Immunomodulatory fusion proteins and uses thereof

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 produce a modulating signal in a host cell, such as a T cell. The 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 disease.

Description

[0001] This application is a divisional application with application number 201880030106.2, application date March 16, 2018, and invention name “Immunomodulatory fusion protein and its use”.

[0002] Statement concerning sequence listing

[0003] The sequence table associated with this application is provided in text format to replace a paper copy and is incorporated by reference into the specification of this article. The name of the text file containing the sequence table is 360056_447WO_SEQUENCE_LISTING.txt. The text file is 322KB, created on March 5, 2018, and submitted electronically through EFS-Web. Background Art

[0004] When tumor-reactive T cells were found in a population of tumor-infiltrating lymphocytes (TILs), T cell-based immunotherapy began to be developed (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 pre-selected for tumor reactivity, clonal expansion of tumor-reactive T cells induced by anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and finally the return of the expanded cell population to patients with tumors (together 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 has only resulted in complete remissions in a small fraction of patients with melanoma, and is rarely effective in other cancers (Besser et al., Clin. Cancer Res. 16: 2646, 2010).

[0005] The isolation of tumor-reactive T cell clones has led to the development of another immunotherapy approach - the production of recombinant T cell receptors (TCRs) specific for specific antigens, which can be introduced into T cells, for example using a vector delivery system, to confer specificity for the desired target, such as tumor-associated peptides presented by major histocompatibility complex (MHC) molecules expressed on tumor cells (called human leukocyte antigen (HLA) molecules in humans). Another approach introduces synthetic receptors, called chimeric antigen receptors (CARs), which typically contain an antigen binding domain that, for example, can bind to tumor-specific or associated antigens in the case of anti-tumor therapy, connected 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. Unlike the administration of TILs, the basic procedure for engineered TCR or CAR T cell immunotherapy is typically to genetically modify human T cells with a transgene encoding a tumor targeting moiety, amplify the recombinant T cells ex vivo, and return the amplified recombinant T cells to the patient.

[0006] Adoptive T cell therapy using T cells expressing recombinant TCR has been shown to have promising clinical benefits, especially in some B cell cancers. However, effective T cell activation usually 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 usually downregulated. As a result, for T cells expressing recombinant TCRs specific for cancer antigens, exogenous stimulation by IL-2 is usually required.

[0007] When TCR engages a specific peptide presented in MHC on an antigen presenting cell (APC), activation of T cells is initiated (Rossy et al., Frontiers in Immunol. 3: 1-12, 2012). The interaction point between T cells and APC becomes an immune synapse, which includes three concentric supramolecular activation clusters (SMAC), including central cSMAC, peripheral pSMAC and distal dSMAC (Rossy et al., Frontiers in Immunol. 3: 1-12, 2012). In cSMAC, costimulatory receptors can recruit signaling molecules to amplify TCR signals. Such costimulatory receptors may include CD28, and in some cases form microclusters with TCR to reduce the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). Transmembrane proteins expressed by T cells entering cSMAC may be limited by the size of the extracellular domain. For example, CD45 has a large extracellular domain and is generally excluded from the immunological synapse, preventing its ability to inhibit TCR signaling (James and Vale, Nature 487:64-69, 2012).

[0008] 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

[0009] In certain aspects, the disclosure relates to fusion proteins comprising an extracellular component containing a binding domain that specifically binds a target, an intracellular component composed of an intracellular signaling domain, and a hydrophobic component connecting 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 immune synapse.

[0010] 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 specific distance, for example, in some embodiments, a distance that is less than or less than about a certain distance. In some aspects, the distance of the fusion protein::target complex (or typically the extracellular portion thereof) 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 equal to or about 15 nm. In some embodiments, it is or is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nm, for example, is or is about 14 or 15 nm. In some aspects, the distance is a distance similar to the distance between membranes in an immune synapse, or is the same, approximately the same, or substantially the same distance as the distance between the nearest membrane portion (e.g., residues of the extracellular domain of the TCR) and the nearest membrane portion (residues of the MHC, e.g., residues of the HLA, such as residues of MHC I or MHC II) relative to the TCR-peptide / MHC complex, 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 a 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, wherein the first cell and the second cell each express a binding partner on their surface, and when the cells are close to each other, the binding partner can form a complex between the membranes. In some aspects, the distance is the same, approximately the same, or substantially the same distance spanned by the extracellular portion of the complex formed between the TCR and the homologous interaction with the MHC molecule. In some aspects, such as when the fusion protein comprises a binding domain from a molecule that is normally capable of entering the immune synapse or co-localizing with an antigen receptor, the distance is similar or identical to 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, such as when the fusion protein comprises a binding domain from a molecule that is normally not capable of entering the immune synapse or is normally not capable of co-localizing with an 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.

[0011] In some embodiments, the binding domain within the extracellular component of the fusion protein of the present disclosure includes a target binding portion of a molecule (e.g., an inhibitory molecule, such as an immunosuppressive molecule, such as an immunosuppressive receptor or an immune checkpoint molecule) that can deliver an inhibitory signal. In some aspects, this molecule is a glycoprotein, a member of the checkpoint family. In certain embodiments, a fusion protein from a glycoprotein, a member of the checkpoint family, or not B7 or a B7 binding molecule or not a CD28-B7-superfamily member (e.g., not CD28, CTLA4, ICOS, or other B7 family binding molecules) exemplary glycoprotein, a member of the checkpoint family includes 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 in the extracellular component of the fusion protein of the present disclosure includes 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 delivering a stimulation signal (e.g., a costimulatory signal) to a lymphocyte (e.g., a T cell, e.g., a costimulatory region of CD28, 4-1BB, ICOS or other costimulatory molecules). In some aspects, when the extracellular binding moiety is from a checkpoint or an immunosuppressive molecule, the intracellular portion of the fusion protein does not include an intracellular signaling domain of an inhibitory molecule such as a checkpoint or an immunosuppressive molecule. In some aspects, the fusion protein does not include a major signaling domain, such as a CD3 ζ signaling domain or other domains capable of delivering a major signal to a T cell.

[0012] In some aspects, the extracellular component or its binding portion contains or is a binding domain capable of specifically binding to a molecule or extracellular domain of CD200, such as a binding portion of CD200R or its variant. In some embodiments, the binding domain is or includes a binding region capable of specifically binding to a molecule or extracellular domain of CD47, such as a SIRP extracellular domain or its CD47 binding region, such as a SIRPα extracellular domain or its CD47 binding region. In some embodiments, the binding domain can bind to PD-L1 or PD-L2 or LAG3 molecules. Exemplary targets can be one or more proteins whose expression increases or is upregulated in certain cells or tissues, and the certain cells or tissues are associated with a disease or condition treated or improved with the fusion proteins and compositions provided herein, such as tumor cells or tumor microenvironments, or with receptors that are typically upregulated on immune cells (e.g., lymphocytes that infiltrate diseased tissues such as tumors).

[0013] In some embodiments, the extracellular component further includes one or more additional regions or domains, for example, from such a molecule: a molecule different from the derived binding domain or a molecule that shares identity with the binding domain. One or more additional extracellular domains may include a spacer, such as a spacer from an immunoglobulin molecule, which may include all or part of a hinge, or a constant region domain such as a CH2 or CH3 domain, or from another cell surface molecule such as a co-stimulatory receptor, such as CD28. In some aspects, additional extracellular domains may include a multimerization domain, such as a dimerization domain or sequence that may promote homodimerization or heterodimerization with another molecule, such as multimerization of two or more fusion proteins. In some embodiments, such domains include a portion of the extracellular domain of the CD28 molecule, which includes at least the most transmembrane end cysteine, and is 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 thereof, such as having at least 90%, 95% or 99% identity thereto. In some aspects, such a domain may be included to assist or promote multimerization. In some embodiments, the fusion protein contains an extracellular component including a CD200 binding domain, such as an extracellular portion of CD200R (or a portion thereof, such as a binding domain thereof), such as an extracellular portion of CD200R having an amino acid sequence as shown in SEQ ID NO: 25, or an extracellular portion of CD200R encoded by a nucleic acid molecule as shown in SEQ ID NO: 2, or a CD200 binding portion thereof or a variant thereof or a binding portion thereof. In some aspects of such embodiments, the extracellular portion of the fusion protein also includes a portion of the extracellular domain of CD28, such as up to about 9 to about 12 amino acids thereof (e.g., 9 amino acids or 12 amino acids), and in some aspects, includes the most membrane-proximal cysteine ​​residues of the extracellular region of CD28. In some such embodiments, the length of the CD200R portion of the extracellular region is reduced in length corresponding to the number of additional residues in the CD28-derived portion, e.g., by about 9 to about 12 amino acids (e.g., 9 amino acids or 12 amino acids), or 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 identical to the distance spanned by the extracellular portion of the complex between a human CD200R (e.g., CD200R) and CD200, or the distance spanned by the extracellular portion of the complex between a TCR in cognate interaction with an MHC molecule (e.g., MHC I or MHC II) in binding to a cognate peptide-MHC complex, or the distance of an immune synapse.In some aspects, the fusion protein further comprises a transmembrane domain, such as a CD28 transmembrane, such as a 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 promote intermolecular interactions. In some embodiments, the protein further comprises a CD28 intracellular signaling domain, such as a costimulatory domain of CD28, such as a domain capable of recruiting one or more adapter molecules to CD28 in response to ligation. In some aspects, the CD28 intracellular domain comprises or is a sequence encoded by the nucleotide sequence of SEQ ID NO: 5 or a portion thereof or a functional variant.

[0014] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0015] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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).

[0016] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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).

