some embodiments, the extracellular portion of CD28 comprises the amino acid sequence shown in SEQ ID NO.: 32. In other embodiments, the multimerization domain contained in the extracellular component of the fusion protein comprises the extracellular portion of CD137 (4-1BB) extending from the CD137 (4-1BB) transmembrane domain (e.g., in the range from 1 to about 50 amino acids). In certain embodiments, the multimerization domain and the hydrophobic component are from different proteins. For example, the multimerization domain contained in the extracellular component of the fusion protein comprises the extracellular portion of CD28 extending from the CD137 transmembrane domain, or comprises the extracellular portion of CD137 extending from the CD28 transmembrane domain. In any of the above embodiments, the multimerization domain may further comprise a glycosylation site.

[0148] In some embodiments, the fusion protein may comprise a linker or a linking amino acid that, for example, links an extracellular component to a multimerization domain, or links an extracellular component to a hydrophobic component, or links a hydrophobic component to an intracellular component. In some embodiments, the linker is Gly x Ser y , where x and y are independently integers from 1 to 5.

[0149] In some embodiments, the extracellular component is or comprises a molecule or a portion thereof that forms a multimer, and the intracellular component is or comprises a molecule or a portion thereof that forms the same number of multimers. For example, in some embodiments, the extracellular component is or comprises a molecule or a portion thereof that forms a dimer, and the intracellular component is or comprises a molecule or a portion thereof that also forms a dimer. In some embodiments, the extracellular component is or comprises a molecule or a portion thereof that forms a trimer, and the intracellular component is or comprises a molecule or a portion thereof that also forms a trimer.

[0150] A target molecule can be found on or associated with a cell of interest (“target cell”) that is specifically bound by a binding domain contained in the fusion protein of the present disclosure. Exemplary target cells include immune cells, cancer cells, cells associated with an autoimmune disease or disorder or an inflammatory disease or disorder, and infectious organisms or cells (e.g., bacteria, viruses, virus-infected cells), or any antigen-presenting cell complexed with MHC or human leukocyte antigen (HLA). Cells of an infectious organism, such as a parasite of a mammal, are also considered target cells. In some embodiments, the target is an immunosuppressive ligand. In some embodiments, the target is selected from CD47, CD58, CD80, CD86, CD95L (FasL), CD200, CD270 (HVEM), CD274 (PD-L1), or GAL9.

[0151] Extracellular component

[0152] The intracellular component contained in the fusion protein of the present disclosure will have an intracellular signaling domain capable of transmitting a functional signal to the cell, such as an activation domain or a co-stimulatory domain. In certain embodiments, the intracellular signaling domain will indirectly facilitate a cellular response by associating with one or more other proteins that directly facilitate the cellular response. The intracellular signaling domain can include one, two, three, or more receptor signaling domains, co-stimulatory domains, or combinations thereof. Any intracellular component containing an activation domain, a co-stimulatory domain, or both from any of a variety of signaling molecules (e.g., signaling receptors) can be used in the fusion protein of the present disclosure.

[0153] As used herein, an "intracellular signaling domain" from a cell surface receptor or ligand includes the full-length intracellular domain, a portion containing the intracellular signaling domain, or a functional (signaling) fragment thereof. In certain embodiments, the intracellular signaling domain comprises a mutant intracellular domain or a functional (signaling) fragment thereof that has higher signaling activity compared to a wild-type or reference intracellular signaling domain.

[0154] As used herein, a "costimulatory molecule" refers to a receptor or cell surface molecule that can transmit a signal to a T cell to positively regulate T cell activation (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). By background, activation and proliferation of T cells require two signals mediated by binding of the T cell antigen-specific receptor (TCR) and a costimulatory signal, most typically CD80 and CD86 binding to CD28 (Ledbetter et al., Blood 75:1531, 1990).

[0155] Intracellular signaling domains or functional fragments thereof useful in the fusion proteins of the present disclosure can be from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the intracellular signaling domain or functional fragment thereof does not include a primary signal. In some embodiments, the intracellular signaling domain does not include CD3ζ.

[0156] In some embodiments, the intracellular signaling domain of the fusion protein of the present disclosure comprises CD28. CD28 signaling promotes the proliferation of T cells stimulated via the TCR (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). Due to cysteine residues near the transmembrane domain, CD28 forms a disulfide-linked homodimer (Lazar-Molnar et al., Cell Immunol. 244:125–129, 2006). In certain embodiments, the CD28 signaling domain comprises the full-length intracellular portion of the CD28 protein, the full-length mature intracellular portion of the CD28 protein, a signaling fragment of the intracellular portion of the CD28 protein, or a signaling fragment of the intracellular portion of the CD28 protein with the CD28 transmembrane domain or a fragment thereof, or any combination thereof.

[0157] In some embodiments, the intracellular signaling domain of the fusion protein comprises the intracellular signaling domain of CD137 (4-1BB). CD137 is a co-stimulatory molecule, and the binding of CD137 to its ligand (4-1BBL or CD137L) is associated with T cell activation and proliferation (Cheuk et al., Cancer Gene Therapy 11:215-226, 2004). In certain embodiments, the CD137 signaling domain comprises the full-length intracellular portion of the CD137 protein, the full-length mature intracellular portion of the CD137 protein, a signaling fragment of the intracellular portion of the CD137 protein, or a signaling fragment of the intracellular portion of the CD137 protein with the CD137 transmembrane domain or a fragment thereof, or any combination thereof.

[0158] In certain embodiments, the intracellular signaling domain comprises a lymphocyte receptor signaling domain or comprises an amino acid sequence having one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In additional embodiments, the intracellular signaling domain comprises a cytoplasmic portion associated with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or a signaling domain thereof, i.e., a protein comprising multiple ITAMs, co-stimulatory factors, or any combination thereof.

[0159] In some exemplary embodiments, the present disclosure provides a fusion protein having: an extracellular component comprising the extracellular portion of CD200R that specifically binds to CD200; an intracellular component comprising the intracellular portion of CD28; and a hydrophobic component that links the extracellular and intracellular components, provided that the fusion protein::target complex spans a distance similar to the distance between membranes in an immunological synapse.

[0160] In certain embodiments, the intracellular component of the fusion protein of the present disclosure comprises CD28, CD137 (4-1BB), or both. For example, in some embodiments, the intracellular component comprises the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 5. In some other embodiments, the intracellular component comprises the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 13. In some embodiments, the intracellular component comprises two intracellular signaling domains, such as CD28 and CD137 (4-1BB). In some other embodiments, the intracellular component comprises the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 5 and the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.: 13.

[0161] Hydrophobic component

[0162] The hydrophobic portion contained in the single-chain fusion protein of the present disclosure will allow the fusion protein of the present disclosure to associate with the cell membrane such that a portion of the fusion protein will be extracellular and a portion will be intracellular (e.g., the intracellular signaling domain). The hydrophobic component will generally be placed within the phospholipid bilayer of the cell membrane. In certain embodiments, one or more linker amino acids may be placed between the intracellular signaling domain and the hydrophobic portion and link the intracellular signaling domain to the hydrophobic portion.

[0163] In certain embodiments, the hydrophobic domain is a transmembrane domain, such as a transmembrane domain derived from an integral membrane protein (e.g., a receptor, a cluster of differentiation (CD) molecule, an enzyme, a transporter, a cell adhesion molecule, etc.). In some embodiments, the hydrophobic domain comprises a transmembrane domain found in or derived from an integral membrane protein, wherein the transmembrane domain has been modified by adding, deleting, or substituting one or more amino acids with at least one different amino acid, or any combination thereof, such as charged or hydrophilic residues that promote intermolecular interactions. Thus, the term "hydrophobic domain" includes transmembrane domains having modifications, such as those that can reduce hydrophobicity.

[0164] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), TIM3, CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LPA5, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM or Zap70. In certain embodiments, the hydrophobic portion is the transmembrane domain from CD28, CD4, CD8, CD27 or CD137 (4-1BB).

[0165] In certain embodiments, the transmembrane domain comprises the CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the CD28 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 4. In further embodiments, the transmembrane domain is the CD28 transmembrane domain consisting of or comprising the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 4. In additional embodiments, the transmembrane domain is the CD28 transmembrane domain having at least 90%, 95% or higher sequence identity to the amino acid sequence of SEQ ID NO.: 27. In additional embodiments, the transmembrane domain is the CD28 transmembrane domain consisting of or comprising the amino acid sequence shown in SEQ ID NO.: 27.

[0166] In certain embodiments, the transmembrane domain comprises the CD137 (4-1BB) transmembrane domain. In further embodiments, the transmembrane domain comprises or consists of the CD137 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 197. In specific embodiments, the transmembrane domain is the CD137 transmembrane domain comprising or consisting of the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 197.

[0167] In certain other embodiments, the transmembrane domain comprises the CD200R transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the CD200R transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 3. In further embodiments, the transmembrane domain is the CD200R transmembrane domain comprising or consisting of the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 3.

[0168] In certain embodiments, the transmembrane domain comprises the SIRPα transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the SIRPα transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 18. In further embodiments, the transmembrane domain is the SIRPα transmembrane domain comprising or consisting of the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 18.

[0169] In certain embodiments, the transmembrane domain comprises the CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the CD2 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 63. In further embodiments, the transmembrane domain is the CD2 transmembrane domain comprising or consisting of the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 63.

[0170] In certain embodiments, the transmembrane domain comprises the Fas transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the Fas transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 77. In further embodiments, the transmembrane domain is the Fas transmembrane domain consisting of or comprising the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 77.

[0171] In certain embodiments, the transmembrane domain comprises the TIM3 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the TIM3 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 169. In further embodiments, the transmembrane domain is the TIM3 transmembrane domain consisting of or comprising the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 169.

[0172] In certain embodiments, the transmembrane domain comprises the LAG3 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of the LAG3 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity to the polynucleotide of SEQ ID NO.: 155. In further embodiments, the transmembrane domain is the LAG3 transmembrane domain consisting of or comprising the amino acid sequence encoded by the nucleic acid molecule as shown in SEQ ID NO.: 155.

[0173] Nucleic Acids and Host Cells

[0174] In certain aspects, the present invention provides nucleic acid molecules encoding one or more of the fusion proteins described herein, which may be immunomodulatory fusion proteins (IFPs). Such nucleic acid molecules can be inserted into a suitable vector (e.g., a viral vector or a non-viral plasmid vector) for introduction into a host cell of interest (e.g., hematopoietic progenitor cells, T cells).

[0175] As used herein, the terms "recombinant" or "non-naturally occurring" refer to a biological, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or is modified by the introduction of an exogenous nucleic acid molecule, where such alteration or modification is introduced by genetic engineering. Genetic alterations include, for example, modifications that introduce an expressible nucleic acid molecule encoding a protein, fusion protein, or enzyme, or other nucleic acid molecule addition, deletion, substitution, or other functional disruption of the cell's genetic material. Additional modifications include, for example, non-coding regulatory regions, where the modification alters the expression of a gene or operon. In certain embodiments, cells obtained from a subject, such as T cells, can be converted into non-naturally occurring or recombinant cells (e.g., non-naturally occurring or recombinant T cells) by introducing a nucleic acid encoding a fusion protein described herein, and the cells thereby express the fusion protein.

[0176] In certain embodiments, a nucleic acid molecule encoding a fusion protein can be codon-optimized to enhance or maximize expression in certain types of cells such as T cells (Scholten et al., Clin. Immunol. 119:135-145, 2006).

[0177] In an exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28 construct (huCD200Rtm-CD28), wherein the extracellular component comprises the extracellular domain of CD200R, the hydrophobic component comprises the transmembrane domain of CD200R, and the intracellular component comprises the intracellular signaling domain of CD28. For example, in one embodiment, a nucleic acid molecule shown in SEQ ID NO.: 1 is provided. In certain embodiments, the present disclosure provides huCD200Rtm-CD28 that comprises a polynucleotide that is at least 80% or at least 90% identical to the polynucleotide sequence of SEQ ID NO.: 1. In other embodiments, the present disclosure provides huCD200Rtm-CD28 that comprises or consists of the polynucleotide sequence of SEQ ID NO.: 1.

[0178] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28 construct (huCD200R-CD28tm), wherein the hydrophobic component comprises the transmembrane domain of CD28. For example, in one embodiment, a nucleic acid molecule shown in SEQ ID NO.: 6 is provided. In certain embodiments, the present disclosure provides huCD200R-CD28tm, which comprises a polynucleotide having at least 80% or at least 90% identity to the polynucleotide sequence of SEQ ID NO.: 6. In other embodiments, the present disclosure provides huCD200R-CD28tm, which comprises or consists of the polynucleotide sequence of SEQ ID NO.: 6. In a further embodiment, a huCD200R-CD28tm extracellular domain is provided, which has an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the amino acid sequence shown in SEQ ID No.: 29.

[0179] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28 construct, wherein the extracellular portion comprises a truncated extracellular domain of CD200R and the extracellular portion of CD28. For example, the CD200R extracellular domain is truncated by about 9 to about 15 amino acids. Exemplary CD200R-CD28 constructs of the present disclosure include those truncated by 9 amino acids (e.g., huCD200R-9aas-CD28Cys, SEQ ID NO.:7), truncated by 12 amino acids (e.g., huCD200R-12aas-CD28Cys, SEQ ID NO:10), or truncated by 15 amino acids (e.g., huCD200R-15aas-CD28Cys, SEQ ID NO.:183). In some embodiments, the extracellular portion of CD28 comprises the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.:9. In certain embodiments, the extracellular portion of CD28tm includes an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.:32. In a further embodiment, the transmembrane portion of CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO.:32. In certain other embodiments, the huCD200R-9aas-CD28Cys protein includes an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.:30. The exemplary huCD200R-9aas-CD28Cys protein comprises or consists of the amino acid sequence shown in SEQ ID NO.:30. In certain other embodiments, the huCD200R-12aas-CD28Cys protein includes an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.:33. The exemplary huCD200R-12aas-CD28Cys protein comprises or consists of the amino acid sequence shown in SEQ ID NO.:33.In certain other embodiments, the huCD200R-15aas-CD28Cys protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.: 184. Exemplary huCD200R-15aas-CD28Cys proteins comprise or consist of the amino acid sequence shown in SEQ ID NO.: 184.

[0180] In one exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28-4-1BB construct (huCD200R-9aas-CD28Cystm-41BBic or huCD200R-12aas-CD28Cystm-41BBic), wherein the intracellular component comprises the intracellular signaling domain of CD137 (4-1BB). For example, in one embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.: 12 or SEQ ID NO.: 14.

[0181] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28-4-1BB construct (huCD200R-9aas-CD28Cys tm ic 41BBic or huCD200R-12aas-CD28Cys tm ic-41BBic), wherein the intracellular component comprises the intracellular signaling domains of CD28 and CD137 (4-1BB). For example, in one embodiment, the nucleic acid of the present disclosure has the nucleotide sequence shown in SEQ ID NO.: 9 or SEQ ID NO.: 15.

[0182] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a SIRPα-CD28 construct. For example, the present disclosure includes nucleic acid molecules shown in SEQ ID NO.: 16 (huSIRPαtm-CD28) or SEQ ID NO.: 19 (huSIRPα-CD28tm).

[0183] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a SIRPα-CD28 construct, wherein the extracellular component comprises a truncated extracellular domain of SIRPα and the extracellular portion of CD28. For example, the SIRPα extracellular domain is truncated by about 8 to about 15 amino acids. An exemplary SIRPα-CD28 construct has a CD95 (Fas) extracellular domain truncated by 12 amino acids (e.g., huSIRPα-12aas-CD28Cys, SEQ ID NO.: 20).

[0184] In one exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a SIRPα-CD28-4-1BB construct (huSIRPα-12aas-CD28Cystm-41BBic), wherein the intracellular component comprises the intracellular signaling domain of CD137 (4-1BB). For example, in one embodiment, the nucleic acid of the present disclosure has the nucleotide sequence shown in SEQ ID NO.: 22.

[0185] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a SIRPα-CD28-4-1BB construct (huSIRPα-12aas-CD28Cys tm ic-41BBic), wherein the intracellular component comprises the intracellular signaling domains of CD28 and CD137 (4-1BB). For example, in one embodiment, the nucleic acid of the present disclosure has the nucleotide sequence shown in SEQ ID NO.: 23.

[0186] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a PD-1-CD28 construct. For example, the present disclosure includes a nucleic acid molecule shown in SEQ ID NO.: 97 (huPD1-CD28Cys).

[0187] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a PD-1-CD28 construct, wherein the extracellular component comprises a truncated extracellular domain of PD-1 and the extracellular portion of CD28. For example, the PD-1 extracellular domain can be truncated by about 10 to about 25 amino acids. Exemplary PD-1-CD28 constructs have a PD-1 extracellular domain truncated by 12 amino acids (e.g., huPD1-12aas-CD28Cys, SEQ ID NO.: 99), 15 amino acids (e.g., huPD1-15aas-CD28Cys, SEQ ID NO.: 101), or 21 amino acids (e.g., huPD1-21aas-CD28Cys, SEQ ID NO.: 103).

[0188] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD2-CD28 construct. For example, the present disclosure includes the nucleic acid molecule shown in SEQ ID NO.:69 (huCD2-CD28Cys).

[0189] In other exemplary embodiments, the present disclosure provides a nucleic acid molecule encoding a Fas-CD28 construct. For example, the present disclosure includes the nucleic acid molecule shown in SEQ ID NO.:83 (huFas-CD28Cys).

[0190] In other exemplary embodiments, the present disclosure provides a nucleic acid molecule encoding a Fas-CD28 construct, wherein the extracellular component comprises a truncated extracellular domain of Fas and the extracellular portion of CD28. For example, the Fas extracellular domain can be truncated by about 7 to about 15 amino acids. Exemplary Fas-CD28 constructs have a CD95 (Fas) extracellular domain truncated by 7 amino acids (e.g., huFas-7aas-CD28Cys, SEQ ID NO.:85) or 12 amino acids (e.g., huFas-12aas-CD28Cys, SEQ ID NO.:87).

[0191] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a Fas-4-1BB construct, wherein the extracellular component comprises the complete extracellular domain or a truncated extracellular domain of CD95 (Fas), and the intracellular component comprises the signaling domain of CD137 (4-1BB). In a further embodiment, the nucleic acid molecule encodes a Fas-4-1BB construct, wherein the hydrophobic component further comprises the transmembrane portion of CD95 (Fas) or CD137 (4-1BB). For example, in certain embodiments, the Fas-4-1BB construct is encoded by a nucleic acid molecule having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide sequence of SEQ ID NO.: 185. In some embodiments, the Fas-4-1BB construct is encoded by a polynucleotide comprising or consisting of the polynucleotide sequence of SEQ ID NO.: 185. In other embodiments, the Fas-4-1BB construct is encoded by a nucleic acid molecule having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide sequence of SEQ ID NO.: 187. In some embodiments, the Fas-4-1BB construct is encoded by a polynucleotide comprising or consisting of the polynucleotide sequence of SEQ ID NO.: 187. In certain other embodiments, the Fas-4-1BB construct is the huFastm-41BB protein, and the huFastm-41BB protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.: 186. The exemplary huFastm-41BB protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 186. In certain other embodiments, the Fas-4-1BB construct is the huFas-41BBtm protein, and the huFas-41BBtm protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID No.: 188. The exemplary huFas-41BBtm protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 188.

[0192] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a Fas-CD28-41BB construct, wherein the extracellular component comprises the extracellular domain of CD95 (Fas), the hydrophobic component comprises the transmembrane domain of CD28, and the intracellular component comprises the signaling domain of CD137 (4-1BB). In some embodiments, the extracellular component comprises the truncated extracellular domain of CD95 (Fas) and the extracellular portion of CD28. For example, the extracellular domain of CD95 (Fas) is truncated by about 7 to about 15 amino acids. Exemplary Fas-CD28-41BB constructs have an extracellular domain of CD95 (Fas) truncated by 7, 9, 12, or 15 amino acids. For example, in some embodiments, the extracellular domain of CD95 (Fas) may comprise the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.:73 or SEQ ID NO.:75. The extracellular portion of CD28 may comprise a multimerization domain. For example, the extracellular portion of CD28 may comprise the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO.:9.

[0193] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a TIM3-CD28 construct. For example, the present disclosure includes the nucleic acid molecule shown in SEQ ID NO.:173 (huTIM3-CD28Cys). Also within the scope of the present disclosure is a TIM3-CD28 fusion protein, wherein the extracellular component comprises the truncated extracellular domain of TIM3 and the extracellular portion of CD28. For example, the extracellular domain of TIM3 may be truncated by about 8 to about 15 amino acids (e.g., huTIM3-12aas-CD28Cys, SEQ ID NO.:175, truncated by 12 amino acids).

[0194] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a LAG3-CD28 construct. For example, the present disclosure includes the nucleic acid molecule shown in SEQ ID NO.:163 (huLAG3-CD28Cys). Also within the scope of the present disclosure is a LAG3-CD28 fusion protein, wherein the extracellular component comprises the truncated extracellular domain of LAG3 and the extracellular portion of CD28. For example, the extracellular domain of LAG3 may be truncated by about 8 to about 15 amino acids (e.g., huLAG3-12aas-CD28Cys, SEQ ID NO.:159, truncated by 12 amino acids).

[0195] Vectors encoding the core virus are referred to herein as "viral vectors". A large number of viral vectors are available for use in the compositions of the present invention, including those identified for human gene therapy applications (see Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna (ovine lentivirus). Methods for transducing mammalian host cells with viral particles containing a chimeric antigen receptor transgene using retroviral and lentiviral viral vectors and packaging cells are known in the art and have been previously described, e.g., in U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0196] In certain embodiments, the viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a fusion protein or a non-endogenous nucleic acid sequence encoding a fusion protein specific for a target. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a transduction marker. Transduction markers for viral vectors are known in the art and include selectable markers that can confer drug resistance or detectable markers such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In certain embodiments, the viral vector further contains a genetic marker for transduction, which includes green fluorescent protein (GFP), the extracellular domain of human CD2, or truncated human EGFR (huEGFRt; see Wang et al., Blood 118:1255, 2011). When the viral vector genome contains multiple nucleic acid sequences to be expressed in a host cell as separate transcripts, the viral vector can also contain additional sequences between the two (or more) transcripts, thereby allowing bicistronic or polycistronic expression. Examples of such sequences for viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0197] Other vectors can also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-deficient HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0198] Other vectors recently developed for gene therapy applications can also be used in conjunction with the compositions and methods of the present invention. These vectors include vectors derived from baculovirus and alphavirus (Jolly, D J. 1999. Emerging Viral Vectors. pp. 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors). In some embodiments, the viral or plasmid vector also contains a genetic marker for transduction (e.g., green fluorescent protein, huEGFRt).

[0199] In some embodiments, a vector encoding a fusion protein as disclosed herein can encode more than one fusion protein. For example, the vector can encode two different fusion proteins (e.g., a first fusion protein comprising the extracellular domain of PD-1 and a second fusion protein comprising the extracellular domain of TIM3).

[0200] In some embodiments, a vector encoding a fusion protein as disclosed herein can further comprise an antigen-specific TCR. In some embodiments, the antigen-specific TCR is exogenous. In some embodiments, the antigen-specific TCR is specific for an HLA (MHC) class I-restricted antigen. In some embodiments, the antigen is a cancer-specific antigen. Embodiments in which the cancer-specific antigen comprises WT-1, mesothelin, or cyclin A1 are also within the scope of the present disclosure. In other embodiments, a vector encoding a fusion protein as disclosed herein also encodes a ligand, which can be CD200, CD47, PD-L1, or CD58. In other embodiments, a vector encoding a fusion protein as disclosed herein also encodes an siRNA for reducing endogenous receptor expression. In some specific embodiments, the endogenous receptor is CD200R, SIRPα, CD279 (PD-1), CD95 (Fas), or CD2.

[0201] In some embodiments, the host cell capable of expressing the fusion protein of the present disclosure on the cell surface is an immune cell. In some embodiments, the host cell capable of expressing the fusion protein of the present disclosure on the cell surface is a T cell, including primary cells or cell lines derived from humans, mice, rats, or other mammals. If obtained from a mammal, T cells can be obtained from many sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, T cells lacking endogenous expression of TCRα and β chains are used. Such T cells may naturally lack endogenous expression of TCRα and β chains, or may have been modified to block expression (e.g., T cells from transgenic mice that do not express TCRα and β chains or cells that have been engineered to inhibit TCRα and β chain expression) or knockout the TCRα chain, TCRβ chain, or both genes. In some embodiments, T cells can be engineered to express a TCR specific for a particular antigen.

[0202] In certain embodiments, the host cell transfected to express the fusion protein of the present disclosure is a functional T cell, such as a virus-specific T cell, a tumor antigen-specific cytotoxic T cell, a naive T cell, a memory stem T cell, a central or effector memory T cell, a γδ T cell, or a CD4+CD25+ regulatory T cell. In further embodiments, the nucleic acid molecule encoding the fusion protein of the present disclosure is introduced into bulk CD8+ T cells, naive CD8+ T cells, CD8+ T CM cells, CD8+ T EM cells, or any combination thereof. In still further embodiments, the nucleic acid molecule encoding the fusion protein of the present disclosure is introduced into bulk CD4+ T cells, naive CD4+ T cells, CD4+ T CM cells, CD4+ T EM cells, or any combination thereof. In other embodiments, the nucleic acid molecule encoding the fusion protein of the present disclosure is introduced into a T cell population enriched for naive CD8+ T cells and CD8+ T CM cells. In some other embodiments, the nucleic acid molecule encoding the fusion protein of the present disclosure is introduced into a T cell population enriched for naive CD4+ T cells and CD4+ T CM cells. In any of the above embodiments, the T cells further comprise a nucleic acid molecule encoding an engineered antigen-specific T cell receptor (TCR), an engineered antigen-specific high-affinity TCR, an exogenous co-stimulatory molecule, a chimeric antigen receptor (CAR), or any combination thereof.

[0203] In certain embodiments, the host cell transfected to express the fusion protein of the present disclosure is a functional natural killer cell.

[0204] One or more growth factor cytokines that promote the proliferation of T cells expressing the fusion proteins of the present disclosure can be added to the culture used for amplifying T cells. The cytokines can be human or non-human. Exemplary growth factor cytokines that can be used to promote T cell proliferation include IL2, IL15, and the like.

[0205] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure are CD4 + T cells that also express an antigen-specific high-affinity TCR specific for HLA (MHC) class I-restricted antigens (see Soto et al., Cancer Immunol Immunother. 62:359–369, 2013).

[0206] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure also express a recombinant TCR specific for a cancer antigen. In some embodiments, the cancer antigen is WT1. "WT1" refers to Wilm tumor 1, a transcription factor that contains four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA-binding domain at the N-terminus. WT1 plays a crucial role in the normal development of the urogenital system and is mutated in a small fraction of patients with Wilm tumors. High expression of WT1 has been observed in a variety of cancers, including breast cancer, ovarian cancer, acute leukemia, hemangioma, melanoma, colon cancer, lung cancer, thyroid cancer, bone and soft tissue sarcoma, and esophageal cancer. Alternative splicing of WT1 has been noted.

[0207] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure also express a recombinant TCR specific for mesothelin. "Mesothelin" (MSLN) refers to the gene encoding a precursor protein that is cleaved into two products, megakaryocyte potentiating factor and mesothelin. Megakaryocyte potentiating factor acts as a cytokine and can stimulate colony formation in bone marrow megakaryocytes. Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein that can act as a cell adhesion protein. The protein is overexpressed in epithelial mesothelioma, ovarian cancer, and certain squamous cell carcinomas. Alternative splicing results in multiple transcript variants.

[0208] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure also express a recombinant TCR specific for cyclin-A1.

[0209] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure also express a CAR.

[0210] In some other embodiments, host cells expressing the fusion proteins as disclosed herein further comprise a ligand, which can be CD200, CD47, PD-L1 or CD58. In still some other embodiments, host cells expressing the fusion proteins as disclosed herein further express siRNA for reducing endogenous receptor expression. In some specific embodiments, the endogenous receptor is CD200R, SIRPα, CD279 (PD-1), CD95 (Fas) or CD2.

[0211] In some embodiments, host cells expressing the fusion proteins as disclosed herein can express more than one fusion protein. For example, the host cells can express two different fusion proteins (e.g., a first fusion protein comprising the extracellular domain of PD-1 and a second fusion protein comprising the extracellular domain of TIM3).

[0212] Use

[0213] Diseases that can be treated with cells expressing the fusion proteins as described in the present disclosure include cancer, infectious diseases (viral, bacterial, protozoal infections), immune diseases (e.g., autoimmunity) or aging-related diseases (e.g., senescence). Adoptive immunotherapy and gene therapy are promising treatment methods for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2011).

[0214] A variety of cancers, including solid tumors and leukemias, are suitable for the compositions and methods disclosed herein. Exemplary cancer types that can be treated include breast cancer, prostate cancer, and colon cancer; all forms of lung bronchogenic carcinoma; myelogenous leukemia; melanoma; liver cancer; neuroblastoma; papilloma; APUDoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart disease; and carcinomas (e.g., Walker, basal cell, basal squamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, sclerosing, bronchiolar, bronchial, squamous cell, and transitional cell). Other types of cancers that can be treated include histiocytic disorders; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. In addition, the following cancer types that can be treated are also contemplated: adenoma; cholangiocarcinoma; cholesteatoma; cylindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; ovarian gynandroblastoma; liver cancer; hidradenoma; islet cell tumor; Leydig cell tumor of testis; papilloma; Sertoli cell tumor of testis; thecoma; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; gangliocytoma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; nonchromaffin paraganglioma. Cancer types that can be treated also include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomus tumor; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangioleiomyoma; lymphangiosarcoma; pinealoma; sarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; tumor; multiple neurofibroma and cervical dysplasia.

[0215] Exemplary proliferative diseases suitable for fusion protein T cell therapy are B cell cancers, including B cell lymphomas (such as various forms of Hodgkin's disease, non-Hodgkin lymphoma (NHL), or central nervous system lymphoma), leukemias (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B cell blast transformation of chronic myelogenous leukemia, and acute myelogenous leukemia (AML)), and myelomas (such as multiple myeloma). Additional B cell cancers include small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, mucosa-associated extranodal marginal zone B cell lymphoma (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, B cell proliferations of undetermined malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0216] Inflammatory and autoimmune diseases include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myopathy, toxic epidermal necrolysis, systemic sclerosis and scleroderma, CREST syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus erythematosus, lupus myelitis, lupus encephalitis, juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham chorea, cytokine- and T lymphocyte-mediated immune responses associated with acute and delayed hypersensitivity reactions, tuberculosis, sarcoidosis, granulomatous diseases including Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including allergic vasculitis / vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, central nervous system (CNS) inflammatory disorders, multiple organ damage syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, seronegative spondyloarthropathy, Reiter's disease, stiff-person syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), allergic purpura, autoimmune thrombocytopenia, testicular and ovarian autoimmune diseases including autoimmune orchitis and oophoritis, primary hypothyroidism;Autoimmune endocrine diseases include autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyendocrine syndrome (or polyglandular endocrine syndrome), type I diabetes also known as insulin-dependent diabetes mellitus (IDDM), and Sheehan syndrome; autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonia (NSIP), Guillain-Barré syndrome, large-vessel vasculitis (including polymyalgia rheumatica and giant cell (temporal) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac sprue (gluten enteropathy), cryoglobulinemia, hepatitis-associated cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan syndrome, Wiskott-Aldrich syndrome, and thromboangiitis obliterans.;

[0217] In certain embodiments, methods of treating a subject with a fusion protein disclosed herein include treating acute myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (AML), and chronic myeloid leukemia.

[0218] In certain embodiments, methods of treating a subject with a fusion protein disclosed herein include treating pancreatic cancer.

[0219] In certain embodiments, methods of treating a subject with a fusion protein disclosed herein include treating ovarian cancer.

[0220] In certain embodiments, methods of treating a disease with a fusion protein described herein are provided, wherein the disease includes a solid tumor expressing CD200 or a tumor infiltrated by CD200 + myeloid cells, and the fusion protein has an extracellular component comprising an extracellular portion of CD200R.

[0221] In certain embodiments, methods of treating a disease with a fusion protein disclosed herein are provided, wherein the disease includes acute myeloid leukemia (AML), and the fusion protein has an extracellular component comprising an extracellular portion of CD200R, SIRPα, CD95 (Fas), CD279 (PD-1), or CD2.