[0017] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0018] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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 to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0020] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0021] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0022] In some embodiments, the present disclosure relates to a fusion protein comprising an extracellular component comprising a binding domain that specifically binds to a target, an intracellular component comprising an intracellular signaling domain, and a hydrophobic component connecting 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 immune 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.

[0023] In some embodiments, the 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 connecting 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 a size of or spans a distance of (i) up to the distance between two cell membranes of an immune synapse, (ii) up to about or substantially the same distance as the distance spanned by the extracellular portion of the complex, wherein the complex is formed by specific binding of the fusion protein to the target (fusion protein::target complex) between a 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, said complex being between a natural 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 a CD95 (Fas) extracellular domain or a functional fragment thereof, and the intracellular component is or comprises a CD137 (4-1BB) intracellular signaling domain or a functional portion thereof.

[0024] In some embodiments, the present disclosure relates to a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds to a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component connecting the extracellular and intracellular components, wherein the binding domain is an inhibitory molecule binding domain or has at least 95% identity with an inhibitory molecule binding domain, and the intracellular signaling domain is a co-stimulatory or stimulatory molecule binding domain or comprises at least 95% identity with an inhibitory 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 from CD137 (4-1BB) or a functional portion thereof.

[0025] In some embodiments, the present disclosure relates to a fusion protein comprising: (a) an extracellular component comprising an amino acid sequence encoded by a nucleic acid molecule shown in SEQ ID NO.:71, (b) a hydrophobic component comprising an amino acid sequence encoded by a nucleic acid molecule shown in SEQ ID NO.:197, and (c) an intracellular component comprising an amino acid sequence encoded by a nucleic acid molecule shown in SEQ ID NO.:13.

[0026] 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.

[0027] In certain aspects, the disclosure relates to nucleic acid molecules encoding fusion proteins as described herein.

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

[0029] In certain other aspects, the present disclosure relates to a host cell comprising a nucleic acid molecule or vector encoding a fusion protein as described herein.

[0030] In certain other aspects, provided are methods of increasing immune cell activity, comprising administering to a subject in need of increased immune cell activity an effective amount of a host cell as described herein.

[0031] In other aspects, the disclosure relates to methods of enhancing or prolonging an immune response, comprising administering to a subject in need of enhanced or prolonged immune cell activity an effective amount of a host cell described herein.

[0032] In yet other aspects, the present disclosure provides methods of stimulating an antigen-specific T cell response, the method comprising administering an effective amount of a host cell described herein to a subject in need of enhanced immune cell activity.

[0033] In other aspects, the present disclosure relates to methods of inhibiting an immunosuppressive signaling pathway comprising administering to a subject in need thereof an effective amount of a host cell described herein.

[0034] In other aspects, the disclosure relates to methods of treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of a host cell described herein.

[0035] In other aspects, the present disclosure relates to methods of inhibiting immune resistance of cancer cells, comprising administering to a subject in need thereof an effective amount of a host cell described herein.

[0036] In other aspects, the present disclosure relates to methods of treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of a host cell described herein, wherein the administered host cell is capable of proliferating in an immunosuppressive tumor microenvironment.

[0037] 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 a host cell described herein.

[0038] These and other aspects of the invention will become apparent upon reference to the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A and 1B showed that in primary mouse CD8 +CD200R-CD28 constructs expressed at high levels on T cells. (A) Schematic diagram of exemplary CD200R-CD28 constructs. Construct "I" comprises CD200R extracellular ("EC") and transmembrane ("TM") domains and CD28 intracellular ("IC") signaling domain (CD200Rtm-CD28). Construct "II" comprises 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 near-transmembrane terminal cysteine ​​to promote multimerization and enhance CD28 signaling. To account for any additional extracellular amino acids (e.g., from one to about 50 amino acids; e.g., the exemplary murine construct disclosed herein comprises an additional nine (9) amino acids, and the exemplary human construct disclosed herein comprises twelve (12) amino acids), some constructs have truncated portions of the extracellular or intracellular domains (e.g., CD200R retaining N-linked glycosylation sites). 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 co-localize with the TCR in cSMAC and transmit a strong co-stimulatory signal. (B) Mouse CD200R-CD28 construct detected by anti-CD200R antibody in TCR gag Transgene expression on T cells. The control vector contained green fluorescent protein (GFP).

[0040] Figures 2A to 2G The CD200R-CD28 construct was shown to respond to CD200 in vitro + Tumor target cells promote proliferation, accumulation and effector function, and accumulate in immune synapses. Anti-CD3, anti-CD28 and recombinant human IL-2 (100 U / ml) were used to stimulate naive TCR in vitro. gag Splenocytes of 100 μg / mL mice were transduced with retroviral supernatant for 2 days. Cells were restimulated with irradiated FBL and splenocytes every 7 days and cultured with rhIL-2 (50 U / mL) for up to 3 stimulations. T cells were used for assays 5-7 days after the last stimulation. (A) CD200R-CD28 and GFP control TCR measured by CellTrace Purple dilution gag T cell proliferation. Using CD200 - FBL (above) or CD200 + T cells were stimulated with FBL (lower panel) for 3 days. (B) After weekly stimulation with irradiated CD200 +FBL and splenocyte stimulation cycles, with untransduced TCR gag During T cell co-culture, transduced TCR gag Preferential expansion / survival of T cells. (C) Enrichment of transduced T cells. Compared with wild-type T cells transduced with an empty GFP control vector, the number of T cells transduced with irradiated CD200 + Repeated restimulation of tumor cells enriched for cells transduced with CD200R-9aas-CD28Cys. (D) Increased CD200R and CD200 signal intensity at T cell:FBL synapses. Lipid rafts are increased at the immune synapse (I). CD200R-9aas-CD28Cys fusion protein colocalizes with lipid rafts, indicating that the fusion protein is concentrated within the immune synapse (III, IV). (E) CD200R-CD28 + CD8 + T cells showed higher in vitro lysis CD200 + FBL cell competence. Target tumor cells were labeled with different dilutions of the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as indicated. Effector TCRs transduced with the indicated CD200R-CD28 fusion proteins or empty vector control gag T cells and CD200 + FBL(CFSE hi ) and nonspecific EL4 (CFSE lo ) 1:1 mixture of control target was incubated for 5 hours at the indicated effector to target ratios. The percentage of FBL was determined by flow cytometry as a percentage of the sum of FBL and control tumor cells. The percentage of lysis 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 CFSE assay in (G). Target tumor cells were labeled with different dilutions of the fluorescent dye CellTrace Violet (CTV) or CFSE. EL4 cells (CTV + )、CD200 + FBL(CFSE hi ) and nonspecific EL4 (CFSE lo ) 1:1:1 mixture of control target. (G) CFSE cytotoxicity assay. TCR was transduced with CD200R-CD28 receptor or GFP control vector gag T cells. Effector TCR gag T cells and CD200 - FBL or CD200 +A 1:1 mixture of FBL and nonspecific EL4 control target was incubated for 4 h at the indicated effector to target ratios. The percentage of FBL was determined by flow cytometry as a percentage of the sum of FBL and control tumor cells. The percentage of lysis was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells.

[0041] 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 FBL-bearing mice. Transduced TCRs were generated as described in Example 2 gag T cells. (A) Experimental principle diagram. C57BL / 6 mice were injected with 4×10 6 CD200 + Five days later, CD200R-9aas-CD28Cys (Thy1.1 homozygous) and eGFP control (Thy1.1 heterozygous) TCR gag T cells were expressed at 4 × 10 per mouse 6 The concentration of 10 cells was co-injected into B6 mice bearing FBL treated with cyclophosphamide. IL-2 (2×10 4 U / dose). On day 8 after T cell transfer, mice were euthanized and spleens and inguinal lymph nodes were collected. (B) Response to FBL, CD200R-9aas-CD28Cys TCR gag T cells accumulate in the spleen. (LN = lymph node; Sp1 = 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. gag Compared with T cells, CD200R-9aas-CD28Cys TCR gag T cells expressed less CD62L, suggesting an effector T cell phenotype. (D) + Comparison of surface proteins of T cells, T cells transduced with empty vector, and endogenous T cells 15 days after transfer. gag Compared with T cells, CD200R-9aas-CD28Cys TCR gag T cells expressed similar levels of cell surface proteins.

[0042] Figures 4A to 4DThis study shows that adoptive immunotherapy with CD200R-CD28-transduced T cells can eradicate disseminated leukemia. (A) Schematic diagram of the experiment. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, 10 5 CD200R-CD28tm, CD200R-CD28Cys, CD200R-9aas-CD28Cys or eGFPTCR were injected intraperitoneally at a dose of 10 cells / mouse gag T cells were given to Cy-treated FBL-bearing mice. In one group of mice, IL-2 (2 × 10 4 U / dose). (B) Representative examples of expression of cell surface proteins in CD200R-CD28tm transduced T cells and non-transduced T cells on the day of IL-2 injection as determined by injection flow cytometry. (C) Survival of mice treated in the presence of IL-2 injection. (C) Survival of mice treated in the absence of IL-2 injection. CD200R-9aas-CD28Cys TCR in the absence of IL-2 injection gag Transfer of T cells significantly improved the survival rate (P < 0.05, log-rank Mantel-Cox test).

[0043] Figures 5A to 5C T cells expressing CD200R-9aas-CD28Cys did not induce detectable autoimmune liver damage or infiltrate normal tissues. (A) Schematic diagram of the experiment. Gag Mice were injected with 4 × 10 6 CD200 + FBL cells. Five days later, 10 5 CD200R-9aas-CD28Cys and eGFP TCR were injected intraperitoneally at a dose of 10 cells / mouse gag T cells were given to cyclophosphamide-treated FBL-bearing mice. In one group of mice, IL-2 (2 × 10 4 U / dose). Liver damage was assessed three and seven days after transfer by quantifying serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). (B) For mice that received no T cells, control T cells expressing GFP, or T cells expressing CD200R-9aas-CD28Cys, AST and ALT levels measured three and seven days after transfer did not change due to treatment. (C) T cell infiltration of normal tissues was assessed. Using an antibody specific for the T cell marker CD3, limited presence of T cells in liver tissue was observed (left panel), whereas CD200R-9aas-CD28Cys TCRgag or control TCR gag There were no significant differences between recipients of T cells (right panel).