[0222] In certain embodiments, provided are methods of treating a disease with a fusion protein disclosed herein, wherein the disease includes solid tumors, and the fusion protein has an extracellular component comprising an extracellular portion of TIM3, CD223 (LAG3), CD95 (Fas), CD279 (PD-1), or CD2.

[0223] In certain embodiments, provided are methods of treating a disease with a fusion protein disclosed herein, wherein the disease includes breast cancer, ovarian cancer, colon cancer, prostate cancer, or multiple myeloma, and the fusion protein has an extracellular component comprising an extracellular portion of CD200R. In some such embodiments, the CD200R-9aas-CD28Cys fusion protein is encoded by a polynucleotide that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the polynucleotide sequence shown in SEQ ID NO.: 7. In certain embodiments, the CD200R-9aas-CD28Cys fusion protein is encoded by a polynucleotide comprising or consisting of the polynucleotide sequence shown in SEQ ID NO: 7. In other embodiments, the CD200R-9aas-CD28Cys fusion protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence shown in SEQ ID No.: 30. In further embodiments, the CD200R-9aas-CD28Cys fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 30.

[0224] In certain embodiments, provided are methods of treating a disease with a fusion protein disclosed herein, wherein the disease includes ovarian cancer, pancreatic cancer, or AML, and the fusion protein has an extracellular component comprising the extracellular portion of CD95 (Fas). In some such embodiments, the Fastm-4-1BB fusion protein is encoded by a polynucleotide that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the polynucleotide sequence shown in SEQ ID NO.: 185. In certain embodiments, the Fastm-4-1BB fusion protein is encoded by a polynucleotide comprising or consisting of the polynucleotide sequence shown in SEQ ID NO: 185. In other embodiments, the Fastm-4-1BB fusion protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence shown in SEQID No.: 186. In further embodiments, the Fastm-4-1BB fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 186.

[0225] In certain embodiments, provided are methods of treating a disease with a fusion protein disclosed herein, wherein the disease includes ovarian cancer, pancreatic cancer, or AML, and the fusion protein has an extracellular component comprising the extracellular portion of CD95 (Fas). In some such embodiments, the Fas-4-1BBtm fusion protein is encoded by a polynucleotide that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the polynucleotide sequence shown in SEQ ID NO.: 187. In certain embodiments, the Fas-4-1BBtm fusion protein is encoded by a polynucleotide comprising or consisting of the polynucleotide sequence shown in SEQ ID NO: 187. In other embodiments, the Fas-4-1BBtm fusion protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence shown in SEQID No.: 188. In further embodiments, the Fas-4-1BBtm fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 188.

[0226] Infectious diseases include diseases associated with infectious agents, including any of a variety of bacteria (such as pathogenic Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, Vibrio cholerae, Listeria spp., Rickettsia spp., Chlamydia spp., etc.), mycobacteria, and parasites (including any known parasitic members of protozoa). Infectious viruses include eukaryotic viruses, such as adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus (such as HPV), paramyxovirus, picornavirus, rhabdovirus (such as rabies virus), orthomyxovirus (such as influenza virus), poxvirus (such as vaccinia virus), reovirus, retrovirus, lentivirus (such as HIV), flavivirus (such as HCV, HBV), etc. In certain embodiments, the fusion proteins of the present disclosure are used to treat infections by cytoplasmic pathogens that are processed and presented by HLA (MHC) class I molecules.

[0227] The fusion proteins of the present disclosure can be administered to a subject in a cell-binding form (e.g., gene therapy of a target cell population (mature T cells (such as CD8 + or CD4 + T cells) or other cells of the T cell lineage)). In a specific embodiment, the cells of the T cell lineage expressing the fusion protein administered to the subject are syngeneic, allogeneic, or autologous cells.

[0228] A pharmaceutical composition comprising the fusion proteins of the present disclosure can be administered in a manner suitable for the disease or disorder to be treated (or prevented) as determined by a person skilled in the medical art. The appropriate dosage, appropriate duration, and frequency of administration of the composition will be determined by factors such as the symptoms of the patient, the size, type, and severity of the disease, the specific form of the active ingredient, and the method of administration. The present disclosure provides a pharmaceutical composition comprising cells expressing a fusion protein as disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, etc. and combinations thereof.

[0229] In some embodiments, the present disclosure relates to methods of increasing immune cell activity, enhancing or prolonging an immune response, stimulating an antigen-specific T cell response, inhibiting an immunosuppressive signaling pathway, treating cancer or a tumor, inhibiting cancer cell immune resistance, or treating an infection, comprising administering to a subject in need thereof an effective amount of a host cell expressing a fusion protein as described herein. In further embodiments, the host cells for any of the above methods further express an engineered antigen-specific TCR, an engineered antigen-specific high-affinity TCR, a CAR, a costimulatory molecule, or any combination thereof. In a specific embodiment, a method of treating leukemia is provided, comprising co-expressing a fusion protein as disclosed herein and a recombinant antigen-specific TCR.

[0230] In some embodiments, provided is a method for inducing or enhancing a class I HLA response in CD4 + T cells, comprising administering to a subject in need thereof an effective amount of CD4 + T cells expressing a fusion protein as described herein. In further embodiments, a host cell for inducing or enhancing a class I HLA response in CD4 + T cells further expresses an engineered antigen-specific TCR, an engineered antigen-specific high-affinity TCR, a CAR, a co-stimulatory molecule, or any combination thereof.

[0231] In any of the above embodiments, the method is effective without administration of exogenous IL-2.

[0232] In some embodiments, provided is a method for increasing cytokine production in immune cells (e.g., CD4 + T cells or CD8 + T cells) of a subject, comprising administering to a subject in need thereof an effective amount of a fusion protein as disclosed herein or a vector encoding the fusion protein.

[0233] In some embodiments, provided is a method for increasing cytokine production in immune cells (e.g., CD4 + T cells or CD8 + T cells), comprising contacting the immune cells with a fusion protein as disclosed herein or a vector encoding the fusion protein. In further embodiments, the immune cells further express an engineered antigen-specific TCR, an engineered antigen-specific high-affinity TCR, a CAR, a co-stimulatory molecule, or any combination thereof. In some embodiments, the increased cytokines include interferon (IFNγ), tumor necrosis factor (TNFα), or interleukin-2 (IL-2)

[0234] In other embodiments, a subject of any of the above methods is further treated with adjuvant therapies, such as chemotherapy. Exemplary chemotherapeutic agents include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen mustard oxide, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens such as calusterone, dromostanolone propionate, cyclothiocarb, mesandrostane, and testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acetoglucuronide; aldehyde phosphoramide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclofenac; diaziquone; elformithine; vinyl acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone ; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllotoxin; 2-ethylhydrazide; procarbazine; PSKTM; razoxane; sizoxane; spirogermanium; tricosporic acid; triimine; 2,2',2"-trichlorotriethylamine; ethyl carbamate; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol; TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere TM , Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; norvinblastine; nosocomial; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins, capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0235] In some embodiments, the adjuvant therapy is a vaccine, an inhibitor of an immunosuppressive signal, a B-Raf inhibitor, a MEK inhibitor, a tyrosine kinase inhibitor, a cytotoxic agent, a chemotherapeutic agent, or any combination thereof. In some embodiments, the inhibitor of an immunosuppressive signal is an antibody or siRNA. In some embodiments, the antibody or siRNA is specific for PD-1, PD-L1, PD-L2, CTLA4, LAG3, KIR, CD244, B7-H3, B7-H4, BTLA, HVEM, GAL9, TIM3, A2aR, or a combination thereof.

[0236] Example

[0237] Example 1

[0238] CD200R-CD28 fusion protein construct

[0239] Use Figure 1A The schematic illustration in shows exemplary fusion proteins described herein. Exemplary fusion proteins include immunomodulatory fusion proteins (IFPs) that include the extracellular domain of CD200R or a portion thereof and the intracellular signaling domain of CD28 or a portion thereof ( Figure 1A , constructs I-V). The hydrophobic component may include the transmembrane domain of CD200R ( Figure 1A , construct I) or the transmembrane domain of CD28 ( Figure 1A , constructs II-V) or a portion thereof. In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the hydrophobic component (e.g., Figure 1A construct III, CD200R-CD28Cys; construct IV, CD200R-3aas-CD28Cys; and construct V, CD200R-9aas-CD28Cys). The extracellular component may comprise all or a portion of the extracellular domain of CD200R. In some embodiments, the extracellular component comprises the entire extracellular domain of CD200R ( Figure 1A , constructs I-III). In other examples, the extracellular component comprises the first 235 amino acids from the N-terminus of CD200R (retaining the N-linked glycosylation site) (e.g., Figure 1A , construct IV, CD200R-3aas-CD28Cys) or the first 229 amino acids (e.g., Figure 1A, construct V, CD200R-9aas-CD28Cys). The size of the extracellular component that can be manipulated by modulating the fusion protein construct may affect the ability of the fusion protein to enter the immune synapse and co-localize with the TCR in the cSMAC to deliver a strong co-stimulatory signal. Additionally, the CD200R-CD28 construct has the ability to convert the inhibitory signal normally derived from the binding of CD200R to its target into a positive signal generated by the CD28 intracellular signaling domain.

[0240] Exemplary nucleic acid molecules encoding the CD200R-CD28 fusion protein comprise the following elements (5' to 3'): extracellular component (CD200R)-polymerization domain (CD28 cysteine)-hydrophobic component (CD28 transmembrane)-intracellular component (CD28 intracellular). In some embodiments, the nucleic acid molecule encoding the CD200R-CD28 fusion protein comprises the nucleic acid molecule shown in any one of SEQ ID NOs: 47-51 or 1, 6, 7, 10, 12, 14 or 15.

[0241] The nucleic acid encoding the construct was ordered from Invitrogen or generated in the laboratory by PCR and then directionally TOPO cloned into the pENTR TM / vector (Invitrogen) and transferred to the retroviral vector pMP71-attR using technology (Invitrogen). In certain embodiments, the nucleic acid molecule encoding the IFP of the present disclosure was codon optimized prior to cloning into the pMP71-attR retroviral vector.

[0242] Example 2

[0243] Transgenic expression of the CD200R-CD28 construct

[0244] Based on the murine C57BL / 6 Friend virus-induced erythroleukemia (FBL) and TCR gag preclinical disseminated leukemia mouse model of transgenic mice was used to determine whether the CD200R-CD28 chimeric receptor could improve T cell function.

[0245] TCR transgenic mice were generated to produce CD8 gag T cells specific for the gag epitope (TCR + ). C57BL / 6 (B6) mice were purchased from Jackson Laboratories. TCR gag transgenic mice express a TCR transgenic specific for the Friend virus gag epitope in CD8 + T cells ( et al., J. Immunol. 166:2863 - 2870, 2001). All animal studies were approved by the University of Washington Animal Care and Use Committee protocol (protocol number 2013 - 01). Murine B6 Friend virus - induced erythroleukemia (FBL) expresses the gag epitope encoded by F - MuLV (peptide CCLCLTVFL (SEQ ID NO.:213)).

[0246] A murine gene - based CD200R - CD28 chimeric construct was inserted into the pMP71 retroviral vector and used to transduce primary murine splenocytes stimulated with anti - CD3 and anti - CD28 antibodies. The construct was designed as described in Example 1 and ordered from Invitrogen or generated in the laboratory by PCR. The construct was then directionally TOPO - cloned into the pENTR TM / vector (Invitrogen) and transferred into the retroviral vector pMP71 - attR using the technique (Invitrogen). The retroviral packaging cell line Plat - E (Morita et al., 2000, Gene Therapy 7:1063 - 1066, 2000; Cell Biolabs, Inc.) was transduced with the retroviral vector using an effector transduction reagent (Qiagen). Viral supernatants were collected on days 2 and 3 and then used to transduce TCR gag T cells.

[0247] One day prior to transfection, TCR gag T cells were stimulated with anti - CD3 / CD28 and 100 U / mL rhIL - 2. TCR gag T cell transduction was performed by spinfection at 1000 g for 90 minutes in a 12 - well plate in the presence of IL - 2 and polybrene. Similar to T cell transduction, FBL cells were transfected with CD200 by polybrene - mediated spinfection and then sorted to generate a homogeneous population.

[0248] Five days after transduction, construct expression in CD8 + T cells was analyzed by anti - CD200R antibody staining and flow cytometry ( Figure 1B ). A vector encoding green fluorescent protein (GFP) was used as a control. The transduction efficiency ranged from 4 - 36%, and the mean fluorescence intensity (MFI) of transduced cells was similar between constructs.

[0249] Example 3

[0250] The CD200R-CD28 construct promotes the proliferation, accumulation, and effector function of transduced T cells in vitro

[0251] Evaluate the ability of the CD200R-CD28 constructs described in Examples 1 and 2 to promote TCR gag T cell proliferation, accumulation, and effector function.

[0252] Expansion of effector cells in vitro

[0253] As previously described, effector cells are generated in vitro (Stromnes et al., J. Clin. Invest. 120:3722-34, 2010). Irradiated antigen-presenting splenocytes (5×10 6 ), irradiated FBL (3×10 6 ), and TCR gag tg cells (10 6 ) are cultured with IL-2 (50 U / mL) in 10 mL of medium (IMDM supplemented with non-essential amino acids, 2 μM glutamine, 100 U / mL penicillin / streptomycin, 10% FBS, and 50 μM 2-mercaptoethanol). Five to seven days after the last stimulation, the T cells are restimulated weekly and evaluated by flow cytometry.

[0254] In vitro T cell proliferation assay

[0255] TCR gag T cells transduced as in Example 2. To evaluate in vitro T cell proliferation, TCR gag T cells are stained with CellTrace Violet (CTV, Life Technologies) according to the manufacturer's protocol. CTV-labeled Tg T cells (10 - ) and GFP control T cells are stimulated with titrated amounts of CD200 + FBL or CD200 5 FBL cells. Three days later, the CTV dilution of TCR gag T cells is evaluated by flow cytometry.

[0256] Flow cytometry results show the number of TCR + T cells after stimulation with titrated amounts of CD200-FBL cells (upper) or CD200 gag FBL (lower) as Figure 2A shown. Four out of the five CD200R-CD28 constructs tested significantly improve TCR gag T cell proliferation (blue line) in response to CD200 + FBL compared to GFP control-transduced T cells (red line).

[0257] In vitro T cell accumulation assay

[0258] To determine whether enhanced proliferation also results in increased accumulation of transduced cells, the proportion of transduced cells in the total TCR + population was measured during multiple stimulation cycles with irradiated CD200 gag FBL.

[0259] Some constructs promoted the accumulation of transduced T cells, including CD200R-CD28tm, CD200R-CD28Cys, CD200R-3aas-CD28Cys, and CD200R-9aas-CD28Cys( Figure 2B ). Among these constructs, CD200R-9aas-CD28Cys showed the greatest increase in transduced T cells during multiple stimulations, resulting in more than a 3-fold expansion after 3 stimulations.

[0260] In vitro T cell enrichment assay

[0261] Transduced and untransduced CD8 + or CD200 - T cells were restimulated with irradiated FBL cells to determine whether restimulation would enrich the transduced CD200R-9aas-CD28Cys IFP + T cell population. Compared to wild-type T cells, repeated restimulation with irradiated CD200 + tumor cells enriched cells transduced with IFP, indicating that targeting a target that recognizes the ligand of CD200R-9aas-CD28Cys IFP can enhance the response( + ). Figure 2C )

[0262] In vitro co-localization assay

[0263] Transduced T cells were imaged by microscopy to determine whether CD200R-9aas-CD28Cys IFP co-localizes with cognate ligands in the immunological synapse (IS) during T cell activation. CTxB was used to stain lipids in the cell membrane, which are enriched at the synapse( Figure 2D , Figure I). Labeled antibodies targeting CD200( Figure 2D , Figure II) expressed by FBL cells or CD200R( Figure 2D , Figure III) expressed by T cells were used to visualize the position of the molecules relative to the IS. CD200 ligand and CD200R co-localize within the IS( Figure 2D , Figure IV), indicating that the construct is of an appropriate size to be accommodated by the immunological synapse.

[0264] (G) CFSE-based cytotoxicity assay.