[0044] Figures 6A to 6D Show that the 4-1BB co-stimulatory signaling domain promotes the accumulation and effector function of transduced T cells in vitro and promotes CD200 responses in tumor-bearing recipients of transduced T cells + Survival of tumor target cells. (A) Schematic representation of CD200R-CD28 ("V"), -4-1BB ("VI"), and -CD28-4-1BB ("VII") constructs. (B) After weekly exposure to irradiated CD200 + After stimulation of FBL and splenocytes, transduced TCR gag T cells relative to untransduced TCR gag Expansion of 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 lytic CD200 relative to controls + The ability of FBL cells to express FBL was determined by flow cytometry as a percentage of the total FBL and control tumor cells. The percentage of lysis was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells. (D) CD200R-41BB-transduced T cells also promoted survival relative to controls. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, 10 5 CD200R-9aas-CD28, CD200R-9aas-4-1BB, CD200R-9aas-CD28-4-1BB or eGFP TCR were injected intraperitoneally at a dose of 10 cells / mouse gag T cells were administered to cyclophosphamide-treated FBL-bearing mice.

[0045] Figures 7A to 7D 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 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 show low levels of endogenous CD200 expression. (C) Proliferation of T cells as shown by CFSE. Cells that proliferate in response to antigens show reduced CFSE fluorescence intensity. Compared with T cells transduced with C4 alone, T cells transduced with both C4 and IFP show enhanced proliferation of target cells expressing low levels of CD200. (D) Cytokine production in response to exposure to CD200dim tumor cells measured by flow cytometry. T cells transduced with both C4 and IFPCD200Rtm-CD28 show increased cytokine production relative to control T cells transduced with TCR C4 alone.

[0046] Figures 8A to 8E It is shown that a fusion protein comprising an extracellular component of SIRPα and a co-stimulatory signaling domain of CD28 promotes the accumulation and proliferation of transduced T cells in vitro. (A) Schematic diagram of an exemplary SIRPα-CD28 construct. 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 cysteine ​​near the transmembrane end to promote polymerization and enhance the signaling of CD28. In order to illustrate the additional extracellular amino acids (e.g., the additional nine (12) amino acids of the mouse construct, or the twelve (12) amino acids of the human construct), some constructs have truncated portions of the extracellular or intracellular domains (e.g., SIRPα retaining N-linked glycosylation sites). Construct IV has a truncated portion of SIRPα with 6 amino acids truncated to retain the N-linked glycosylation site. For example, construct V has a truncated portion of SIRPα with 9 amino acids truncated. For example, construct VI has a truncated portion of SIRPα with 23 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 co-localize with TCR in cSMAC and transmit a strong co-stimulatory signal. (B) In weekly exposure to irradiated SIRPα + After stimulation of FBL and splenocytes, transduced TCR gag T cells relative to untransduced TCR gagExpansion of T cells. SIRPα-CD28 constructs promoted accumulation of transduced T cells in vitro, with SIRPα-9aas-CD28Cys showing enhanced accumulation. (C) Proliferation of T cells transduced with SIRPα-CD28 constructs in the CellTrace Purple (CTV) dilution proliferation assay. T cells expressing SIRPα-CD28 constructs, which were engineered to maintain distance between T cells and tumor cells, showed enhanced proliferation relative to untransduced T cells. (D) SIRPα-CD28 transduced to express SIRPαtm-CD28 or SIRPα-9aas-CD28Cys constructs + After co-culture with T cells, CD47 + In contrast, tumor cells were not eradicated when cultured with T cells that received an empty vector or a truncated SIRPα lacking its intracellular domain. (E) Quantification of CD47 killing + Tumor cell killing Results of the assay. CD47 transduction with mCherry + FBL tumor cells. Loss of red signal indicates tumor cell killing. Tumor cell killing was tested at effector:target ratios of 10:1, 2:1, and 0.4:1. Even at the lowest effector:target ratio, SIRPα-CD28 + T cells also kill CD47 + Tumor cells.

[0047] Fig.9A and 9BFusion proteins comprising the extracellular component of PD-1 and the co-stimulatory signaling domain of CD28 are shown to promote cytokine production in vitro. (A) Schematic diagram 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 adjacent to the proximal transmembrane end cysteine ​​to promote multimerization and enhance CD28 signaling. To account for additional extracellular amino acids (e.g., an additional nine (12) amino acids for a mouse construct, or twelve (12) amino acids for a 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 co-localize with the TCR in cSMAC and transmit a strong co-stimulatory signal. (B) In response to stimulation of FBL cells endogenously expressing 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. The intracellular expression of the effector cytokines IFNγ and TNFα by stimulated T cells was assessed by flow cytometry.

[0048] Fig.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 showed high transduction efficiency and expression of both proteins.

[0049] Figures 11A to 11CFusion proteins comprising the extracellular components of Fas and the co-stimulatory signaling domain of CD28 are shown to accumulate in vitro following stimulation with irradiated FBL cells. (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 adjacent to the proximal transmembrane cysteine ​​to promote multimerization and enhance CD28 signaling. To account for additional extracellular amino acids (e.g., an additional nine (12) amino acids for the mouse construct, or twelve (12) amino acids for the human construct), construct IV has a truncated portion of Fas in which the Fas extracellular domain is truncated by 9 amino acids. Constructs "I", "II" and "V" maintain a short spatial distance between cells (e.g., between T cells and antigen presenting cells) and can co-localize with the TCR in cSMAC and deliver a strong co-stimulatory signal. (B) TCR transduced with Fas constructs gag T cell accumulation upon multiple stimulation of irradiated FBL cells. All constructs promoted T cell accumulation relative to control T cells. (C) Constructs expressing Fas-CD28 instead of full-length (FL) Fas promoted T cell survival or expansion after multiple stimulation in vitro.

[0050] Fig. 12A and 12BThe structure and expression of fusion proteins comprising the LAG3 extracellular component and the CD28 co-stimulatory signaling domain are shown. (A) Schematic diagram of exemplary LAG3-CD28 constructs. Construct "I" comprises the LAG3 extracellular ("EC") and transmembrane ("TM") domains and the CD28 intracellular ("IC") signaling domain (LAG3tm-CD28). Construct "II" comprises 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., an additional nine (12) amino acids for a mouse construct, or twelve (12) amino acids for a human construct), construct IV has a truncated portion of LAG3, wherein the LAG3 extracellular domain is truncated by 9 amino acids. Constructs "I", "II" and "V" maintain a short spatial distance between cells (e.g., between T cells and antigen presenting cells) and can co-localize with the TCR in cSMAC and deliver a strong co-stimulatory signal. (B) As determined by anti-LAG3 antibody staining and flow cytometry, mouse CD8 + T cells express LAG3-CD28 constructs. In contrast to control T cells receiving an empty vector, T cells transduced to express LAG3-CD28 constructs (LAG3tm-CD28, LAG3-CD28tm, LAG3-CD28Cys, LAG3-9aas-CD28Cys) showed expression of the construct.

[0051] Fig.13A and 13BThe structure and expression of fusion proteins comprising TIM3 extracellular components and CD28 costimulatory signaling domains are shown. (A) Schematic diagram of an exemplary TIM3-CD28 construct. Construct "I" comprises TIM3 extracellular ("EC") and transmembrane ("TM") domains and CD28 intracellular ("IC") signaling domains (TIM3tm-CD28). Construct "II" comprises the transmembrane and intracellular domains of the extracellular domain of TIM3 and CD28 (TIM3-CD28tm). Constructs "III" and "IV" also incorporate a portion of the extracellular domain of CD28 (the portion being adjacent to the transmembrane proximal cysteine) to promote multimerization and enhance CD28 signaling. To illustrate additional extracellular amino acids (e.g., additional nine (12) amino acids for a mouse construct, or twelve (12) amino acids for a human construct), construct IV has a truncated portion of TIM3, wherein the TIM3 extracellular domain is truncated by 9 amino acids. Constructs "I", "II" and "V" maintain a short spatial distance between cells (e.g., between T cells and antigen presenting cells) and can co-localize with the TCR in cSMAC and deliver a strong co-stimulatory signal. (B) Mouse CD8 + T cells express TIM3-CD28 constructs. In contrast to control T cells receiving an empty vector, T cells transduced to express TIM3-CD28 constructs (TIM3tm-CD28; TIM3-CD28tm; TIM3-CD28Cys; TIM3-9aas-CD28Cys) typically show expression of the construct.

[0052] Fig.14A and 14B showed that in primary mouse CD8 +CD200R-CD28 constructs expressed at high levels on T cells. (A) Schematic diagram of representative CD200R-CD28 constructs. Construct "I" comprises CD200R extracellular ("EC") and transmembrane ("TM") domains and CD28 intracellular ("IC") signaling domain (CD200Rtm-CD28). Construct "II" comprises 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 near-transmembrane terminal cysteine ​​to promote multimerization and enhance CD28 signaling. To account for any additional extracellular amino acids resulting from incorporating a portion of the extracellular domain of CD28 (e.g., from one to about 50 amino acids; e.g., the exemplary mouse constructs disclosed herein comprise additional three (3) or nine (9) amino acids, and the exemplary human constructs disclosed herein comprise additional nine (9) or twelve (12) amino acids), some constructs have truncated portions of the extracellular or intracellular domains (e.g., CD200R retaining an N-linked glycosylation site). For example, construct IV has a truncated portion of CD200R that is truncated by 3 amino acids. For example, construct V has a truncated portion of CD200R that is truncated by 9 amino acids. Constructs "I", "II" and "V" maintain spatial distance between cells (e.g., between T cells and antigen presenting cells) and, as indicated by the dotted lines, can co-localize with the TCR in the cSMAC and deliver a strong co-stimulatory signal. (B) Mouse CD200R-CD28 constructs detected by anti-CD200R antibodies in the TCR gag Transgene expression on T cells.