[0265] Increased CD28 signaling also promotes effector function (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). The increased killing of target tumor cells by T cells transduced with CD200R-CD28 fusion protein was tested. FBL and control EL4 tumors were incubated with 2.5 μM (CFSEhi) or 0.25 μM (CFSElo) CFSE in PBS for 10 minutes at room temperature. Excess dye was removed by washing the tumor cells in serum-containing medium. A 1:1 mixture of EL4 and FBL tumor cells was incubated with TCR gag effector T cells expanded in vitro in 96-well round-bottom plates at 37 °C and 5% CO 2 2 for 4 hours. Specific FBL lysis was determined by analyzing the %CFSEhi (FBL) in the total CFSE-positive cells (FBL + EL4) remaining in the wells by flow cytometry.

[0266] compared to TCR gag T cells transduced with empty vector, TCR gag T cells transduced with CD200R-CD28 construct showed higher ability to lyse FBL tumors in vitro ( Figure 2E 、 2G ). Target tumor cells were labeled with different dilutions of the fluorescent dye CellTrace Violet (CTV) or CFSE to generate a 1:1:1 mixture of EL4 cells (CTV + ), CD200 + FBL (CFSEhi) and non-specific EL4 (CFSElo) control targets ( Figure 2F ). In addition, control GFP-transduced TCR gag T cells lysed CD200 - FBL and CD200 + FBL with equal efficiency ( Figure 2G ). In contrast, compared to control T cells, TCR gag T cells transduced with CD200R-9aas-CD28Cys showed increased killing of CD200 + FBL cells, lysing more than 40% of CD200 + FBL at the lowest tested E:T ratio ( Figure 2G ).

[0267] In summary, these data indicate that the CD200R-CD28 construct serves to increase the accumulation and lytic activity of transduced T cells in response to tumor cell stimulation.

[0268] Example 4

[0269] CD200R-9AAS-CD28CYS transduced T cells showed enhanced in vivo accumulation in response to recognition of FBL

[0270] As previously described, B6 mice were injected intraperitoneally (i.p.) with 4 × 10 6 viable FBL leukemia cells (Stromnes et al., J. Clin. Invest. 120:3722 - 34, 2010). Five days after allowing FBL to disseminate, at least 6 hours prior to transfer of effector T cells, the mice received an intraperitoneal injection of 180 mg / kg cyclophosphamide (Cy, commercially available and used). For survival studies, 10 5 TCR gag T cells that had been stimulated 1 - 3 times in vitro previously were transferred into tumor-bearing mice. For assessment of short-term proliferation and accumulation, 2 × 10 6 each of the fusion protein-transduced T cells and GFP control-transduced T cells were co-injected into tumor-bearing mice, and the mice were euthanized for analysis at 8 days. The tumor burden of the mice was monitored regularly, and if evidence of tumor progression indicated that death would occur within 24 - 48 hours, the mice were euthanized.

[0271] To assess whether CD200R-9aas-CD28Cys fusion protein-transduced T cells exhibit greater proliferation and accumulation in vivo in response to recognition of FBL, a mixed population of fusion protein-transduced and control cells was transferred into tumor-bearing mice, and the ratio of the cells was compared by ex vivo analysis 8 days after transfer ( Figure 3A ). By using homologous markers, 1.2 - 1.4 times more transduced T cells than control cells were detected in the spleen and lymph nodes relative to the injected proportion ( Figure 3B ). The transduced CD200R-9aas-CD28Cys + TCR gag T cells showed decreased CD62L expression 3 days after transfer into tumor-bearing mice, indicating an effector T cell phenotype ( Figure 3C ). By day 15, the transduced T cells and control T cells showed similar phenotypes, including the absence of exhaustion markers ( Figure 3D)。Similar to the in vitro findings, T cells expressing CD200R-9aas-CD28Cys showed increased accumulation in vivo in response to tumor stimulation. In addition, they exhibited a protein expression pattern consistent with the effector T cell phenotype for at least 3 days after transfer to tumor-bearing mice.

[0272] Example 5

[0273] CD200R-CD28 + Adoptive immunotherapy with T cells has shown greater activity in the treatment of disseminated leukemia

[0274] Adoptive immunotherapy with CD200R-CD28-transduced T cells mediated enhanced therapeutic activity in a preclinical murine model of disseminated leukemia.

[0275] Mice were injected with a lethal dose of CD200 + FBL leukemia, and five days later, the group of mice receiving Cy treatment received an additional treatment of 10 5 T cells ( Figure 4A ). As Figure 1A shown, the contribution of the CD28 cysteine bond to the efficacy mediated by the CD200R-CD28 construct was evaluated by comparing T cells transduced with CD200R-CD28tm, CD200R-9aas-CD28Cys, and GFP control constructs. IL-2 was given as an additional therapeutic agent to one group of mice for 10 days to boost T cell activity (Stromnes et al., J. Clin. Invest. 120:3722-34, 2010). Before injection, various surface proteins of the T cells were evaluated by flow cytometry. The transduced and control TCR gag T cells exhibited similar phenotypes, indicating that transduction did not alter the phenotype of the cells before injection ( Figure 4B ).

[0276] In a small group of mice receiving IL-2 injection, the T cells improved survival, but no significant difference in survival was detected among mice receiving different groups of T cells ( Figure 4C ). However, in the group of mice not receiving IL-2 injection, the survival rate of mice receiving T cells transduced with the CD200R-CD28 construct, the size of which was suitable within the immunological synapse ( Figure 4D ), was significantly improved. Most mice receiving GFP control vector-transduced T cells or T cells transduced with the maximal extracellular domain (CD200R-CD28Cys IFP) without receiving T cells did not survive beyond 30 days ( Figure 4C and 4D, respectively black solid line, dashed line, and orange line). In contrast, 71% of the mice receiving CD200R-CD28tm + T cells and 83% of the mice receiving CD200R-9aas-CD28Cys + T cells survived more than 100 days after treatment ( Figure 4C and 4D , green line and red line respectively). These data indicate that transducing T cells with the CD200R-CD28 construct provides sufficient co-stimulation to overcome the dependence of T cell immunotherapy on exogenous IL-2 injection, and the CD200R-CD28 construct spans a distance similar to the distance between membranes in the immune synapse. In addition, although there are differences in proliferation and accumulation between the CD200Rtm-CD28 and CD200R-9aas-CD28Cys constructs tested in mice not receiving exogenous IL-2 injection, both IFPs effectively enhanced T cell immunotherapy, thus significantly improving the clinical outcomes obtained from other progressive leukemias.

[0277] Example 6

[0278] CD200R-9AAS-CD28CYS + T cells do not cause spontaneous reactions with endogenous tissues and do not show infiltration of normal tissues in vivo

[0279] To determine whether transduction of TCR gag T cells can sufficiently lower the activation threshold to result in autoimmunity against endogenous tissues, autoimmunotoxicity was evaluated in transgenic mice under the control of the albumin promoter, which were engineered to express FBL in hepatocytes gag tumor Ag, as a self-antigen ( Figure 5A ). TCR gag effectors were generated in vitro and 10 6 strains were transferred to cyclophosphamide-treated Alb: Gag mice with disseminated leukemia. Three and seven days after transfer, liver injury was evaluated by quantifying the serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Treatment of mice with control or CD200R-9aas-CD28Cys + TCR gag cells by adoptive transfer did not affect the serum levels of AST or ALT on day 3 or 7 after transfer, indicating that CD200R-9aas-CD28Cys does not induce detectable autoimmune liver injury in Alb: Gag mice ( Figure 5B ).

[0280] Compared to control T cells, IFP-transduced T cells did not show an increase in normal tissue infiltration. Mice were euthanized on day 7 after transfer, and liver sections were stained with an antibody against the T cell marker CD3 to quantify T cell infiltration. A restricted presence of T cells in the liver tissue was observed, CD200R-9aas-CD28Cys + or control TCR gag There was no significant difference between the receptors, indicating that there was no increase in lymphocyte infiltration due to IFP expression ( Figure 5C ).

[0281] Example 7

[0282] The 4-1BB costimulatory signaling domain promotes the accumulation of transduced T cells in vitro

[0283] The costimulatory receptor 4-1BB is upregulated on activated T cells, which promotes T cell survival and cytokine production (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). To evaluate whether the intracellular signaling domain of 4-1BB with or without the intracellular signaling domain of CD28 could induce an increase in T cell proliferation and accumulation, 4-1BB (CD200R-9aas-4-1BB), or a combination of 4-1BB and CD28 (CD200R-9aas-CD28-4-1BB) was used to generate IFP by the method described in Example 2 ( Figure 6A ). TCR gag T cells were transduced as in Example 2 and TCR gag effector cells were generated in vitro as in Example 3.

[0284] As observed with CD200R-9aas-CD28Cys, T cells transduced with the 4-1BB construct accumulated in multiple rounds of in vitro stimulation ( Figure 6B ). These data indicate that 4-1BB IFP also promotes T cell proliferation and survival.

[0285] Using the CFSE-based cytotoxicity assay described in Example 3, TCR gag T cells transduced with CD200R-4-1BB showed enhanced ability to lyse FBL tumors in vitro ( Figure 6C ). CD200R-41BB-transduced T cells also promoted survival ( Figure 6D ).

[0286] Example 8

[0287] Co-expression of CD200RTM-CD28 enhances the function of WT1-specific TCR primary T cells

[0288] The human CD200Rtm-CD28 construct (SEQ ID NO.: 1) was generated to determine whether IFP expression enhanced T cell function in human primary T cells. By ligating the gene with the P2A element, the construct was combined with the HLA-A2 restricted WT1 126 specific TCR “C4” β and α chains ( Figure 7A ). The first P2A sequence was codon-optimized to prevent genetic recombination with the second P2A sequence. To generate lentivirus, 293T / 17 cells (3×10 6 cells / plate) were transduced with the human construct in pRRLSIN and packaging vectors pMDLg / pRRE, pMD2-G, and pRSV-REV using Effectene (Qiagen). The medium was changed on day 1 post-transfection, and the virus-containing supernatant was collected on days 2 and 3 and aliquots were frozen for later use.

[0289] The Jurkat human T cell subline lacking endogenous TCR was used to test the expression of IFP and TCR. These Jurkat T cells were transduced by centrifuging at 1000g for 90 minutes with 2 ml of retroviral supernatant at 32 °C for 2×10 6 cells. Transduction of the three-gene construct in the Jurkat human T cell line resulted in high expression of IFP and expression of TCR, and the expression of the TCR was similar to the MFI of T cells transduced with TCR only ( Figure 7A ).

[0290] To transduce primary human T cells, peripheral blood mononuclear cells (PBMC) were harvested from HLA-A2 + donors. CD8 + T cells were purified using Miltenyi beads and stimulated with human T cell Expander CD3 / CD28 Dynabeads (Life Technologies) and 50 IU / ml IL-2. Four hours after stimulation, the T cells were transduced as described above for Jurkat T cells. The T cells were restimulated every 10 - 14 days using a rapid expansion protocol (REP) as previously described (Ho et al., J Immunol Methods 310:40 - 52, 2006).

[0291] The human cell line T2 was used as an APC because it lacks TAP and thus cannot present endogenous peptides, while low-level expression of MHC I allows presentation of exogenously loaded peptides. The expression of CD200 on T2 cells was evaluated by flow cytometry ( Figure 7B ). T2 cells exhibited low levels of endogenous CD200 expression ( Figure 7B ).

[0292] Using WT1 126 T2 cells pulsed with WT1 stimulate transduced T cells. Despite low levels of CD200 expression on target cells, CD200Rtm-CD28 transduced T cells showed enhanced proliferation compared to T cells transduced with C4 TCR alone ( Figure 7C ). Additionally, when exposed to CD200dim tumor cells, stimulated CD200Rtm-CD28 transduced T cells (i.e., IFP + T cells) produced elevated levels of IFNγ and IL-2 compared to control T cells ( Figure 7D ).

[0293] Overall, these results indicate that primary T cells transduced to express the human CD200Rtm-CD28 construct and WT1 126 specific TCR β and α chains exhibit enhanced proliferation and increased cytokine production compared to T cells transduced with the TCR construct alone.

[0294] Example 9

[0295] The SIRPα-CD28 fusion protein construct promotes the accumulation of transduced T cells in vitro.

[0296] Exemplary fusion proteins as described herein also include IFP, which comprises the extracellular domain or portion thereof of SIRPα and the intracellular signaling domain of CD28 ( Figure 8A)。The hydrophobic component may include the transmembrane domain of SIRPα or CD28 or a portion thereof. In some exemplary SIRPα-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the hydrophobic component (e.g., SIRPα-CD28Cys, SIRPα-6aas-CD28Cys, SIRPα-9aas-CD28Cys, and SIRPα-9aas-CD28Cys). The extracellular component may comprise all or part of the extracellular domain of SIRPα. In some embodiments, the extracellular component comprises the entire extracellular domain of SIRPα. In other instances, the extracellular component comprises the first 367 amino acids (e.g., SIRPα-6aas-CD28Cys) or the first 364 amino acids (e.g., SIRPα-9aas-CD28Cys) or the first 350 amino acids (SIRPα-23aas-CD28Cys) from the N-terminus of SIRPα. The size of the extracellular component may affect the ability of the fusion protein to enter the immunological synapse and co-localize with the TCR in the cSMAC to deliver a strong co-stimulatory signal. In some instances, the extracellular component comprises a truncated SIRPα, which may alter the size of the extracellular component. For example, to account for additional extracellular amino acids (e.g., an additional 9 or 12 amino acids) in the extracellular domain of the fusion protein, SIRPα-6aas-CD28 has a truncated portion of SIRPα that retains the native N-linked glycosylation site. In another instance, SIRPα-23aas-CD28 has a truncated portion of SIRPα that lacks the entire stalk region of the SIRPα extracellular domain. Additionally, the SIRPα-CD28 construct has the ability to convert the signal initiated by SIRPα binding to its target into a positive (e.g., co-stimulatory) signal generated by the CD28 intracellular signaling domain.

[0297] Generate an IFP using the SIRPα extracellular component using the method described in Example 2 ( Figure 8A ). Transduce TCR gag T cells as in Example 2 and generate TCR gag effector cells in vitro as in Example 3. Similar to T cell transduction, transduce FBL cells with CD47 or mCherry by polybrene centrifugation transfection and then sort to generate a homogeneous population.

[0298] As observed with CD200R-9aas-CD28Cys, T cells transduced with the SIRPα construct accumulate during multiple rounds of stimulation in vitro ( Figure 8B ). These data indicate that the SIRPα-CD28 IFP also promotes T cell proliferation and survival.

[0299] To assess T cell proliferation in vitro, a CTV dilution proliferation assay was performed as described in Example 2. As observed with CD200R-9aas-CD28Cys, T cells transduced with SIRPα constructs engineered to maintain the T cell-tumor cell synaptic distance showed enhanced proliferation compared to control T cells( Figure 8C ). Additionally, after 3 days of co-culture with SIRPα-CD28 + T cells, CD47 + tumor cells were efficiently killed, but not control T cells or T cells transduced with SIRPα constructs lacking an intracellular signaling domain( Figure 8D ). To further assess the lytic ability of SIRPα-CD28 + T cells, a detection method was used to quantify the killing of CD47 + FBL. A total of 10 5 m of Cherry + CD47 + FBL was co-cultured in 24-well plates with titers of human T cells transduced with the SIRPα-CD28 construct. The plates were incubated in a (Essen BioScience) at 70 hours. Images were captured hourly to monitor the killing of tumor cells, which was determined by the loss of the red signal. Even when tested at the lowest effector-to-target ratio, SIRPα-CD28 + T cells killed CD47 + tumor cells.

[0300] Example 10

[0301] The PD-1-CD28 fusion protein construct promotes cytokine production in transduced T cells in vitro

[0302] Exemplary fusion proteins as described herein also include IFPs that contain the extracellular domain of PD-1 or a portion thereof and the intracellular signaling domain of CD28( Figure 9A)。The transmembrane component may include the transmembrane domain of PD-1 or CD28 or a portion thereof. In some exemplary PD-1-CD28 fusion proteins, the transmembrane component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the transmembrane component, to facilitate interchain dimerization (e.g., PD1-CD28Cys, PD1-9aas-CD28Cys, and PD1-21aas-CD28Cys). The extracellular component may comprise all or part of the extracellular domain of PD-1 or may be truncated (e.g., -9aas in murine constructs, -12aas or -15aas in human constructs; lacking the stalk region of PD-1, -21aas) to maintain a shorter spatial distance between cells and to facilitate ligand-bound receptor entry into the immunological synapse. Additionally, the PD1-CD28 construct has the ability to convert the inhibitory signal normally derived from the binding of PD1 to its target into a positive (e.g., co-stimulatory) signal generated by the intracellular signaling domain of CD28.