[0053] Figures 15A to 15N The CD200R-CD28 construct promoted the expression of CD200 in vitro + Proliferation, accumulation, and effector function of T cells stimulated by tumor target cells and accumulated in immune synapses. Unless otherwise indicated, all results are representative of at least 2 experiments with similar results. In vitro stimulation of naive TCRs with anti-CD3, anti-CD28, and recombinant human IL-2 (rhIL-2, 100 U / ml) gag Splenocytes of mice were transduced with retroviral supernatant for 2 days. Cells were restimulated with irradiated FBL and splenocytes every 7 days and cultured with rhIL-2 (50 U / mL) for up to 3 stimulations. T cells were used for assays 5-7 days after the last stimulation. (A) CD200R-CD28 and GFP control TCR measured by CellTrace Purple (CTV) dilution gag T cell proliferation, relative to unstimulated cells (shaded). -FBL (above) or CD200 + FBL (bottom) stimulated T cells for 3 days. (B) Schematic diagram of truncated CD200R (trCD200R). (C) CD28 signaling domain is required for co-stimulation. trCD200R construct detected by anti-CD200R antibody in TCR gag Transgene expression on T cells. (D) trCD200R (blue line) and GFP control (red line) TCR measured by CellTrace Purple dilution gag T cell proliferation. Using CD200 + FBL stimulated T cells for 3 days. (E) + FBL restimulation enriched CD200R IFP-transduced T cells. - (left picture) or CD200 + (Right) Transduced TCRs during one cycle of FBL and splenocyte restimulation gag T cells in the presence of non-transduced TCR gag Enrichment of mixed populations of T cells. TCR transduced with CD200R-CD28 (upper panel) or GFP control (lower panel) gag T cells. (F, G) After weekly exposure to irradiated CD200 + During the stimulation cycle of FBL and splenocytes, transduction of TCR gag T cells in the presence of non-transduced TCR gag Enrichment in mixed populations of T cells. *P<0.05, **P<0.01 (t test). (H) CD200R-9aas-CD28Cys + CD8 + T cells showed higher in vitro lysis CD200 + FBL cell competence. Target tumor cells were labeled with the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). TCR was transduced with CD200R-9aas-CD28Cys or mock-transduced cells (black symbols) gag T cells. Effector TCR gag T cells and CD200 + A 1:1 mixture of FBL and nonspecific EL4 control target was incubated for 4 hours at the indicated effector to target ratios. The percentage of FBL was determined by flow cytometry as a percentage of the total FBL and control tumor cells. The percentage of lysis was determined by dividing the percentage of FBL incubated with T cells by the percentage of FBL incubated without T cells. (I) Pie chart depicts the percentage of TCRs in response to FBL stimulation at a 1:1 ratio. 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 cells transduced with the GFP control. (KM) CD200R-9aas-CD28Cys fusion protein colocalizes 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 immune synapse. gag The in vitro expanded effector T cells were combined with FBL at an E:T ratio of 10:1 at 37°C for 20 minutes. The conjugate was loaded onto a μ-Slide VI.4 chamber (Ibidi) and left for another 15 minutes. The fixed cells were stained and observed by microscopy. Figure 15K In the figure, the upper panel shows the contacted cells, while the lower panel shows the uncontacted cells. (N) TCR transduced with CD200R-9aas-CD28Cys (red line), CD200R-CD28Cys (blue line) and GFP control (black line) gag LCKY394 expression of T cells stimulated for 10 min as marked.

[0054] Figures 16A to 16E It was shown that T cells transduced with CD200R-9aas-CD28Cys preferentially accumulated in response to tumor attack in vivo and enhanced adoptive immunotherapy of disseminated leukemia. Transduced TCRs were generated as described in Example 15 gag T cells. C57BL / 6 mice were injected with 4×10 6 CD200 + Five days later, CD200R-9aas-CD28Cys (Thy1.1 homozygous) and eGFP (Thy1.1 heterozygous) TCR gag T cells were expressed at 4 × 10 per mouse 6 10 cells were co-injected into Cy-treated B6 mice with FBL. IL-2 (2×10 4 U / dose). On day 8 after T cell transfer, mice were euthanized and spleens and inguinal lymph nodes were collected. (A) Relative to empty vector control TCR gag T cells, responding to FBL, CD200R-9aas-CD28CysTCR gag T cells accumulate in the spleen. (B) The phenotype of IFP-transduced T cells is similar to that of controls. TCR transduced (red line) or not transduced (blue line) with CD200R-CD28 5 days after in vitro stimulation gagExpression of surface markers on T cells. (C) Responses to FBL, CD200R-9aas-CD28Cy and empty vector control TCR gag T cells accumulated in lymph nodes (LN) and spleen (Spl). + Thy1.2 + )TCR gag The percentage of T cells was calculated using CD200R-9aas-CD28Cys TCR gag Fold increase of T cells (Thy1.1 homozygous). (D, E) CD200R-9aas-CD28Cys TCR on day 8 (D) and day 15 (E) gag T cells (blue line), control TCR gag Surface marker expression on T cells (red line) and endogenous T cells (shadow). 15 days after transfer, compared with control TCR gag Compared with T cells, CD200R-9aas-CD28CysTCR gag T cells expressed similar levels of cell surface proteins.

[0055] Fig.17A and 17B Survival rates of mice treated in the presence (A) or absence (B) of IL-2 injection are shown. C57BL / 6 mice were injected with 4×10 6 CD200 + FBL cells. Five days later, 10 5 CD200R-9aas-CD28Cys and eGFP TCR were injected intraperitoneally at a dose of 10 cells / mouse gag T cells were given to Cy-treated mice with FBL (indicated by arrows). IL-2 was given every 2 days for a total of 10 days in one group of mice (2×10 4 U / dose). In the absence of IL-2 injection, CD200R-9aas-CD28Cys TCR gag Transfer of T cells significantly increased survival (P<0.05, log-rank Mantel-Cox test) (B). In (A), data are from experiment 1 (n=3-4 mice / group). In (B), data are from 3 independent experiments in total (n=6-10 mice / group).

[0056] Figures 18A to 18EFigure 3 shows that human primary T cells transduced to express WT1-specific TCR and CD200Rtm-CD28 fusion protein displayed enhanced proliferation against target cells expressing CD200 and increased cytokine production in response to tumor cells expressing CD200. (A) CD34 from healthy donor leukapheresis (upper panel) or leukemic blasts (lower panel) + CD200 expression on cells. (B) WT1 126 Specific TCR, TCR C4 and CD200Rtm-CD28 expression in primary human T cells. The figure shows constructs combining 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. 126 T2 cells stimulated by TCR C4 , or use TCR C4 and CD200Rtm-CD28 transduced T cells. (E) Cytokine production in response to exposure to T2 cells as measured by flow cytometry. 126 4. Titering of Pulsed T2 Cells Stimulated with TCR Alone C4 Transduced T cells (above) or with TCR C4 T cells transduced with both TCR and IFP targeting CD200 (lower panel). C4 Control T cells transduced with TCR C4 T cells transduced with both PD-1 and IFPCD200Rtm-CD28 showed increased cytokine production.

[0057] Figures 19A to 19C CD200R-CD28 constructs co-expressed with WT1-specific TCR in primary human T cells are shown. (A) Schematic diagram of representative CD200R-CD28 constructs. (B) Diagram showing constructs binding IFP, TCRα and TCRβ chains. (C) WT1 126 Specific TCR, TCR C4 and expression of CD200R-CD28 fusion protein in primary human T cells.

[0058] Figures 20A to 20D Shown are the results of an assay for enrichment of T cells expressing the CD200R-CD28 construct.

[0059] Figures 21A to 21K Effector function assays (cytokine production, cytotoxicity) of T cells expressing the CD200R-CD28 construct are shown.

[0060] Figures 22A to 22DResults of in vivo studies using T cells expressing the Fas IFP construct for immunotherapy are shown. (A) Study design. (B-C) 4 × 10 6 Firefly luciferase was performed in C57BL / 6 mice at the indicated time points after 100 tumor cells were grown (day 0) and after cyclophosphamide treatment. + In vivo bioluminescence imaging of FBL tumors without further manipulation or adoptive transfer 10 6 GFP-transduced or Fas-CD28-transduced TCR gag Transgenic CD8 + T cells (day 5). Fig. 22C The two mice shown are representative of n=4 mice. (D) Biodistribution of FBL tumor cells quantified by IVIS imaging. 4×10 6 FBL tumors in C57BL / 6 mice at the indicated time points after 10 tumor cells were grown (day 0) and after cyclophosphamide treatment, followed by no other treatment (white circles) or adoptive transfer for 10 6 GFP-transduced (black circles) or Fas-CD28-transduced (red circles) TCR gag Transgenic CD8 + T cells (day 5).

[0061] Figures 23A to 23D It is shown that a fusion protein comprising a Fas extracellular component and a 4-1BB co-stimulatory signaling domain accumulates and proliferates in vitro after stimulation with tumor cells, and also reduces Fas-induced cell death. (A) Schematic diagram of an exemplary Fas-4-1BB construct. The construct comprises a Fas extracellular ("EC") domain and a 4-1BB transmembrane ("TM") and intracellular ("IC") signaling domain ("Fas-4-1BBtm"). (B) Co-expression of transgenic TCR and Fas-4-1BBIFP (Fas-4-1BBtm) in mouse T cells. By using a transgenic TCR encoding only gag , or encoding TCR gag Plat-E cells were transfected with DNA constructs containing Fas-4-1BBtm (SEQ ID NO.: 187) and Fas-4-1BBtm (SEQ ID NO.: 187) to produce retroviral supernatant. Naive P14 T cells were stimulated with anti-CD3 and anti-CD28 and then transduced with retroviral supernatant for 2 days. Five days after stimulation, transduced T cells were stained with specific antibodies against TCR and Fas and analyzed by flow cytometry. (C) As measured by CellTrace purple (CTV) dilution, TCR alone was expressed in 96 cells, while Fas was expressed in 104 cells, while Fas was expressed in 104 cells, while Fas was expressed in 104 cells, while Fas was expressed in 104 cells. gag , or use TCR gagProliferation of T cells transduced with Fas-4-1BBtm. Transduced P14 T cells were stained with CellTrace violet (CTV) proliferation dye and were either not stimulated (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) Activity of the cell death Fas signaling pathway in: (i) expressing transgenic TCR gag but lacks Fas expression in T cells; (ii) expressing transgenic TCR gag wild-type T cells; and (iii) expressing a transgenic TCR gag and Fas-4-1BBtm T cells. gag or TCR gag P14 T cells were stimulated and transduced with +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.

[0062] Figures 24A to 24B Figure 3 Fas-4-1BB T cells control tumor growth and promote survival in the ID8 model of ovarian cancer. (A) Quantitative killing of ID8 ovarian tumor cells by Fas-4-1BB T cells. The results of the assay were as follows. Mouse 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 Analysis of quantified ID8 cell growth. Loss of red signal indicates tumor cell killing. (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 grown and spread for 6 weeks. After cyclophosphamide treatment, mice received 10 7 T cells and 5.0×10 8 Mice were treated every two weeks until euthanasia.

[0063] Figures 25A to 25DFas-4-1BB T cells show greater persistence and promote survival in the KPC mouse model of pancreatic cancer. (A) Ultrasound images of healthy mice with normal pancreas (left) and pancreatic tumors in "enrolled" mice (KPC genetically engineered mice) (right). (B) Schematic diagram of the experiment. 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 found. Mice were randomly assigned to treatment groups, treated with cyclophosphamide, and mice receiving TCR-T cells were injected with mesothelin-specific T cells and mesothelin peptide-pulsed spleen cells after cyclophosphamide, 10 each. 7 (C) Mice surviving 28 days after the last T cell infusion were bled and the persistence of the transferred T cells was assessed by flow cytometry of T cells with innate markers. (D) Survival of KPC mice treated with (i) anti-mesothelin TCR cells or (ii) anti-mesothelin TCR + Fas-4-1BBtm cells.

[0064] Fig.26 Shown with (i) TCR gag T cells or (ii) using TCR gag +Fas-4-1BBtm T cell-treated AML model (FBL injection) mice were injected with FBL cells. Five days later, the mice were treated with or without 10 6 In the case of T cells, mice were treated with cyclophosphamide. DETAILED DESCRIPTION

[0065] 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 costimulatory signals in human T cells, wherein the T cells can be optionally transformed into TCRs with preferred antigen-specificity. These immunomodulatory fusion proteins (IFPs) can interact with commonly expressed targets or with targets that are usually upregulated or overexpressed in abnormal cells (e.g., cancer cells). Such IFPs have an extracellular binding domain and an intracellular signaling domain. By transducing T cells with engineered TCRs (e.g., high-affinity TCRs) and fusion proteins of the present disclosure that generate activation signals, certain embodiments of T cells may no longer require exogenous costimulation when interacting with, for example, tumor cells.

[0066] In certain aspects, the present disclosure provides host cells (e.g., immune cells such as T cells, dendritic cells, NK cells, etc.) comprising IFP, vectors encoding IFP, and methods of activating T cells comprising IFP for various therapeutic applications, including treating a disease (e.g., cancer, infectious disease) in a subject.

[0067] Before describing the present invention in more detail, it may be helpful to understand the present invention to provide definitions of certain terms used herein. Other definitions are set forth throughout the present invention.

[0068] In this specification, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the range, and when appropriate, include its fraction (e.g., one tenth and one hundredth of an integer), unless otherwise specified. In addition, unless otherwise specified, any numerical range associated with any physical feature described herein, such as polymer subunits, size or thickness, should be understood to include any integer in the range. Unless otherwise specified, the term "about" used herein refers to ±20% of the range, value or structure shown. It should be understood that the terms "one" and "one" used herein refer to "one or more" in the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two or any combination thereof in the alternatives. As used herein, the terms "including", "having" and "comprising" are used synonymously, and their terms and variants are intended to be interpreted as non-restrictive.

[0069] 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 substantially affect the basic features of the claimed invention. For example, a protein domain, region, module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, modules) consists essentially of a specific amino acid sequence (e.g., amino or carboxyl terminus or amino acids between domains) when the amino acid sequence of the domain, region, module or protein includes extensions, deletions, mutations or a combination thereof, which, in combination, contribute up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) to the length of the domain, region, module or protein, and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as not more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) the activity of the domain, region, module or protein (e.g., the target binding affinity of the binding protein).

[0070] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, molecule or activity that is not native to a host cell or subject, or is native to a host or host cell but has been altered or mutated so that the structure, activity, or both are different between the native and mutated molecules. In certain embodiments, heterologous, non-endogenous or exogenous molecules (e.g., receptors, ligands) may not be endogenous to a host cell or subject, but nucleic acids encoding such molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, etc., wherein the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homologous" refers to a molecule or activity found in a host cell, species or strain, or derived from a host cell, species or strain. For example, a heterologous or exogenous molecule or gene encoding the molecule may be homologous to a natural host or host cell molecule or gene encoding the molecule, respectively, but may have an altered structure, sequence, expression level, or a combination thereof. Non-endogenous molecules may be from the same species, a different species, or a combination thereof.

[0071] As used herein, the term "endogenous" or "native" refers to a gene, protein, compound, molecule or activity that is normally present in a host or host cell and has not been altered by an engineered process.

[0072] As used herein, a "binding domain" (also referred to as a "binding region" or "binding portion") refers to a molecule, such as a peptide, oligopeptide, polypeptide or protein, that has the ability to specifically and non-covalently associate, associate or combine with a target molecule (e.g., CD200, CD47, CD19, CD20, CD22, ROR1, mesothelin, PD-L1, PD-L2, PSMA, WT-1, cyclin-A1). The binding domain includes any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner of a biological molecule 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 T cell receptor (TCR), or a functional binding domain or an antigen binding fragment thereof. Exemplary binding domains include receptor extracellular domains (e.g., those of CD200R, PD-1, CTLA4, BTLA, CD2, Fas) or binding portions thereof, ligands (e.g., cytokines such as IL35, chemokines) or binding portions thereof, single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab) or binding portions thereof, 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 biological molecules.

[0073] As used herein, "specific binding" refers to the association or association of a binding domain or fusion protein thereof with a target molecule with an affinity or Ka (i.e., the equilibrium association constant for a specific binding interaction, in units of 1 / M) that is equal to or greater than 10 5 M-1, or binds to such a target molecule without significantly associating or associating 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 a domain with 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 "Low affinity" binding domains are those with a Ka of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 Alternatively, affinity can be defined in units of M (e.g., 10 -5 M to 10 -13 M). In certain embodiments, a binding domain may have "enhanced affinity", which refers to a selected or engineered binding domain that binds to a target antigen more strongly than a wild-type (or parent) binding domain. For example, the enhanced affinity may be due to a higher Ka (equilibrium association constant) for the target antigen than for the wild-type binding domain, or due to a Kd (dissociation constant) for the target antigen that is less than the Kd (dissociation constant) for the wild-type binding domain, or due to a lower dissociation rate (Koff) for the target antigen than for the wild-type binding domain. A variety of assays are known for identifying binding domains of the invention that specifically bind to 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. NY Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173, 5,468,614, or their equivalents).

[0074] As used herein, "fusion protein" refers to a polypeptide having at least two different domains in a single chain, wherein the domains are not naturally present together in the protein. PCR, recombinant engineering, etc. can be used to construct nucleic acid molecules encoding fusion proteins, or protein synthesis methods can be used to prepare such fusion proteins. The fusion protein may further include other components (e.g., covalently bound), such as tags or bioactive molecules. In certain embodiments, the fusion protein expressed or produced by a host cell (e.g., a T cell) is located on the cell surface, wherein the fusion protein is anchored to the cell membrane, and a portion of the fusion protein is located outside the cell (e.g., containing a binding domain) and a portion of the fusion protein is located inside the cell (e.g., containing a signaling domain).

[0075] As used herein, "hydrophobic component" refers to any amino acid sequence having a thermodynamically stable three-dimensional structure in a cell membrane, and is generally within the length range of about 15 amino acids to about 30 amino acids. The structure of the hydrophobic component may include an alpha helix, a beta barrel, a beta fold, a beta helix, or any combination thereof. In certain embodiments, the hydrophobic component includes a "transmembrane domain" from a known transmembrane protein, which is a part of a transmembrane protein that can be inserted into or across a cell membrane. In further embodiments, the hydrophobic component or transmembrane domain may be placed between and connected to the extracellular and intracellular portions of the fusion protein. In addition, the hydrophobic component may be modified to include a charged region or hydrophilic residues to promote intermolecular interactions.