[0303] Generate an IFP comprising the extracellular component of SIRPα using the method described in Example 2 ( Figure 9A ). Transduce TCR gag T cells as in Example 2 and generate TCR gag effector cells in vitro as in Example 3.

[0304] Generate murine PD1-CD28 IFP using constructs I-IV and VII ( Figure 9A ). In the presence of brefeldin A (to retain the cytokines produced), restimulate PD1-CD28 + T cells with FBL cells endogenously expressing the PD-1 ligands (PD-L1 and PD-L2). After 5 hours, fix the cells and treat them with the BD Cytofix / Cytoperm kit to allow intracellular staining of effector cytokines (IFNγ and TNFα). Transduction with each of the five PD1-CD28 constructs enhanced the production of intracellular cytokines compared to control T cells ( Figure 9B ).

[0305] Generate human PD1-CD28 IFP using constructs I-III and V-VII ( Figure 9A ). Generate a vector comprising PD1-CD28IFP and C4 TCR as described above. Transduce Jurkat T cells as described above. T cells transduced with TCR and PD1-12aas-CD28Cys or PD1-15aas-CD28Cys exhibited high transduction efficiency and expression of both proteins ( Figure 10 ).

[0306] Example 11

[0307] The FAS-CD28 fusion protein construct promotes the accumulation and enhanced function of transduced T cells in vitro.

[0308] Exemplary fusion proteins as described herein also include an IFP that comprises the extracellular domain or a portion thereof of Fas and the intracellular signaling domain of CD28 ( Figure 11A ). The transmembrane component can include a domain or a portion thereof of Fas or CD28. In some exemplary Fas-CD28 fusion proteins, the transmembrane component includes the transmembrane domain of CD28, and the extracellular component also includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the transmembrane component (e.g., Fas-CD28Cys and Fas-9aas-CD28Cys). The extracellular component can comprise all or a portion of the extracellular domain of Fas or can be truncated to maintain a short spatial distance (-9aas) between cells upon receptor-ligand interaction. Additionally, the Fas-CD28 construct has the ability to convert the signal initiated by the binding of Fas to its target into a positive (e.g., co-stimulatory) signal generated by the CD28 intracellular signaling domain.

[0309] An IFP comprising the extracellular component of Fas was generated using the method described in Example 2 ( Figure 11A ). TCR gag T cells were transduced as in Example 2 and TCR gag effector cells were generated in vitro as in Example 3.

[0310] To determine whether the expression of the Fas-CD28 IFP results in an increase in the accumulation of transduced cells, the proportion of transduced cells in the total TCR gag population in the mixed population was measured in multiple cycles of stimulation with irradiated FBL as described in Example 3. All constructs promoted the accumulation of transduced T cells relative to control T cells ( Figure 11B ). However, the expression of the Fas-CD28 construct but not full-length (FL) Fas promoted the survival or expansion of T cells after multiple stimulations in vitro ( Figure 11C ).

[0311] Example 12

[0312] LAG3-CD28 fusion protein construct

[0313] Exemplary fusion proteins as described herein also include an IFP that comprises the extracellular domain or a portion thereof of LAG3 and the intracellular signaling domain of CD28 ( Figure 12A)。The transmembrane component can include the domain or a portion thereof of LAG3 or CD28. In some exemplary LAG3-CD28 fusion proteins, the transmembrane component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the transmembrane component (e.g., LAG3-CD28Cys and LAG3-9aas-CD28Cys). The extracellular component can contain all or part of the extracellular domain of LAG3 or can be truncated to maintain a short spatial distance between cells upon receptor-ligand interaction (e.g., -9aas). Additionally, the LAG3-CD28 construct has the ability to convert the inhibitory signal normally derived from the binding of LAG3 to its target into a positive (e.g., co-stimulatory) signal generated by the intracellular signaling domain of CD28.

[0314] Generate an IFP using the extracellular component of LAG3 using the method described in Example 2 ( Figure 12A )。As described, transduce T cells with LAG3-eGFP. Five days after transduction, analyze the construct expression of CD8 + T cells by anti-LAG3 antibody staining and flow cytometry ( Figure 12B )。A vector encoding only green fluorescent protein (GFP) is used as a control. All constructs exhibit expression of LAG3 ( Figure 12B )。

[0315] Example 13

[0316] TIM3-CD28 fusion protein construct

[0317] Exemplary fusion proteins as described herein further include an IFP that contains the extracellular domain or a portion thereof of TIM3 and the intracellular signaling domain of CD28 ( Figure 13A )。The transmembrane component can include the domain or a portion thereof of TIM3 or CD28. In some exemplary TIM3-CD28 fusion proteins, the transmembrane component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the transmembrane component (e.g., TIM3-CD28Cys and TIM3-9aas-CD28Cys). The extracellular component can contain all or part of the extracellular domain of TIM3 or can be truncated to maintain a short spatial distance between cells (e.g., -9aas). Additionally, the TIM3-CD28 construct has the ability to convert the inhibitory signal normally derived from the binding of TIM3 to its target into a positive signal generated by the intracellular signaling domain of CD28.

[0318] Generate a new IFP using the extracellular component of TIM3 using the method described in Example 2 ( Figure 13A)。As described, T cells were transduced with the GFP-TIM3 construct. Five days after transduction, CD8 was analyzed by anti-TIM3 antibody staining and flow cytometry + of the construct expression in T cells ( Figure 13B ). A vector encoding only green fluorescent protein (GFP) was used as a control. Most constructs showed similar TIM3 expression ( Figure 13B ).

[0319] Example 14

[0320] The CD200R-CD28 fusion protein construct can be expressed by primary T cells

[0321] In a further example, the schematic diagram in Figure 14A illustrates the exemplary fusion proteins described herein. The exemplary fusion proteins include immunomodulatory fusion proteins (IFPs) that comprise the extracellular domain or a portion thereof of CD200R and the intracellular signaling domain or a portion thereof of CD28 ( Figure 14A , constructs I-V). The hydrophobic component can include the transmembrane domain or a portion thereof of CD200R ( Figure 14A , construct I) or CD28 ( Figure 14A , constructs II-V). In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the hydrophobic component (e.g., Figure 14A construct III, CD200R-CD28Cys; construct IV, CD200R-3aas-CD28Cys; and construct V, CD200R-9aas-CD28Cys). The extracellular component can comprise all or a portion of the extracellular domain of CD200R. In some examples, the extracellular component comprises the entire extracellular domain of CD200R ( Figure 14A , constructs I-III). In other examples, the extracellular component comprises the first 235 amino acids from the N-terminus of CD200R (retaining the N-linked glycosylation site) (e.g., Figure 14A , construct IV, CD200R-3aas-CD28Cys) or the first 229 amino acids (e.g., Figure 14A , construct V, CD200R-9aas-CD28Cys). The CD200R-CD28 constructs disclosed herein have the ability to convert an inhibitory signal normally derived from the binding of CD200R to its target into a positive signal generated by the CD28 intracellular signaling domain.

[0322] The size of the extracellular component that can be manipulated by modulating the fusion protein construct may affect the ability of the fusion protein to enter the immunological synapse and co-localize with the TCR in the cSMAC to deliver a strong co-stimulatory signal. CD28 signaling naturally occurs in the immunological synapse, where CD28 is recruited to amplify TCR signals and lower the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013; Yokosuka et al., Immunity 29:589-601, 2008). In the immunological synapse, the spatial distance between the T cell and the APC is shortest, and molecules with large extracellular domains are excluded. Thus, constructs that are closest to the intercellular space of the immunological synapse may be able to co-localize with the TCR within the immunological synapse and deliver an effective co-stimulatory signal. Constructs III and IV extend the CD28 transmembrane domain into the extracellular space to incorporate juxtamembrane cysteines (CD28Cys), which promote CD28 homodimerization and enhance native CD28 signaling (Lazar-Molnar et al., Cell Immunol. 244:125-129, 2006). To illustrate the nine amino acid increase in length of the extracellular CD28 domain, the extracellular domain of CD200R in CD200R-9aas-CD28Cys was truncated by nine amino acids, which is equivalent to the number added by the CD28 extracellular domain. Similarly, the extracellular CD200R of CD200R-3aas-CD28Cys was truncated by 3 amino acids. The truncated extracellular CD200R was truncated from the C-terminus to preserve the N-linked glycosylation site. Thus, in theory, the murine constructs CD200Rtm-CD28, CD200R-CD28tm, and CD200R-9aas-CD28Cys optimally maintain the required short spatial distance between the T cell and the APC to co-localize with the TCR in the immunological synapse.

[0323] Exemplary nucleic acid molecules encoding a CD200R-CD28 fusion protein comprise the following elements (5’ to 3’): extracellular component (CD200R)-polymerization domain (CD28 cysteine)-hydrophobic component (CD28 transmembrane)-intracellular component (CD28 intracellular). In some embodiments, the nucleic acid molecule encoding the CD200R-CD28 fusion protein comprises a nucleic acid molecule as set forth in any one of SEQ ID NOs: 47-51 or 1, 6, 7, 10, 12, 14, 15, or 183.

[0324] The nucleic acid encoding the construct was inserted into the pMP71 retroviral vector to transduce primary murine splenocytes stimulated with anti-CD3 and anti-CD28 antibodies. C57BL / 6 (B6) mice were purchased from Jackson Laboratories. TCR gagTransgenic mice express a TCR transgenic specific for the Friend virus gag epitope in CD8 + T cells (Ohlen et al., J. Exp. Med. 195:1407 - 1418, 2002). B6 Friend virus-induced erythroleukemia (FBL) expresses the Friend virus gag epitope (peptide CCLCLTVFL (SEQ ID NO.: 213)) (Teague et al., Nat. Med. 12:335 - 341, 2006). The DNA construct was ordered from Invitrogen or generated in the laboratory by PCR. Using technology, the construct cloned directionally into the vector pENTR / D-TOPO was transferred to the retroviral vector (RV) pMP71-attR. The retroviral packaging cell line Plat-E (Cell-Bio Labs) was transduced with RV using an effector transduction reagent (Qiagen). The viral supernatant was collected on days 2 and 3. One day before transfection, TCR gag T cells were stimulated with anti-CD3 / CD28 and 100 U / mL rhIL-2. In the presence of IL-2 and polybrene, TCR gag T cells were transduced by centrifugation at 1000 g for 90 minutes in a 12-well plate. On day 7 after stimulation, the transduced cells were restimulated in the presence of irradiated splenocytes (5×10 6 ), irradiated FBL (3×10 6 ) and IL-2 (IU / mL).

[0325] Five days after transduction, the IFP expression of CD8 + T cells was analyzed by flow cytometry ( Figure 14B ). Fluorochrome-conjugated antibodies were purchased from eBioscience or Biolegend. The transduction efficiency ranged from 5 - 43%, and the mean fluorescence intensity of the transduced cells was similar between constructs, indicating similar IFP expression.

[0326] Example 15

[0327] The CD200R-CD28 construct can promote the proliferation, accumulation, and effector function of transduced T cells in vitro

[0328] Evaluate the ability of the CD200R-CD28 construct described in Example 14 to promote TCR gag T cell proliferation, accumulation, and effector function.

[0329] In vitro T cell proliferation assay

[0330] CD28 signaling promotes the proliferation and survival of T cells stimulated via the TCR (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). To determine whether CD200R-CD28 IFP can improve proliferation, naive CD8 + TCR transgenic T cells (TCR gag cells) (Stromnes et al., J Clin Invest. 120:3722-3734, 2010)) (as described in Example 14) were transduced with antigen and amplified for 2-3 stimulation cycles in the presence of IL-2 in vitro to generate effector T cells and mimic a human adoptive immunotherapy protocol. Effector T cells were labeled with CellTrace Violet (CTV) and stimulated with FBL that does not naturally express CD200 (CD200 - FBL) ( Figure 15A , upper panel) or FBL cell lines transduced to express CD200 (CD200 + FBL) for 3 days, and then evaluated by flow cytometry ( 5 , lower panel). Figure 15A At a low 25:1 T cell to FBL ratio, GFP control-transduced T cells (

[0331] , blue line) showed minimal proliferation in response to CD200 Figure 15A or CD200 - or CD200 + FBL. In contrast, four of the five tested constructs ( Figure 15A , red lines) significantly improved proliferation in response to CD200 + FBL, but not in response to CD200 - FBL. T cells transduced with the largest extracellular domain CD200R-CD28Cys did not improve proliferation.

[0332] To test whether the increased proliferation transmitted by the interaction of CD200R with leukemia-expressed CD200 reflects enhanced adhesion and / or decoy binding rather than costimulation, a truncated non-signaling version of the construct containing only the extracellular CD200R and the transmembrane domain of CD28 ("trCD200R"; Figure 15B ) was generated. Transduced TCR gag T cells expressing the construct ( Figure 15C ) did not show enhanced proliferation to CD200 + FBL ( Figure 15D ), indicating the essential role of the CD28 costimulatory signal.

[0333] In vitro T cell enrichment assay

[0334] It was expected that after stimulation with CD200 + FBL, the expression of IFP targeting CD200 would lead to enrichment of IFP + T cells relative to IFP - T cells. The proportion of transduced cells in the total TCR - or CD200 + population was evaluated after multiple stimulation cycles with irradiated CD200 gag or CD200 - FBL. Analysis of the cell composition after 3 cycles of stimulation with CD200 + or CD200 gag FBL showed no change in the fraction of TCR Figure 15E T cells expressing the GFP control ([[]] + ). In contrast, after stimulation with CD200 gag T cells expressing IFP were enriched, while they were not enriched under CD200 - FBL stimulation (CD200R-9aas-CD28Cys, [[[]] Figure 15F ). As predicted, although several constructs promoted the accumulation of transduced T cells, the size of this construct was suitable for the immune synapse and included the dimeric cysteine motif CD200R-9aas-CD28Cys, which produced the largest relative increase after 3 stimulations in 3 separate experiments, resulting in an average >3-fold enrichment (P<0.05)([[[]] Figure 15G , showing fold enrichment, 3 stimulations / stimulation 1 time, for eGFP, CD200Rtm-CD28, CD200R-CD28tm, CD200R-3aas-CD28cys, and CD200R-9aas-CD28cys transduced T cells).

[0335] CFSE-based cytotoxicity assay.

[0336] CD28 signaling promotes effector function (Chen and Flies, Nat.Rev.Immunol. 13:227-242, 2013). T cells transduced to express CD200R-9aas-CD28Cys were tested for increased killing of tumor target cells.

[0337] FBL and control EL4 tumors were incubated with 2.5 μM (hi) or 0.25 μM (lo) CFSE in PBS at room temperature for 10 minutes, respectively. Excess dye was removed by washing the tumor cells in serum-containing medium. Nonspecific EL4 control targets and CD200+ 1:1 mixture of FBL tumor cells with titrated amounts of CD200R-9aas-CD28Cys-transduced or GFP-transduced TCR gag Effector T cells (i.e., effector-to-target (E:T) ratio in the range) were incubated for 5 hours in a 96-well round-bottom plate at 37 °C and 5% CO2. The total remaining CFSE-positive cells (FBL + EL4) in the wells were analyzed by flow cytometry for %CFSEhi (FBL) to determine specific FBL lysis.