[0076] As used herein, an "intracellular signaling domain" is an intracellular portion of a molecule, such as a molecule used in a fusion protein of the present invention, that can directly or indirectly promote a response, such as a co-stimulatory, positive, or biological or physiological response activated in a cell, when receiving an appropriate signal. In certain embodiments, the intracellular signaling domain is part of a protein or protein complex that receives a signal when bound, or it can itself bind directly to a target molecule to transmit a signal to other components in the cell. When the intracellular signaling domain comprises one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM), a kinase domain, a co-stimulatory domain, 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 a cellular response. In some embodiments, the intracellular signaling domain or its functional fragment may 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 its functional fragment does not comprise CD3ζ.

[0077] As used herein, "multimerization domain" refers to a polypeptide molecule or region that preferentially interacts or associates with another polypeptide molecule or region, directly or indirectly, wherein the interaction of the multimerization domain substantially contributes to or effectively promotes multimerization (i.e., forming 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 bridge bonds), ionic bonds, metallic bonds, electrostatic interactions, salt bridges, dipole-dipole forces, hydrogen bonds, van der Waals forces, hydrophobic interactions, or any combination thereof. The polymer is stable under appropriate conditions (e.g., physiological conditions, in an aqueous solution suitable for expressing, purifying or storing recombinant or engineered proteins, or under non-denaturing or non-reducing electrophoresis conditions). Exemplary multimerization domains may comprise one or more disulfide bonds, zinc finger motifs, leucine zipper motifs, helix-turn-helix, helix-loop-helix, and the like.

[0078] In certain embodiments, the fusion protein may include 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, so that the resulting polypeptide structure maintains specific binding affinity for the target molecule or maintains signal transduction activity (e.g., effector domain activity) or both. Exemplary linkers include Gly x Ser y 1 to about ten repeats of the compound, wherein x and y are independently an integer from 1 to 5.

[0079] "Linking amino acids" or "linking amino acid residues" refer 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 component, or at one or both ends of a hydrophobic component. The linking amino acids can be generated by the construct design of the fusion protein (e.g., amino acid residues generated by using restriction enzyme sites during the construction of a nucleic acid molecule encoding a fusion protein). In certain embodiments, the linking amino acids form a linker, such as a Gly having 1 to about ten repeats. x Ser y Those wherein x and y are independently integers from 1 to 5.

[0080] As used herein, "immune system cell" refers to any cell of the immune system that originates from hematopoietic stem cells in the bone marrow, which give rise to two major lineages, namely myeloid progenitor cells (which give rise to myeloid cells, such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (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, gamma delta T cells, regulatory T cells, natural killer cells and dendritic cells. Macrophages and dendritic cells can be called "antigen presenting cells" or "APCs", which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptors on the surface of the APCs complexed with peptides interact with the TCR on the surface of the T cells.

[0081] A "T cell" is an immune system cell that matures in the thymus and produces the T cell receptor (TCR). T cells can be naive (not exposed to antigen; unlike T CM Compared with normal T cells, the expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA increased, while the expression of CD45RO decreased), memory T cells (T M T cells (cells that have experienced antigen and survived long term) and effector cells (cells that have experienced antigen, cytotoxic). MThey can be further divided into central memory T cells (increased expression of TCD62L, CCR7, CD28, CD127, CD45RO, and CD95 and decreased expression of CD54RA compared with naive T cells) and effector memory T cells (T EM , with naive T cells or T CM Compared with WT T cells, the expression of CD62L, CCR7, CD28, and CD45RA is reduced, while the expression of CD127 is increased). E ) refers to antigen-experienced CD8 + Cytotoxic T lymphocytes, T CM Compared to WT T cells, they have reduced expression of CD62L, CCR7, and CD28 and are positive for granzymes and perforins. 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.

[0082] "T cell receptor" (TCR) refers to a molecule found on the surface of a T cell (or T lymphocyte), which associates with CD3 and is generally responsible for identifying antigens bound to major histocompatibility complex (MHC) molecules. TCR has a disulfide-bonded 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, TCR consists of a heterodimer of variable γ chains and δ chains (also referred to as TCRγ and TCRδ, respectively). Each chain of 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, TCR can be from various animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs can be cell-bound (ie, have a transmembrane region or domain) or in soluble form.

[0083] "Major histocompatibility complex molecule" (MHC molecule), which is used interchangeably and understood to also refer to the human corresponding human leukocyte antigen (HLA molecule), refers to a glycoprotein that delivers peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a transmembrane α chain (with three α domains) and a non-covalently associated β2 microglobulin. MHC class II molecules are composed 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 originating from the cytosol to the cell surface, where the peptide:MHC complex (or peptide:human HLA) is composed of CD8 + T cell recognition. MHC (HLA) class II molecules deliver peptides originating from the vesicle system to the cell surface, where they are recognized by CD4 + T cells recognize MHC molecules that can be derived from a variety of animal species, including humans, mice, rats, or other mammals.

[0084] "Nucleic acid molecules" or "polynucleotides" can be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules can be double-stranded or single-stranded, and if single-stranded, can be coding strands or non-coding strands (antisense strands). Coding molecules can have coding sequences identical to those known in the art, or can have different coding sequences that, due to redundancy or degeneracy of the genetic code or by splicing, can encode the same polypeptide.

[0085] Variants of nucleic acid molecules or polynucleotides of the present disclosure are also contemplated. Variant polynucleotides have at least 80%, preferably at least 85%, 90%, 95%, 99% or 99.9% identity with one of the polynucleotides of the defined sequence described herein, or hybridize with one of those polynucleotides in the defined sequence under stringent hybridization conditions (0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at about 42°C). Polynucleotide variants retain the ability to encode a binding domain or fusion protein thereof having the functions described herein.

[0086] The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents 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, NY, 1989).

[0087] More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide or other denaturing agents) may 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).

[0088] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector may be, for example, a plasmid, a cosmid, a virus, or a bacteriophage. An "expression vector" is a vector that, when present in the appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector.

[0089] A "retrovirus" is a virus with an RNA genome. "Gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0090] "Lentivirus" refers to a genus of retroviruses that can infect 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).

[0091] In the context of two or more polypeptide or nucleic acid molecule sequences, the terms "identical" or "percent identity" means that when compared and aligned for maximum correspondence over a comparison window or over a 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 the same or have a specified percentage of amino acid residues or nucleotides that are the same over a 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.

[0092] "Treatment" or "treatment" or "improvement" 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). Typically, an appropriate dose or treatment regimen comprising a host cell expressing a fusion protein of the present disclosure and an optional adjuvant or auxiliary therapy is administered in sufficient amounts to induce a therapeutic or preventive benefit. Therapeutic or preventive / preventive benefits include improved clinical outcome, alleviation or alleviation of symptoms associated with the disease, reduced incidence of symptoms, improved quality of life, longer disease-free state, reduced extent of disease, stable disease state, delayed disease progression, remission, survival, prolonged survival, or any combination thereof.

[0093] A "therapeutically effective amount" or "effective amount" of a cell or fusion protein expressing a fusion protein of the present disclosure (e.g., CD200R-CD28, SIRPα-CD28, CD200R-41BB, SIRPα-41BB, CD200R-CD28-41BB, SIRPα-CD28-4-1BB or other such fusion protein) in the context of the disease or condition being treated refers to the amount of the fusion protein or 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.).

[0094] Immunomodulatory fusion protein (IFP)

[0095] In certain aspects, the present disclosure provides fusion proteins, which include extracellular components, hydrophobic components and intracellular components. In certain embodiments, the extracellular component includes a binding domain, such as a binding domain that specifically binds to a target. In certain embodiments, the binding domain is from a molecule that can deliver a negative or inhibitory signal when it is bound to its binding partner or ligand or receptor (such as an immunosuppressive receptor or a checkpoint molecule) in its natural environment, or the target is an inhibitory receptor or ligand or a checkpoint molecule or other inhibitory ligand. In certain embodiments, the intracellular component includes a signaling domain, such as a costimulatory signaling domain or a signaling domain of a molecule that can usually deliver a costimulatory signal or a positive signal to an immune cell. Therefore, in certain aspects, the fusion protein can deliver a positive or costimulatory signal in response to a binding event that can cause an inhibitory signal in a natural environment.

[0096] In some embodiments, the fusion protein allows a specific distance to be reached. For example, in some embodiments, the fusion protein::target complex (e.g., a complex comprising 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, for example, a distance between membranes in an immune synapse, or a 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 a distance spanned by the extracellular portion of a complex formed between a natural molecule and its natural binding partner. In some embodiments, when expressed in an immune cell such as a T cell or entering an immune synapse, the distance or length is sufficient to promote colocalization of the fusion protein with an antigen receptor or other signaling molecule.

[0097] As background, an immune synapse is an interface between cells that can be formed 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 a T cell contacting an antigen presenting cell (APC), an immune synapse can be formed by binding a 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 an APC; HLA class I molecules can be found on the surface of all nucleated cells, while HLA class II molecules can be conditionally expressed on all cell types, but are typically found on APCs). In addition, immune synapses can be organized into supramolecular activation clusters (SMACs), which can affect lymphocyte activation, direct presentation of antigen-HLA (or antigen-MHC) complexes to lymphocytes, and direct secretion of cytokines or lytic granules between cells. The SMAC can include three structures arranged in concentric circles: a central region (cSMAC) containing a large number of TCRs and 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 immune synapse will span about 10nm to about 15nm. For example, protein interactions found in immune synapses, such as TCR::HLA peptide interactions or fusion protein-target interactions, typically span about 14nm between membranes. In certain embodiments, the width of the SMAC in the immune synapse does not exceed 15nm.

[0098] In some embodiments, the extracellular span of the fusion protein::target complex allows it to be localized to a specific compartment of the immune synapse. Some complexes thought to be located in various compartments of the immune synapse are well characterized in terms of the length of their extracellular span. For example, the MHC-TCR complex is thought to have an extracellular span of about 10-15 nm, while more integrin-based complexes are thought to have an extracellular span 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 believed to localize to the cSMAC include TCR and MHC complexes, CD2, CD4, CD8, CD28, and their ligands (Dustin et al., CSH Performances in Biology 2:a002311, 2010); therefore, these molecules complexed with their natural ligands are expected to be of an appropriate size to localize to the cSMAC.

[0099] In some respects, the length or distance or approximate length or distance of the extracellular part (such as the extracellular part of fusion protein, or the complex (such as with its binding partner compound)) of a specific construct or its engineered modification can be determined or modeled by known methods. In some exemplary models, the tertiary structure, binding domain and other features of the protein can be estimated using the amino acid or nucleic acid sequence of input. The tertiary structure of the protein can be used to estimate the size of the extracellular part, flexibility and other features that can be used to determine the approximate length of the extracellular part of the protein or its complex. Generally, the method for the length of the extracellular part of the protein known for modeling or estimating. For example, molbiol-tools.ca and Swiss-Model include multiple tools that can be used to predict protein structure (see also Schwede, T., Structure 21: 1531, 2013).

[0100] In certain embodiments, the fusion proteins of the present disclosure that are complexed, associated or interacted with a target are capable of being located within an immune synapse. In some embodiments, the extracellular portion of the fusion protein::target complex spans the immune synapse. In other embodiments, the fusion protein::target complex is located in a supramolecular activation cluster (SMAC), such as a cSMAC. In further embodiments, the extracellular portion of the fusion protein::target complex spans an immune 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 about 12 nm to about 15 nm, or about 14 nm.

[0101] The distance between the cell membranes of cells interacting in an immune 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).

[0102] In certain embodiments, the fusion proteins disclosed herein include an extracellular portion extending less than 40 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein include an extracellular portion extending less than 30 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein include an extracellular portion extending less than 20 nm from the cell membrane. In some embodiments, the fusion proteins disclosed herein include an extracellular portion extending less than 15 nm from the cell membrane.

[0103] In some embodiments, compared with the distance between the cell membranes that allow entry into the synapse or co-localize with antigen receptors or simulate the length or distance present in natural proteins, the fusion protein provided provides the advantage of having extracellular length or spatial distance. In certain embodiments, when the extracellular portion of the fusion protein includes a domain from another molecule, and the other molecule is from different molecules that obtain a binding domain, the length of the extracellular component comprising the binding domain is reduced, such as truncated compared to the extracellular region of a natural molecule, to provide a similar length or distance. In certain embodiments, the fusion protein described herein includes an extracellular component, and the extracellular component includes an extracellular domain and a second domain (e.g., a joint or an extracellular domain of a second cell surface receptor) of a cell surface receptor. In some such embodiments, in order to maintain an extracellular component that can stay in an immune synapse or span an immune synapse when compounded with a target molecule, one or more domains of the extracellular component can be truncated.

[0104] In some diseases (such as cancer), due to the imbalance between costimulatory signals and inhibitory signals, the amplitude and quality of the T cell response caused by the T cell receptor (TCR) recognition antigen may be disordered (e.g., reduced), which can lead to immune resistance. One advantage of some fusion proteins disclosed herein is that a first signal can be converted into a second signal of different nature. For example, in some embodiments, the fusion protein allows a negative or inhibitory signal to be effectively converted into a positive or costimulatory signal, thereby alleviating or minimizing immune resistance associated with a disease such as cancer. For example, in combination with a target, if combined by its natural binding partner, it will lead to inhibition or delivery of a negative signal, and in some embodiments, the fusion protein provided herein can deliver positive instead, for example, a costimulatory signal, to cells expressing it, such as T cells. In certain embodiments, the fusion protein of the present invention comprises 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 a T cell, cytotoxic T lymphocyte-associated antigen 4 (CTLA4 or CD152), can receive an inhibitory signal when combined with one of the ligands CD80 or CD86 found on an APC. 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 inhibitory signals when bound to one of its ligands PD-L1 (B7-H1, CD274) or PD-L2 (B7-DC, CD73) found on APC. PD-1 limits 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 that contain 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 protein, CTLA4-CD137 fusion protein, CTLA4-CD28-CD137 fusion protein, PD1-CD28 fusion protein, PD1-CD137 fusion protein, or PD1-CD28-CD137 fusion protein.

[0105] The fusion proteins disclosed herein 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 common 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 in 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.

[0106] Another advantage of some fusion proteins disclosed herein 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 costimulatory domains may not produce sufficient costimulatory signals. Co-expression of multiple immunomodulatory fusion proteins, especially those that can remain in the immune synapse, can provide the necessary costimulatory signaling for T cells to avoid anergy and proliferation.

[0107] 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 immune synapse.

[0108] In another aspect, the fusion proteins of the present disclosure allow enrichment of transduced T cells without sorting by restimulation with tumor cells that express a ligand that binds to the fusion protein.

[0109] In an exemplary embodiment, a fusion protein is provided, which 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).

[0110] In another exemplary embodiment, a fusion protein is provided, which comprises (a) an extracellular portion of SIRPα, (b) a transmembrane domain of CD28, and (c) an intracellular signaling domain of CD28. In some embodiments, the fusion protein further comprises an extracellular portion of CD28 extending from the CD28 transmembrane domain. In a further embodiment, 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 an 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 CD137 (4-1BB), and (c) the intracellular signaling domain of CD137 (4-1BB).

[0112] In another exemplary embodiment, a fusion protein is provided, which comprises (a) an extracellular portion of CD95 (Fas), (b) a transmembrane domain of CD28, and (c) an intracellular signaling domain of CD137 (4-1BB). In some embodiments, the fusion protein further comprises an extracellular portion of CD28 extending from the CD28 transmembrane domain.

[0113] In another exemplary embodiment, the present disclosure provides a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds to a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component connecting 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 a size of or spans a distance of (i) up to the distance between two cell membranes of an immune synapse, (ii) up to about or substantially the same distance as the distance spanned by the extracellular portion of the complex, wherein the complex is formed by specific binding of the fusion protein to the target (fusion protein::target complex) and wherein the extracellular portion of ... A method of treating a pluripotent stem cell-derived protein (SPC) comprising a binding domain of a T cell or a plurality of other proteins, wherein the SPC comprises ...

[0114] In another exemplary embodiment, the present disclosure provides a fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds to a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component connecting 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 from CD137 (4-1BB) or a functional portion thereof.

[0115] In another exemplary embodiment, the present disclosure provides a fusion protein comprising: (a) an extracellular component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.:71, (b) a hydrophobic component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.:197, and (c) an intracellular component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.:13.

[0116] 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.

[0117] The components of the fusion proteins of the present disclosure are described in further detail herein.

[0118] Extracellular components

[0119] As described herein, the fusion protein of the present invention generally comprises an extracellular component, which comprises a binding domain that specifically binds to a target. The binding of the fusion protein binding domain to the target can (1) block the interaction between the target and another molecule (e.g., block or interfere with receptor-ligand interaction), (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 when binding to the target (e.g., converting an inhibitory or negative signal to a stimulatory or positive signal), such as in a cell expressing the fusion protein, or any combination thereof. In some embodiments, the fusion protein described herein comprises an extracellular portion, wherein the extracellular portion comprises an extracellular portion of a protein associated with a negative signal.

[0120] Exemplary binding domains disclosed herein can be the extracellular domain of a cell surface receptor or its binding portion, the extracellular domain of a cell surface ligand, a cytokine (e.g., IL35), a chemokine, an antibody-based binding domain, a TCR-based binding domain, an unconventional binding domain, or any combination thereof. For example, the binding domain of the extracellular domain including CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CTLA4 (CD152), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5 is within the scope of the present disclosure. As used herein, the "extracellular domain" from a cell surface receptor or ligand includes a complete 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, which has a higher affinity for the target than a wild-type or reference protein. In certain embodiments, the extracellular domain comprises a variable-like domain or a CDR of a variable-like domain.

[0121] In some embodiments, the fusion protein comprises an extracellular component containing a CD200 binding domain, such as a CD200R extracellular domain or a CD200 binding portion thereof. As background, CD200R is a receptor that binds to the type 1 membrane protein CD200 of the immunoglobulin superfamily (Tonks et al., Leukemia 21:566-568, 2007). It is reported that CD200 is upregulated in various malignancies, including leukemia, multiple myeloma, and various solid tumors (e.g., melanoma, breast cancer, and squamous cell carcinoma). In fact, high levels of CD200 expression are associated with a poor prognosis in acute myeloid leukemia (AML), and CD200R signals have been shown to have an inhibitory effect on T cells (Coles et al., Leukemia 26:2148-2151, 2012). In certain embodiments, the CD200R extracellular domain 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.

[0122] In further embodiments, CD200R is encoded by the nucleic acid molecule set forth in SEQ ID NO.: 2. In certain other embodiments, the CD200R extracellular domain comprises at least 200 amino acids from the N-terminus of CD200R. In some other embodiments, CD200R is encoded by the nucleic acid molecule set forth in SEQ ID NO.: 11. In still other 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 set forth in SEQ ID NO.: 8. In any of the foregoing embodiments, the CD200R, CD200R extracellular domain, or any CD200R fragment thereof used in the fusion protein of the present disclosure is human CD200R. In a further embodiment, a CD200R extracellular domain is provided, which 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 an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID No.: 2.