[0338] TCR transduced with CD200R-9aas-CD28Cys gag T cells kill CD200 + FBL better than control T cells, lysed more than 40% of CD200 at a low E:T ratio (0.3:1) + FBL( Figure 15H )

[0339] In vitro cytokine production assay

[0340] To determine whether T cells transduced with CD200R-9aas-CD28Cys IFP produce increased amounts and diversity of cytokines, which represent additional co-stimulatory functions, the production of multifunctional cytokines was evaluated by flow cytometry. After stimulation with CD200 + FBL, the percentage of CD200R IFP + T cells producing IFNγ, IL-2, and TNFα was higher than that of control T cells. A lower percentage of CD200R IFP + T cells were cytokine-negative cells (27% vs 51%, Figure 15I , blue), while a higher percentage of CD200R IFP + T cells were multifunctional cells producing all 3 cytokines (22% vs 7%, Figure 15I , purple). Based on mean fluorescence intensity (MFI), CD200R IFP + T cells stimulated with CD200 + FBL had increased cytokine / cell( Figure 15J )

[0341] In vitro co-localization assay

[0342] To more closely examine the mechanism underlying the enhanced IFP expression function of T cells, the location of CD200R IFP on the T cell surface was observed by microscopy. After binding to CD80 / 86, native CD28 localizes to the immunological synapse, recruiting signaling molecules that amplify TCR signals (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). To evaluate the movement of IFP after stimulation, the cell membrane was stained with FITC-conjugated cholera toxin B subunit (CTxB) Figure 15K , Figure III), which is enriched at the immunological synapse (Stephan et al., Nat Med. 13:1440-1449, 2007), and used to define the site of immunological synapse components. Antibodies that bind to CD200 on FBL Figure 15K , Figure II) or CD200R on T cells (CD200R-9aas-CD28Cys, Figure 15K , Figure I) were then used to visualize these molecules associated with the immunological synapse. CD200R IFP-transduced, in vitro-expanded effector TCR gag cells were mixed with FBL at an E:T ratio of 15:1 in 15 mL, then incubated at 37 °C for 20 minutes, and then loaded into a μ-Slide VI.4 chamber (ibidi) for 15 minutes. The slides were washed with PBS and fixed with 2% paraformaldehyde for 4 minutes. The cells were then washed, stained, imaged at 60x using a Deltavision Elite fluorescence microscope, and analyzed using Image J (NIH).

[0343] CD200R colocalized with enhanced lipid raft staining at the T cell:target contact area Figures 15K to 15M , Figure IV), indicating that the size of IFP can be regulated by the immunological synapse.

[0344] LCK phosphorylation

[0345] The tyrosine kinase LCK is crucial for TCR signaling. The recruitment of LCK to the TCR signaling complex leads to the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) sequences in the CD3 complex, thereby initiating the TCR signaling cascade (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013). LCK associates with CD28 through a proline motif in the CD28 signaling tail, and T cell expression of CD28 is required for the sustained phosphorylation of the LCK residue Y394 (Holdorf et al., Nat Immunol. 3:259-264, 2002). To determine whether CD200R-CD28IFP expression provides or enhances CD28 signaling, pLCK Y394 ( Figure 15A ) was evaluated in T cells transduced with GFP control, lead construct (CD200R-9aas-CD28Cys), or null construct (CD200R-CD28Cys) that did not promote proliferation. The transduced T cells were stimulated with PMA / ionomycin, FBL, or CD200 + FBL for 10 minutes with or without stimulation, fixed, stained for intracellular pLCK Y394, and analyzed by flow cytometry. The phospho-LCK (Tyr394) antibody was purchased from R&D Systems, and intracellular staining was detected by secondary labeling with anti-mouse PE from BioLegend.

[0346] In response to strong stimulation (PMA / ionomycin) and CD200 - FBL stimulation, these three T cell populations achieved similar phosphorylation of LCK Y394 ( Figure 15N ). T cells transduced with GFP control or IFP with a larger extracellular domain CD200R-CD28Cys showed similar low levels of pLCK Y394 expression in response to CD200 - FBL and CD200 + FBL. However, when stimulated with CD200 + FBL, T cells transduced with CD200R-9aas-CD28Cys showed a sustained increase in the phosphorylation level of LCK Y394 at 10 minutes, indicating that the expression of CD200R-9aas-CD28cys provides an essential function for CD28 co-stimulation.

[0347] Summary

[0348] Collectively, these data indicate that the CD200R-CD28 construct serves to increase the accumulation and lytic activity of transduced T cells in response to tumor cell stimulation. Analysis of a panel of CD200R-CD28 IFP constructs demonstrated that costimulation was most effectively achieved in IFPs containing a dimerization motif and a tumor-T cell distance that facilitated localization to the immunological synapse.

[0349] T cells transduced with such CD200R-CD28 IFP exhibited enhanced proliferation and effector function in response to CD200 + target cells in vitro.

[0350] Example 16

[0351] CD200R-9AAS-CD28CYS-transduced T cells demonstrated enhanced in vivo accumulation in response to recognition of FBL

[0352] In adoptive T cell therapy of malignancies, tumors typically provide limited or no costimulatory signals but instead express ligands for inhibitory receptors. In leukemia, CD200 is a commonly expressed inhibitory ligand and is associated with poor prognosis (Tonks et al., Leukemia 21:566-568, 2007). Accordingly, the ability of TCR gag T cells expressing the most effective CD200R-9aas-CD28Cys IFP in vitro to proliferate and accumulate when encountering CD200 + FBL leukemia in vivo was evaluated.

[0353] Transduced TCR gag T cells were generated as described in Example 15. B6 mice were injected intraperitoneally (i.p.) with 4×10 6 viable CD200 + FBL leukemia. Five days after allowing FBL dissemination, 6 hours prior to transfer of effector T cells, the mice received 180 mg / kg cyclophosphamide (Cy) intraperitoneally to reduce the tumor burden and induce lymphopenia, similar to a human adoptive immunotherapy protocol. To evaluate short-term proliferation and accumulation, 2×10 6 IFP-transduced Thy1.1 + T cells were co-injected into tumor-bearing mice with an equal number of congenitally distinct GFP control-transduced Thy1.1 + ×Thy1.2 + T cells such that each mouse served as its own internal control ( Figure 16A ). Both T cell populations were generated in vitro and expanded with three stimulation cycles by the same method, and on the day of injection, were phenotypically similar 5 days after the third stimulation ( Figure 16B). IL-2 (2 × 10 4 U / dose) was administered every 2 days. On day 8 after T cell transfer, the mice were euthanized and spleens and inguinal lymph nodes were collected.

[0354] For some studies, CD8 TM Mouse CD8 + T cells were isolated using the EasySep + Mouse CD8 T Cell Enrichment Kit (STEMCELL). The tumor burden of the mice was monitored regularly, and if evidence of tumor progression indicated that death would occur within 24 - 48 hours, the mice were euthanized.

[0355] Compared to control cells, T cells expressing IFP were enriched 1.2 - 1.4 - fold in the spleen and lymph nodes ( Figure 16C ). To evaluate possible phenotypic differences acquired by the transferred T cells, a group of mice was euthanized at early (d3) and late (d15) time points to identify effector, memory, and exhaustion markers. T cells were isolated from the spleen by negative selection using unstimulated CD8 + T cell enrichment and evaluated by flow cytometry. Transduced CD200R - 9aas - CD28Cys + TCR gag and control T cells expressed similar surface molecules consistent with the effector T cell phenotype 3 days after transfer ( Figure 16D ). By day 15, persistent IFP + and control T cells were again phenotypically similar and did not express the exhaustion markers PD - 1 or Lag - 3 ( Figure 16E ), indicating that both cell types may still be functional during this period.

[0356] In summary, in an in - vivo study of adoptive therapy for disseminated leukemia, CD200R - CD28 - transduced leukemia - specific T cells eradicated other lethal diseases more effectively than wild - type cells and bypassed the requirement for IL - 2 administration to maintain in - vivo activity.

[0357] Example 17

[0358] CD200R - CD28 + adoptive immunotherapy of T cells showed greater activity in the treatment of disseminated leukemia

[0359] In a preclinical murine model of disseminated leukemia, it was evaluated whether costimulation provided to cells expressing CD200R-9aas-CD28Cys IFP led to enhanced activity of therapeutic T cells. This model requires a T cell response to persist >25 days to eradicate leukemia (Cheever et al., J Immunol. 125:711-714, 1980). As previously described, lethally irradiated (4×10 6 ) CD200 + FBL leukemia cells (Stromnes et al., J Clin Invest. 120:3722-3734, 2010) were injected intraperitoneally into B6 mice. Five days later, the mouse cohort received 180 mg / kg cyclophosphamide (Cy) intraperitoneally and 10 5 TCR gag effector T cells 6 hours later to allow drug metabolism (Cheever et al., 1980). TCR gag T cells were previously stimulated in vitro 1-3 times. The therapeutic efficacy of T cells transduced with CD200R-9aas-CD28Cys was compared to T cells expressing GFP as a control ( Figure 17A , 17B ). This method was initially tested in a small cohort of mice that received IL-2 for 10 days after T cell transfer to enhance and maintain T cell activity (Stromnes et al., J Clin Invest. 120:3722-3734, 2010) ( Figure 17A ).

[0360] With IL-2 injection, immunotherapy with control T cells cured 67% of the mice, while CD200R-9aas-CD28Cys + T cells cured 100% of the mice, without statistical significance ( Figure 17A ). Subsequent studies were performed with a larger cohort and IL-2 injection was omitted. In these studies, only 40% of the mice treated with T cells transduced with the GFP control vector survived beyond 30 days ( Figure 17B , blue line). In contrast, 89% of the mice receiving CD200R-9aas-CD28Cys + T cells survived 100 days after FBL transfer ( Figure 17B , red line, P<0.05). These results indicate that the IFP with CD200R provides a costimulatory signal that can not only enhance T cell immunotherapy for progressive leukemia but also greatly bypass the need to administer IL-2.

[0361] Example 18

[0362] Co-expression of CD200RTM-CD28 enhances the function of WT1-specific TCR primary T cells

[0363] Adoptive therapy with engineered T cells has shown encouraging clinical benefits, particularly in acute lymphoblastic leukemia, in which T cells express chimeric antigen receptors (CARs) specific for the cell surface protein CD19 (Turtle et al., J Clin Invest. 126:2123-2138, 2016; Kalos et al., Sci Transl Med. 3:95ra73, 2011). T cells can alternatively be transduced to express tumor-specific T cell receptors (TCRs), which greatly expand the breadth of target antigens by including intracellular proteins such as transcription factors that typically drive the oncogenic phenotype. WT1-specific CD8 + T cells exhibit anti-leukemic activity after transfer to patients (Chapuis et al., Sci Transl Med. 5:174ra127, 2013), and CD8 + T cells transduced with high-affinity WT1-specific TCRs are being tested in patients with leukemia, lung cancer, or mesothelioma (clinicaltrials.gov NCT01640301, NCT02408016), where WT1 is a transcription factor overexpressed in many malignancies (Yang et al., Leukemia 21:868-876, 2007; Qi et al., Sci Rep. 5:8924, 2015). T cell activation with associated proliferation and survival requires co-stimulatory signals concurrent with triggering of the antigen receptor (Chen et al., Nat Rev Immunol. 13:227-242, 2013). Unlike CARs that incorporate co-stimulatory domains in chimeric signaling proteins, cells with introduced TCRs require independent triggering of co-stimulatory receptors. However, tumor cells typically express few, if any, ligands for co-stimulatory receptors and often upregulate ligands for inhibitory receptors that can interfere with co-stimulation and block T cell activation (Driessens et al., Immunol Rev. 229:126-144, 2009). Thus, strategies to overcome inhibitory signaling and increase co-stimulatory / activation signaling are actively sought to promote T cell anti-tumor activity (Mellman et al., Nature 480:480-489, 2011).

[0364] The 5-year survival rate of acute myeloid leukemia (AML) under current therapies is 26% (Society AC, Cancer Facts & Figures 2016. Atlanta: American Cancer Society, 2016). Since T cells naturally home to the hematopoietic sites where AML is located, T cell therapies have great potential for treating this disease, but the overexpression of inhibitory molecules by AML cells represents a substantial obstacle to success (Geiger & Rubnitz, Discov Med. 19:275-284, 2015). The type I membrane protein CD200, a member of the immunoglobulin superfamily, binds to the T cell inhibitory receptor CD200R (Hatherley et al., Structure 21:820-832, 2013), and increased CD200 expression has been observed in AML and other malignancies, including multiple myeloma, ovarian cancer, and prostate cancer (Siva et al., Cancer Immunol Immunother. 57:987-996, 2008; Stumpfova et al., Cancer Res. 70:2962-2972, 2010; Kawasaki et al., Trends Immunol. 29:464-468, 2008). Importantly for targeted therapy, increased CD200 expression has been reported in cancer stem cells (CSCs) and leukemia stem cells (LSCs), a small subset of cells that initiate and maintain the disease with high proliferative capacity and resistance to radiation and chemotherapy (Snauwaert et al., Oncoimmunology 2:e22943, 2013; Tonks et al., Leukemia 21:566-568, 2007; Ho et al., 58th ASH Annual Meeting, San Diego, CA, 2016; Kawasaki et al., Biochem Biophys Res Commun. 364:778-782, 2007).CD200R signaling inhibits T cell function (Coles et al., Leukemia 26:2148-2151, 2012; Kretz-Rommel et al., The Journal of Immunology 178:5595-5605, 2007) and other immune cells, including natural killer (NK) cells (Coles et al., Leukemia 25:792-799, 2011), and high levels of CD200 expression are associated with poor prognosis in AML patients (Tonks et al., Leukemia 21:566-568, 2007).

[0365] Synthetic biology offers the opportunity not only to engineer T cells with tumor-reactive receptors, but also to eliminate negative signals and replace them with molecules that provide activating signals. To overcome inhibitory CD200R signaling associated with AML and simultaneously provide the missing co-stimulatory signals to CD8 + T cells, an immunomodulatory fusion protein (IFP) was designed that consists of the extracellular domain of CD200R fused to an intracellular T cell co-stimulatory signaling domain so that the IFP can engage leukemic cells expressing CD200 by binding this inhibitory ligand but generate co-stimulatory signals. Fusion proteins containing the extracellular domain of PD-1 have been shown to provide co-stimulatory signals (Prosser et al., Mol Immunol. 51:263-272, 2012), but the principles for designing molecules to generate or even optimize co-stimulatory signals have not been defined.

[0366] The therapy using TCR-transduced T cells was investigated in an AML clinical trial (registered in Clinicaltrials.org as NCT01640301). All clinical studies were conducted in accordance with the principles of the Declaration of Helsinki. Protocol 2498 has been approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board (IRB) and the US Food and Drug Administration (FDA). AML patients were treated with TCR-transduced T cells. Peripheral blasts were obtained from 4 patients who progressed / relapsed after T cell therapy. The AML-maintaining subset of LSCs is located within the CD45 dim CD34 + CD38- population (Bachas et al., Leukemia 26:1313-1320, 2012; Ho et al., 58th ASH Annual Meeting, San Diego, CA, 2016), and CD200 expression was compared with that of CD34 obtained from mobilized leukocytes of 3 healthy donors +cells (used to generate T cells for infusion) were compared. Although CD200 expression was not detected on normal CD34 + cells, CD200 was expressed in the majority of AML blasts from each subject patient (range 42 - 97% + )( Figure 18A ), consistent with previous reports (Tonks et al., Leukemia 21:566 - 568, 2007; Coles et al., Leukemia 29:1952 - 1954, 2015).

[0367] Based on previous murine experimental results (see Examples 14 - 17), a human CD200Rtm - CD28 construct (SEQ ID NO.: 1) was generated to maintain the spatial distance between human T cells and tumor cells at the immunological synapse ( Figure 18B ). Primary human T cells were transduced to determine whether the expression of this IFP could enhance function. The construct was inserted into a single lentiviral vector construct of the β and α chains of a WT1 126 -specific TCR C4 with HLA - A2 restriction by ligating each gene to a P2A element (Stromnes et al., Immunol Rev. 257:145 - 164, 2014) for transducing T cells used in clinical trials for the treatment of AML ( Figure 18B ). The first P2A sequence was codon - optimized to prevent genetic recombination with the second P2A sequence.

[0368] To generate lentivirus, 293T / 17 cells (3×10 6 cells / plate) were transduced with the human construct in the pRRLSIN plasmid and the packaging plasmids pMDLg / pRRE, pMD2 - G, and pRSV - REV using Effectene (Qiagen). The medium was changed on day 1 after transfection, and the virus - containing supernatant was collected on days 2 and 3 and aliquots were frozen for later use. After obtaining informed consent, peripheral blood mononuclear cells (PBMC) were harvested from normal HLA - A2 + donors. CD8 + T cells were purified using Miltenyi beads and stimulated with human T cell Expander CD3 / CD28 Dynabeads (Life Technologies) and 50 IU / ml IL - 2. Four hours after stimulation, 5 - 10×10 6It takes 90 minutes for a single cell to transduce T cells. As previously described, T cells are restimulated every 10 - 14 days with a rapid expansion protocol (REP) (see Hoe et al., J. Immunol. Methods 310:40 - 52, 2006).

[0369] Relative to T cells transduced with TCR alone C4 Human primary T cells transduced to express TCR C4 and the CD200R - CD28 fusion protein exhibit high levels of CD200R expression and equivalent levels of TCR C4 expression.

[0370] To determine whether the CD200R - CD28 IFP can improve the function of transduced human T cells, cells were stimulated with peptide - pulsed T2 lymphoblasts. Relative to primary AML( Figure 18A ) and the CD200 - CML cell line K562 (Coles et al., Leukemia 25:792 - 799, 2011), T2 lymphoblasts naturally express low levels of endogenous CD200( Figure 18C ). In response to WT1 126 pulsed T2 cells, T cells transduced with TCR C4 plus the CD200R - CD28 IFP exhibit enhanced proliferation( Figure 18D ) and increased cytokine production, especially at low E:T ratios( Figure 18E ), indicating that tumor cells express very little CD200 that can provide costimulation.

[0371] In summary, transduction of human primary T cells with human IFP also increases the proliferation and cytokine production in response to CD200 + leukemia cells. The focus of this study was to generate IFPs to target the inhibitory molecule CD200, which is typically upregulated in cancer cells (especially AML and LSC cells) and is known to inhibit T - cell immune responses. In addition to AML, increased CD200 expression has also been reported in other hematological malignancies and solid tumors such as breast cancer, colon cancer, ovarian cancer, and prostate cancer. In certain embodiments, the CD200 IFP can be used to treat hematological malignancies and solid tumors, including breast cancer, colon cancer, ovarian cancer, and prostate cancer. These results suggest that genetic engineering of tumor - specific T cells with an IFP containing the extracellular domain of CD200 can effectively convert the inhibitory signals delivered by leukemia cells into costimulatory signals in a cell - intrinsic manner, thereby obviating the need for global blockade of this inhibitory receptor and the associated risk of promoting endogenous autoreactive T - cell activation. In addition, the IFP can be used to increase sensitivity without manipulating the TCR.

[0372] Example 19

[0373] Co-expression of CD200R-TM-CD28 or CD200R-9AAS-CD28CYS with a WT1-specific TCR enhances the function of primary T cells

[0374] In a further example, use Figure 19A The schematic diagrams in illustrate exemplary fusion proteins described herein. Exemplary fusion proteins include IFPs that comprise the extracellular domain of human CD200R or a portion thereof and the intracellular signaling domain of human CD28 or a portion thereof ( Figure 19A , constructs II-VII). The hydrophobic component may include the transmembrane domain of CD200R ( Figure 19A , constructs I, II, and VIII) or the transmembrane domain of human CD28 ( Figure 19A , constructs III-VII) or a portion thereof. In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component further includes the extracellular portion of human CD28, particularly the extracellular cysteine residue adjacent to the hydrophobic component (e.g., Figure 19A construct IV, CD200R-CD28Cys; construct V, CD200R-9aas-CD28Cys; construct VI, CD200R-12aas-CD28Cys; and construct VII, CD200R-15aas-CD28Cys). Construct "VIII" contains the extracellular domain and the transmembrane domain but does not contain the intracellular signaling domain ( Figure 19A ). The extracellular component may comprise all or a portion of the extracellular domain of human CD200R. In some embodiments, the extracellular component comprises the entire extracellular domain of CD200R ( Figure 19A , constructs II-IV and VIII). In some other examples, the extracellular component comprises the first 234 amino acids from the N-terminus of CD200R (e.g., Figure 19A , construct V, CD200R-9aas-CD28Cys), the first 231 amino acids (e.g., Figure 19A , construct VI, CD200R-12aas-CD28Cys), or the first 228 amino acids (e.g., Figure 19A , construct VII, CD200R-15aas-CD28Cys). The human CD200R-CD28 constructs disclosed herein have the ability to convert inhibitory signals normally derived from CD200R binding to its target into positive signals generated by the CD28 intracellular signaling domain.

[0375] The size of the extracellular component that can be manipulated by modulating the fusion protein construct may affect the ability of the fusion protein to enter the immune synapse and co-localize with the TCR in the cSMAC to deliver a strong co-stimulatory signal. CD28 signaling naturally occurs in the immune synapse, where CD28 is recruited to amplify TCR signaling and lower the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13:227-242, 2013; Yokosuka et al., Immunity 29:589-601, 2008). In the immune synapse, the spatial distance between the T cell and the APC is shortest, and molecules with large extracellular domains are excluded. Thus, constructs that are closest to the intercellular space of the immune synapse may be able to co-localize with the TCR within the immune synapse and deliver an effective co-stimulatory signal. Constructs IV-VII extend the CD28 transmembrane domain into the extracellular space to incorporate juxtamembrane cysteines (CD28Cys), which promote CD28 homodimerization and enhance native CD28 signaling ((Lazar-Molnar et al., Cell Immunol. 244:125-129, 2006). In some embodiments, to illustrate the increased length contributed by additional amino acids in the extracellular CD28 domain, the CD200R extracellular domain portion is truncated an equal number of amino acids, e.g., the CD200R extracellular domain portion of CD200R-9aas-CD28Cys is truncated 9 amino acids, which is equivalent to the number added by the CD28 extracellular domain. Similarly, the extracellular CD200R of CD200R-12aas-CD28Cys is truncated 12 amino acids, and the extracellular CD200R of CD200R-15aas-CD28Cys is truncated 15 amino acids. In constructs V-VII, the truncated extracellular CD200R is truncated from the C-terminus to preserve the N-linked glycosylation site. To represent Figure 19A the fusion proteins shown, theoretically, CD200Rtm-CD28, CD200R-CD28tm, and CD200R-12aas-CD28cys optimally maintain the short spatial distance required between the T cell and the APC to co-localize with the TCR in the immune synapse.

[0376] All clinical studies were conducted in accordance with the principles of the Declaration of Helsinki. Protocol 2498 has been approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board (IRB) and the US Food and Drug Administration (FDA). The experiment was registered at clinicaltrials.org as NCT01640301.

[0377] To generate lentivirus, Effectene (Qiagen) was used with the pRRLSIN plasmid and the human constructs in the packaging plasmids pMDLg / pRRE, pMD2-G, and pRSV-REV ( Figure 19B ) transduced 293T / 17 cells (3×10 6 The medium was changed on day 1 after transfection, and the virus-containing supernatant was collected on days 2 and 3, and aliquots were frozen for future use.

[0378] A human CD200R-CD28 IFP construct was generated to theoretically maintain spatial distance between human T cells and tumor cells at the immune synapse ( Figure 19B ). The constructs were inserted into the WT1 with HLA-A2 restriction by ligating each gene with the P2A element. 126 -Specific TCR C4 The β and α chains of P2A were expressed in a single lentiviral vector construct (Stromnes et al., Immunol Rev. 257: 145-164, 2014), which was used to transduce T cells in a clinical trial for the treatment of AML. The first P2A sequence was codon optimized to prevent genetic recombination with the second P2A sequence.

[0379] After obtaining informed consent, + Peripheral blood mononuclear cells (PBMC) were harvested from donors. CD8 + T cells were stimulated with human T cell Expander CD3 / CD28 Dynabeads (Life Technologies) and 50 IU / ml IL-2. Four hours after stimulation, 5-10 × 10 cells were transfected with 2 mL of lentiviral supernatant by centrifugation at 1000 g at 32 °C. 6 T cells were transduced with 10 cells per 90 minutes. T cells were restimulated every 10-14 days using the rapid expansion protocol (REP) as previously described (Ho et al., J Immunol Methods 310:40-52, 2006).

[0380] Analysis of transduced CD8 + IFP expression in T cells ( Figure 19C ). The results showed that primary human T cells co-expressed CD200R-CD28 IFP and WT1-specific TCR when transduced with constructs encoding IFP and WT1-specific TCR.

[0381] Example 20

[0382] CD200 in use +After cell stimulation, T cells expressing CD200-targeted IFP are relative to IFP - T cell enrichment

[0383] To test with CD200 + Whether the expression of IFP targeting CD200 after cell stimulation will lead to IFP + T cells are relative to IFP - T cell enrichment. Before and after stimulation with CD200 + LCL cells, measure the relative proportion of CD200R + cells.

[0384] As Figure 20A shown, the expression of IFP CD200R-CD28tm ( Figure 19A Structure III in) leads to IFP after restimulation with LCL transduced with CD200 + T cells (CD200R + ) are relative to IFP - T cells (CD200R - ) are enriched.

[0385] T cells transduced with trCD200R ( Figure 19A Structure VIII in) and CD200R-15aas-CD28Cys ( Figure 19A Structure VII in) are not enriched, indicating the lack of costimulation ( Figure 20B and 20C ). However, T cells expressing several other constructs have an increased ratio after CD200 + LCL REP, especially after CD200R-CD28tm ( Figure 19A Construct III in) and CD200R-9aas-CD28Cys ( Figure 19A Construct V in) ( Figure 20C and 20D ).

[0386] Example 21

[0387] Human T cells expressing CD200R-CD28TM and CD200R-9AAS-CD28CYS IFP exhibit greater effector function

[0388] Relative to IFP - cells, human T cells expressing IFP CD200R-9aas-CD28Cys targeting CD200 have increased cytokine production. The TAP-deficient tumor cell line T2 expresses endogenous CD200 ( Figure 21A)。The expression of CD200R-9aas-CD28Cys enhanced cytokine production ([ 1-126 ) of peptide-pulsed T2 cells (pulsed with 1 μg / mL WT Figure 21B ) compared to unstimulated cells and stimulated cells expressing TCR but not IFP.

[0389] To test whether the enhanced effector function associated with CD200R-9aas-CD28Cys expression reflects enhanced adhesion and / or decoy binding rather than costimulation, a truncated non-signaling form construct with only the extracellular domain of CD200R and the transmembrane domain of CD28 was generated ("trCD200R"; Figure 19A , construct VIII; Figure 21C ). Transduced T cells expressing this construct did not show enhanced cytokine production ([ C4 ) compared to cells expressing only TCR Figure 21D ), indicating that CD200R-9aas-CD28Cys provides a costimulatory signal.

[0390] Figure 21E The ability of other CD200R-targeting constructs to increase cytokine production is shown in Figure 21F and 21G . CD200R-CD28tm ([ Figure 21F and 21G , labeled "B") and CD200R-9aas-CD28Cys ([ Figure 21F and 21G , labeled "-9") significantly increased cytokine production. IFP targeting CD200 showed increased multifunctional cytokine production ([ Figure 21I ).

[0391] Effector function was also evaluated in a flow cytometry-based cytotoxicity assay similar to that described in Example 15 ([ Figure 21J ). T cells transduced with CD200R-CD28 constructs lysed targets more efficiently than control T cells ([ Figure 21K , CD200R-CD28tm, labeled "B", CD200R-9aas-CD28Cys, labeled "-9").

[0392] Example 22

[0393] In vivo testing of FAS IFP

[0394] The Fas-CD28 construct was designed as in Example 11 and tested in vivo in a murine model of leukemia ([ Figure 22A ). C57BL / 6 mice were intraperitoneally inoculated with 4×10 6tumor cells (day 0), and treated with cyclophosphamide, and then provided with (1) no other treatment, (2) adoptive transfer of 10 6 GFP-converted TCR gag transgenic CD8 + T cells (day 5), or (3) adoptive transfer of 10 6 Fas-CD28-transduced TCR gag transgenic CD8 + T cells (day 5). Firefly luciferase + In vivo bioluminescence imaging of FBL tumors was used to measure leukemia in mice at various time points.

[0395] T cells transduced with Fas IFP tend to eradicate the disease more rapidly ( Figure 22B ) and provide protection over time ( Figure 22B , 22C ).

[0396] Example 23

[0397] FAS-4-1BB fusion protein construct

[0398] Exemplary fusion proteins as described herein also include IFP, which comprises the extracellular domain of Fas or a portion thereof and the intracellular signaling domain of 4-1BB. The extracellular component may comprise all or part of the extracellular domain of Fas. In some embodiments, the transmembrane component may include the domain of Fas, 4-1BB or CD28 or a portion thereof. In some exemplary Fas-4-1BB fusion proteins, the transmembrane component includes the transmembrane domain of CD28, and the extracellular component also includes the extracellular portion of CD28, particularly the extracellular cysteine residue adjacent to the transmembrane component (e.g., Fas-CD28Cys-4-1BBic and Fas-9aas-CD28Cys-4-1BBic). The extracellular component may comprise all or part of the extracellular domain of Fas, or may be truncated to maintain a short spatial distance (-9aas) between cells upon receptor-ligand interaction. In some other exemplary Fas-4-1BB fusion proteins, the transmembrane component includes the transmembrane domain of 4-1BB (e.g., Fas-4-1BBtm; Figure 23A ). Additionally, the Fas-4-1BB construct has the ability to convert the signal initiated by the binding of Fas to its target into a positive (e.g., co-stimulatory) signal generated by the 4-1BB intracellular signaling domain.

[0399] Fas-4-1BB IFP and the transgenic TCR can be co-expressed in transduced murine T cells. The IFP containing the extracellular component of Fas was generated using the general method described in Example 2. P14 T cells were transduced to co-express Fas-4-1BBtm IFP and the transgenic TCR (TCR gag , specific for an epitope derived from Friend murine leukemia virus-transformed FBL leukemia (Stromnes et al., J Clin Invest. 120:3722-3734, 2010). Retroviral supernatants were generated by transfecting Plat-E cells with DNA constructs encoding only TCR gag or TCR gag and Fas-4-1BBtm. Naive P14 T cells were stimulated with anti-CD3 and anti-CD28 and then transduced with the retroviral supernatants for 2 days. Five days after stimulation, the transduced T cells were stained with specific antibodies against the TCR and against Fas and analyzed by flow cytometry. P14 T cells transduced with the construct encoding TCR gag and Fas-4-1BBtm expressed similar levels of TCR and also expressed high levels of the Fas-4-1BBtm IFP construct ( Figure 23B ).

[0400] It was found that Fas-4-1BB + T cells exhibited enhanced proliferation in vitro. The transduced P14 T cells were stained with CellTrace Violet (CTV) proliferation dye and stimulated with FBL tumor cells at an effector-to-target ratio of 8:1 for 6 days. The T cells were then harvested and analyzed by flow cytometry. Without stimulation, T cells transduced with TCR gag alone showed only lack of proliferation, as did T cells transduced with both TCR gag and Fas-4-1BBtm (TCR + Fas-4-1BBtm) ( Figure 23C , left). When E:T was 8:1, some of the TCR-only T cells showed proliferation; however, all TCR+Fas-4-1BB + T cells showed robust proliferation, supporting increased stimulatory and proliferative capacity ( Figure 23C , right).

[0401] In addition, Fas-4-1BB + T cells showed reduced Fas pathway signaling of cell death, indicating that binding of the extracellular domain of Fas does not lead to activation of the Fas signaling pathway, as expected for the IFP in which the intracellular domain of Fas is replaced by the intracellular domain of 4-1BB. Figure 23DThe activation of the Fas signaling pathway for cell death is shown in: (i) T cells expressing the transgenic TCR gag but lacking Fas expression; (ii) wild-type T cells expressing the transgenic TCR gag ; and (iii) T cells expressing the transgenic TCR gag and Fas-4-1BBtm. P14 T cells were stimulated and transduced with TCR gag or TCR gag +Fas-4-1BB IFP. After 7 days, the active caspase-8 expression of T cells was stained with a fluorescent inhibitor of caspase (FLICA) to measure cell death through the Fas pathway. Fas-deficient T cells (gray) did not show active caspase-8 expression, while TCR-transduced T cells showed elevated expression. The caspase-8 expression activity of TCR+Fas-4-1BB T cells was lower than that of TCR-only T cells, indicating less cell death through the Fas pathway ( Figure 23D ).

[0402] Overall, these data indicate that the Fas-4-1BBtm fusion protein is able to convert the negative / cell death signal transduction associated with Fas binding into a positive co-stimulatory signal.

[0403] Example 24

[0404] The FAS-4-1BB fusion protein enhances control of tumor growth and improves survival in the ID8 ovarian cancer model.

[0405] Fas-4-1BBtm-transduced T cells control tumor growth and promote survival in the ID8 ovarian cancer model.

[0406] The ID8 model is a transplantable murine model of ovarian cancer (Walton et al., Cancer Res 76:6118-29, 2016). Quantification for the killing of ID8 ovarian tumor cells was determined. Murine transduced T cells (TCR or TCR+4-1BB) were co-incubated with red fluorescent ID8 ovarian tumor cells for 2 days, and the growth of ID8 cells was quantified by analysis. Loss of the red signal indicates killing of tumor cells. TCR+Fas-4-1BB T cells showed enhanced control of ID8 tumor cell growth compared to TCR-only T cells, as shown by less red signal ( Figure 24A ).

[0407] In addition, mice treated with T cells transduced with anti-mesothelin TCR + Fas-4-1BBtm had improved survival relative to mice treated with T cells transduced with anti-mesothelin TCR alone. In the ID8 murine ovarian cancer model, 5 × 10 6 ID8 tumor cells were implanted and allowed to disseminate for 6 weeks. After cyclophosphamide treatment, mice receiving 10 7 T cells and 5.0 × 10 8 mesothelin-pulsed splenocytes were then injected with IL-2 for a total of 10 days. Mice were treated every two weeks until they were euthanized according to the endpoint criteria approved by the IACUC. T cells transduced with TCR + Fas-4-1BBtm improved survival relative to TCR-only T cells ( Figure 24B ).

[0408] Example 25

[0409] T cells expressing the FAS-4-1BB fusion protein showed T cell persistence and improved survival in the KPC mouse model of pancreatic cancer

[0410] It has previously been shown that immunotherapy with mesothelin-targeted TCR-T cells can significantly prolong survival in the murine pancreatic KPC tumor model. In this study, the KPC model was used to determine whether immunotherapy using T cells expressing the Fas-4-1BB fusion protein could improve survival.

[0411] An autologous KPC pancreatic cancer model was used to mimic human disease (Lee et al., Curr. Protoc. Pharmacol. 73:14.39.1-14.39.20, 2016). In patients, >90% of pancreatic ductal adenoma (PDA) cases show activating mutations in KRAS, while >75% of patients have mutations in p53. The KPC model uses a pancreas-specific Cre recombinase ("C") to generate mutations in Kras ("K") and p53 ("P") in the pancreatic epithelium. The KPC model (i) recapitulates many key features of the immune microenvironment observed in human PDA, including a strong inflammatory response and effector T cell exclusion, (ii) is the most widely studied genetic model of PDA for evaluating immunotherapy, and (iii) recapitulates the clinical observations in PDA patients treated with several immuno-oncology drugs, including CD40 agonists and anti-PDL1 antibodies. The model has also been used to screen drugs as a predictor of patient treatment response.

[0412] KPC mice were screened by ultrasound to determine when tumors appeared and were enrolled in the study at approximately 8 weeks of age when tumors were detected. Figure 25AShows ultrasound images of the pancreas of healthy mice with normal pancreas and pancreatic tumors in "engrafted" mice (KPC genetically engineered mice). Mice were randomly assigned to treatment groups. Mice were treated with cyclophosphamide, and after cyclophosphamide, mesothelin-specific T cells (cells transduced with anti-mesothelin TCR or anti-mesothelin TCR+Fas-4-1BBtm) and mesothelin peptide-pulsed splenocytes were injected into the mice receiving TCR-T cells, 10 of each. 7 Starting 14 days after engraftment, T cell / APC infusions (without cyclophosphamide) were repeated every 2 weeks for a total of 3 infusions, and IL-2 was not injected. Mice that survived 28 days after the last T cell infusion were bled, and the persistence of transferred T cells was evaluated by detecting innate marker T cells using flow cytometry. At the end of the study, the survival of mice was evaluated according to the endpoint criteria approved by IACUC and the mice were euthanized. A summary of the experimental design used in this example is as Figure 25B shown.

[0413] Twenty-eight days after the third T cell infusion, Fas-4-1BB + T cells showed greater persistence in the blood ( Figure 25C ). All mice (100%) receiving Fas-4-1BB + T cells showed T cell persistence, while T cells with only TCR did not show persistence ( Figure 25C ). The survival rate of mice treated with mesothelin-specific TCR and Fas-4-1BB + T cells was significantly higher than that of adoptive immunotherapy with mesothelin-specific TCR T cells alone (Mantel-Cox test, P<0.05; Figure 25D ).

[0414] Example 26

[0415] Enhanced adoptive immunotherapy with FAS-4-1BB expression in a murine model of AML

[0416] As shown in Example 24 for solid tumors, treatment with Fas-4-1BB + T cells improved the survival of liquid tumors. In a murine AML model (Teague et al., Nature Medicine 12:335-341, 2006; Oda et al., Blood 130:2410-2419, 2017), FBL cells were injected and allowed to disseminate for 5 days. On the fifth day, mice were treated with cyclophosphamide with or without 10 6 T cells. Compared with T cells with only TCR, T cells transduced with TCR+Fas-4-1BBtm increased survival ( Figure 26 ).

[0417] Although specific embodiments of the invention have been shown and described, it will be readily understood that the various embodiments described above can be combined to provide other embodiments, and various changes can be made therein without departing from the spirit and scope of the invention.

[0418] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification or listed in the application data sheet, including but not limited to U.S. Provisional Patent Application Nos. 62 / 128,979, 62 / 473,282, and 62 / 629,663, and PCT International Application PCT / US2016 / 021064, are hereby incorporated by reference in their entirety. Aspects of the embodiments may be modified if concepts from various patents, applications, and publications are needed to provide other embodiments.

[0419] These and other changes may be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the entire scope of equivalents of these claimed rights. Thus, the claims are not limited by the disclosure. Sequence Listing <110> Fred Hutchinson Cancer Research Center K.S. Odar P.D. Greenberg T.M. Schmidt <120> Immunomodulatory Fusion Proteins and Their Uses <130> 360056.447WO <140> PCT <141> 2018-03-16 <150> US 62 / 629,663 <151> 2018-02-12 <150> US 62 / 473,282 <151> 2017-03-17 <160> 213 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 915 <212> DNA <213> Artificial Sequence <220> <223> huCD200Rtm-CD28 construct <400> 1 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctgctgcctg tgcctggcgc caagaagtcc 720 gccaagctgt acatccccta catcatcctg acaatcatca ttctgaccat cgtgggcttc 780 atctggctgc tgcgcagcaa gcggagcaga ggcggccaca gcgactacat gaacatgacc 840 cctagacggc ctggccccac cagaaagcac taccagccct acgcccctcc ccgggacttt 900 gccgcctaca gaagc 915 <210> 2 <211> 728 <212> DNA <213> Artificial sequence <220> <223> huCD200R whole extracellular domain <400> 2 tgctgtgccc ttggagaacc gccaacctgg gcctgctgct gatcctgacc atcttcctgg 60 tggccgccag cagcagcctg tgcatggacg agaagcagat cacccagaac tacagcaagg 120 tgctggccga agtgaacacc agctggcccg tgaagatggc caccaacgcc gtgctgtgct 180 gccctcctat cgccctgcgg aacctgatca tcatcacctg ggagatcatc ctgcggggcc 240 agcccagctg taccaaggcc taccggaaag agacaaacga gacaaaagaa acaaactgca 300 ccgacgagcg gatcacatgg gtgtccagac ccgaccagaa cagcgacctg cagatcagac 360 ccgtggccat cacccacgac ggctactacc ggtgcatcat ggtcaccccc gatggcaact 420 tccaccgggg ataccatctg caggtgctcg tgacccccga agtgaccctg ttccagaacc 480 ggaacagaac cgccgtgtgc aaggccgtgg ccggaaaacc tgccgcccag atctcttgga 540 tccccgaggg cgattgcgcc accaagcagg aatactggtc caacggcacc gtgaccgtga 600 agtccacctg tcactgggag gtgcacaacg tgtccaccgt gacatgccac gtgtcccacc 660 tgaccggcaa caagagcctg tacatcgagc tgctgcctgt gcctggcgcc aagaagtccg 720 ccaagctg 728 <210> 3 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> huCD200R transmembrane domain <400> 3 tacatcccct acatcatcct gacaatcatc attctgacca tcgtgggctt catctggctg 60 ctg 63 <210> 4 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> CD28 transmembrane domain <400> 4 ttctgggtgc tggtggtggt cggaggcgtg ctggcctgct acagcctgct ggtcaccgtg 60 gccttcatca tcttttgggt c 81 <210> 5 <211> 123 <212> DNA <213> Artificial sequence <220> <223> CD28 intracellular domain <400> 5 cgcagcaagc ggagcagagg cggccacagc gactacatga acatgacccc tagacggcct 60 ggccccacca gaaagcacta ccagccctac gcccctcccc gggactttgc cgcctacaga 120 agc 123 <210> 6 <211> 933 <212> DNA <213> Artificial sequence <220> <223> huCD200R-CD28tm construct <400> 6 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctgctgcctg tgcctggcgc caagaagtcc 720 gccaagctgt tctgggtgct ggtggtggtc ggaggcgtgc tggcctgcta cagcctgctg 780 gtcaccgtgg ccttcatcat cttttgggtc cgcagcaagc ggagcagagg cggccacagc 840 gactacatga acatgacccc tagacggcct ggccccacca gaaagcacta ccagccctac 900 gcccctcccc gggactttgc cgcctacaga agc 933 <210> 7 <211> 942 <212> DNA <213> Artificial Sequence <220> <223> huCD200R-9aas-CD28Cys construct <400> 7 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctgctgcctg tgtgtcccag ccctctgttt 720 cccggcccta gcaagccttt ctgggtgctg gtggtggtcg gaggcgtgct ggcctgctac 780 agcctgctgg tcaccgtggc cttcatcatc ttttgggtcc gcagcaagcg gagcagaggc 840 ggccacagcg actacatgaa catgacccct agacggcctg gccccaccag aaagcactac 900 cagccctacg cccctccccg ggactttgcc gcctacagaa gc 942 <210> 8 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> huCD200R - 9aas portion of extracellular domain <400> 8 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctgctgcctg tg 702 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> CD28 Cys Polymerization Domain <400> 9 tgtcccagcc ctctgtttcc cggccctagc aagcct 36 <210> 10 <211> 933 <212> DNA <213> Artificial Sequence <220> <223> huCD200R-12aas-CD28Cys Construct <400> 10 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctgtgtccca gccctctgtt tcccggccct 720 agcaagcctt tctgggtgct ggtggtggtc ggaggcgtgc tggcctgcta cagcctgctg 780 gtcaccgtgg ccttcatcat cttttgggtc cgcagcaagc ggagcagagg cggccacagc 840 gactacatga acatgacccc tagacggcct ggccccacca gaaagcacta ccagccctac 900 gcccctcccc gggactttgc cgcctacaga agc 933 <210> 11 <211> 693 <212> DNA <213> Artificial Sequence <220> <223> huCD200R-12aas portion of extracellular domain <400> 11 atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60 gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag 120 gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180 tgccctccta tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc 240 cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300 accgacgagc ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360 cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac 420 ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480 cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg 540 atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600 aagtccacct gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660 ctgaccggca acaagagcct gtacatcgag ctg 693 <210> 12 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> huCD200R-9aas-CD28Cys tm-41BBic construct <400> 12 atgctgtgcc cttggagaac cgccaacctg ggc...

Claims

1. A fusion protein, which consists of the following components: (a) An extracellular component, wherein the amino acid sequence of the extracellular component is encoded by the nucleic acid molecule shown in SEQ ID NO:71; (b) A hydrophobic component, wherein the amino acid sequence of the hydrophobic component is encoded by the nucleic acid molecule shown in SEQ ID NO:197; and (c) An intracellular component, wherein the amino acid sequence of the intracellular component is encoded by the nucleic acid molecule shown in SEQ ID NO:13, wherein the fusion protein binds to FasL and provides an intracellular 4-1BB signal.

2. The fusion protein according to claim 1, wherein the fusion protein is expressed in T cells and co-localizes with the TCR or CAR expressed by T cells.

3. A fusion protein, which consists of the following components: (a) An extracellular binding domain component having the amino acid sequence shown in SEQ ID NO:72; (b) A hydrophobic component having the amino acid sequence shown in SEQ ID NO:198; and (c) An intracellular component having the amino acid sequence shown in SEQ ID NO:36, wherein the fusion protein binds to FasL and provides an intracellular 4-1BB signal.

4. The fusion protein according to claim 3, wherein the fusion protein is expressed in T cells and co-localizes with the TCR or CAR expressed by T cells.

5. A fusion protein having the amino acid sequence shown in SEQ ID NO:188, wherein the fusion protein binds to FasL and provides an intracellular 4-1BB signal.

6. A nucleic acid molecule encoding the fusion protein according to any one of claims 1-5.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. The vector according to claim 7, wherein the vector is a viral vector.

9. The vector according to claim 8, wherein the viral vector is a lentiviral vector or a retroviral vector.

10. The vector according to claim 7, further encoding an antigen-specific TCR.

11. The vector according to claim 10, wherein the antigen-specific TCR is exogenous to the host cell.

12. The vector according to claim 10, wherein the antigen-specific TCR is specific for an HLA class I-restricted antigen.

13. The vector according to claim 10, wherein the antigen-specific TCR is specific for a cancer-specific antigen.

14. A host cell comprising the nucleic acid molecule according to claim 6.

15. A host cell comprising the vector encoding the vector according to claim 7.

16. A host cell comprising the fusion protein according to any one of claims 1-5.

17. The host cell according to claim 16, wherein the host cell is an immune system cell.

18. The host cell according to claim 17, wherein the immune system cell is a T cell.

19. The host cell according to claim 18, wherein the T cell is a CD4 + T cell or a CD8 + T cell.

20. The host cell according to claim 16, further comprising an antigen-specific TCR.

21. The host cell according to claim 20, wherein the antigen-specific TCR is exogenous to the host cell.

22. The host cell according to claim 20, wherein the antigen - specific TCR is specific for an HLA class I - restricted antigen.

23. The host cell according to claim 20, wherein the antigen - specific TCR is specific for a cancer - specific antigen.

24. The host cell according to claim 16, further comprising a CAR.

25. Use of the fusion protein according to any one of claims 1 - 5 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

26. The use according to claim 25, wherein the cancer is acute myeloid leukemia (AML).

27. The use according to claim 25, wherein the cancer is ovarian cancer.

28. The use according to claim 25, wherein the cancer is pancreatic cancer.

29. Use of the vector according to claim 7 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

30. Use of the host cell according to claim 14 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

31. Use of the host cell according to claim 15 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

32. Use of the host cell according to claim 16 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

33. Use of the host cell according to claim 17 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

34. Use of the host cell according to claim 18 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

35. Use of the host cell according to claim 19 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

36. Use of the host cell according to claim 20 in the preparation of a medicament for the treatment of ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

37. A human T cell expressing a fusion protein, the fusion protein consisting of the following components consisting of: (a) an extracellular component of the CD95 (Fas) extracellular domain that specifically binds CD95L (FasL), wherein the amino acid sequence of the extracellular component of the Fas extracellular domain is encoded by the nucleic acid molecule shown in SEQ ID NO:71; (b) a 4 - 1BB intracellular signaling domain component, wherein the amino acid sequence of the 4 - 1BB intracellular signaling domain component is encoded by the nucleic acid molecule shown in SEQ ID NO:13; and (c) The hydrophobic component of the 4-1BB transmembrane domain, wherein the amino acid sequence of the hydrophobic component of the 4-1BB transmembrane domain is encoded by the nucleic acid molecule shown in SEQ ID NO: 197, wherein the fusion protein binds to FasL and provides 4-1BB intracellular signaling.

38. The human T cell according to claim 37, wherein the human T cell is a CD4 + T cell or a CD8 + T cell.

39. The human T cell according to claim 38, further expressing an antigen-specific TCR.

40. The human T cell according to claim 39, wherein the antigen-specific TCR is exogenous to the human T cell.

41. The human T cell according to claim 39, wherein the antigen-specific TCR is specific for an HLA class I-restricted antigen.

42. The human T cell according to claim 39, wherein the antigen-specific TCR is specific for a cancer-specific antigen.

43. The human T cell according to claim 38, further expressing a CAR.

44. Use of the human T cell according to any one of claims 37-43 in the preparation of a medicament for treating ovarian cancer, pancreatic cancer or acute myeloid leukemia in which cancer cells express FasL.

45. The use according to claim 44, wherein the cancer is acute myeloid leukemia (AML).

46. The use according to claim 44, wherein the cancer is ovarian cancer.

47. The use according to claim 44, wherein the cancer is pancreatic cancer.

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