[0123] In some embodiments, CD200R comprises an amino acid sequence as shown in SEQ ID NO.: 25. In some embodiments, CD200R comprises an amino acid sequence as shown in SEQ ID NO.: 34. In certain embodiments, CD200R comprises an amino acid sequence as shown in SEQ ID NO.: 31. In any of the foregoing embodiments, the CD200R, CD200R extracellular domain, or any CD200R fragment thereof used in the fusion protein of the present disclosure is human CD200R. In further embodiments, a CD200R extracellular domain is provided, which 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 an amino acid sequence as shown in SEQ ID NO: 25.

[0124] In some embodiments, the fusion protein comprises an extracellular component containing a CD47 binding domain, such as a SIRPα extracellular domain 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 the phagocytosis of macrophages. Therefore, downregulation of SIRPα will lead to increased phagocytosis of macrophages. SIRPα is expressed in a variety of human tumor types, including AML, chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), lung, bladder and other solid tumors. In certain embodiments, the SIRPα extracellular domain includes 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.

[0125] In a further embodiment, the SIRPα extracellular domain or a binding portion thereof is encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 17. In certain embodiments, the SIRPα extracellular domain 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 a nucleic acid molecule as set forth in SEQ ID NO.: 21. In any of the foregoing embodiments, the SIRPα, SIRPα extracellular domain, or any SIRPα fragment thereof used in the fusion protein of the present disclosure is human SIRPα. In a further embodiment, a SIRPα extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 17.

[0126] In a further embodiment, the SIRPα extracellular domain 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, SIRPα extracellular domain, or any SIRPα fragment thereof used in the fusion protein of the present disclosure is human SIRPα. In a further embodiment, a SIRPα extracellular domain is provided, which 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.

[0127] In some embodiments, the fusion protein comprises an extracellular component comprising a binding domain that binds to PD-L1, PD-L2, or both. In some embodiments, the fusion protein comprises an extracellular component comprising a PD 1 extracellular domain or a ligand binding portion thereof. In certain embodiments, the PD-1 extracellular domain includes a full-length extracellular portion of a PD-1 protein, a full-length mature extracellular portion of a PD-1 protein, a binding fragment of the extracellular portion of a PD-1 protein, or a binding fragment of the extracellular portion of a PD-1 protein with a portion of a PD-1 transmembrane domain, or any combination thereof. In certain embodiments, the PD-1 extracellular domain 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 PD-1 extracellular domain is encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 91, 93, or 95. In further embodiments, the PD-1 extracellular domain comprises at least from about 90 amino acids to at least about 130 amino acids, as set forth in SEQ ID NO.: 60. In still further embodiments, the PD-1 extracellular domain comprises a 170 amino acid sequence from the N-terminus of the PD-1 extracellular domain, as set forth in SEQ ID NO.: 90. In some embodiments, PD-1 is encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 89. In any of the foregoing embodiments, the PD-1, PD-1 extracellular domain, or any PD-1 fragment thereof used in the fusion protein of the present disclosure is human PD-1. In a further embodiment, a PD-1 extracellular domain is provided, which 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 an amino acid sequence as shown in SEQ ID NO.: 60. In a further embodiment, a PD-1 extracellular domain is provided, which 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 an amino acid sequence as shown in SEQ ID NO.: 90. In a further embodiment, a PD-1 extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 89.

[0128] In certain 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, which 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 protein of the present disclosure is human PD-1.

[0129] In some embodiments, the fusion protein comprises an extracellular component comprising an extracellular domain of CD2. In certain embodiments, CD2 is encoded by a nucleic acid molecule as shown in SEQ ID NO.: 61. In certain embodiments, the extracellular domain of CD2 comprises a full-length extracellular portion of a CD2 protein, a full-length mature extracellular portion of a CD2 protein, a binding fragment of an extracellular portion of a CD2 protein, or a binding fragment of an extracellular portion of a CD2 protein with a portion of a 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 protein of the present disclosure is human CD2. In a further embodiment, a CD2 extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as shown in GenBank Accession No. NM_001767.3. In a further embodiment, a GenBank Accession No. NM_001767.3 extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 61.

[0130] In some embodiments, the CD2 extracellular domain comprises the amino acid sequence set forth 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 set forth 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 protein of the present disclosure is human CD2.

[0131] In some embodiments, the fusion protein comprises an extracellular component, which comprises a binding domain that binds to FasL. In some embodiments, the fusion protein comprises an extracellular component containing a Fas (CD95) extracellular domain. 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; Continuiet al., Leukemia 21: 253–260, 2007; Motz et al., Nature Medicine 20: 607–615, 2014). In addition, 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 the involvement of CD44. In certain embodiments, the Fas extracellular domain includes a full-length extracellular portion of a Fas protein, a full-length mature extracellular portion of a Fas protein, a binding fragment of an extracellular portion of a Fas protein, or a binding fragment of an extracellular portion of a Fas protein with a portion of a Fas transmembrane domain, or any combination thereof. In some embodiments, the Fas extracellular domain is encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 71. In other embodiments, the Fas extracellular domain 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 a nucleic acid molecule as set forth in SEQ ID NO.: 73. In certain other embodiments, Fas is encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 75. In any of the foregoing embodiments, the Fas, Fas extracellular domain, or any Fas fragment thereof used in the fusion protein of the present disclosure is human Fas. In a further embodiment, a Fas extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as set forth in GenBank Accession No. NM_000043.4. In a further embodiment, a Fas extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule as set forth in SEQ ID NO.: 71.

[0132] In some embodiments, the Fas extracellular domain comprises the amino acid sequence set forth in SEQ ID NO.: 72. In some embodiments, the Fas extracellular domain comprises the amino acid sequence set forth in SEQ ID NO.: 74. In certain embodiments, the Fas extracellular domain comprises the amino acid sequence set forth 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 protein 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 set forth in SEQ ID NO.: 72.

[0133] In some embodiments, the fusion protein comprises an extracellular component comprising an extracellular domain of LAG3 (CD223). In certain embodiments, the LAG3 extracellular domain comprises a full-length extracellular portion of a LAG3 protein, a full-length mature extracellular portion of a LAG3 protein, a binding fragment of an extracellular portion of a LAG3 protein, or a binding fragment of an extracellular portion of a LAG3 protein with a portion of a LAG3 transmembrane domain, or any combination thereof. For example, in some embodiments, the LAG3 extracellular domain comprises approximately 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 protein of the present disclosure is human LAG3. In a further embodiment, a LAG3 extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule shown in GenBank Accession No. NM_002286.5.

[0134] In further embodiments, LAG3 is encoded by the nucleic acid molecule set forth in SEQ ID NO.: 153. In certain other embodiments, the LAG3 extracellular domain 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 set forth in SEQ ID NO.: 161. In any of the foregoing embodiments, the LAG3, LAG3 extracellular domain or any LAG3 fragment thereof used in the fusion protein 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 an amino acid sequence encoded by the nucleic acid molecule set forth in SEQ ID NO.: 153.

[0135] In some embodiments, LAG3 comprises the amino acid sequence set forth in SEQ ID NO.: 154. In some embodiments, LAG3 comprises the amino acid sequence set forth in SEQ ID NO.: 162. In any of the foregoing embodiments, the LAG3, LAG3 extracellular domain, or any LAG3 fragment thereof used in the fusion protein 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 set forth in SEQ ID NO.: 154.

[0136] In some embodiments, the fusion protein comprises an extracellular component containing a TIM3 extracellular domain. In certain embodiments, the TIM3 extracellular domain comprises a full-length extracellular portion of a TIM3 protein, a full-length mature extracellular portion of a TIM3 protein, a binding fragment of the extracellular portion of a TIM3 protein, or a binding fragment of the extracellular portion of a TIM3 protein with a portion of a TIM3 transmembrane domain, or any combination thereof. In any of the foregoing embodiments, the TIM3, TIM3 extracellular domain, or any TIM3 fragment thereof used in the fusion protein of the present disclosure is human TIM3. In a further embodiment, a TIM3 extracellular domain is provided, which 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 an amino acid sequence encoded by a nucleic acid molecule shown in GenBank accession number NM_032782.4.

[0137] In a further embodiment, TIM3 is encoded by the nucleic acid molecule shown in SEQ ID NO.: 167. In certain other embodiments, the TIM3 extracellular domain 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, TIM3 extracellular domain or any TIM3 fragment thereof used in the fusion protein of the present disclosure is human TIM3. In a further embodiment, a TIM3 extracellular domain is provided, which 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 an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO.: 167.

[0138] 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, the TIM3, TIM3 extracellular domain, or any TIM3 fragment thereof used in the fusion protein of the present disclosure is human TIM3. In a further embodiment, a TIM3 extracellular domain is provided, which 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.

[0139] The binding domain can be any peptide that specifically binds to a target of interest. The sources of the binding domain include antibody variable regions (which can be in the form of antibodies, sFv, scFv, Fab, scFv-based grababodies or soluble VH domains or domain antibodies) from different species, including humans, rodents, birds or sheep. Other sources of binding domains include variable regions of antibodies from other species, such as camelids (from camels, dromedaries, or llamas; Ghahroudie 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., Immunogen. 55:39, 2003). 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 heavy chain variable region, that is, these functional antibodies are only homodimers of heavy chains (called "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).

[0140] Alternative sources of unconventional binding domains of the present invention include sequences encoding random peptide libraries or sequences encoding engineered diverse amino acids that can replace loop regions of non-antibody scaffolds, such as scTCRs (see, e.g., Lake et al., Int. Immunol. 11:745, 1999; Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Pat. No. 8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388, 1985), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498), designed ankyrin repeat proteins (DARPins) (Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., J. Mol. Biol. 334:514, 2003; U.S. Pat. No. 8,361,794). 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 small proteins (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), a tetratricopeptide repeat domain (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), a leucine-rich repeat domain (Stumpp et al., J. Mol. Biol. 332:471, 2003), a lipocalin domain (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), mAbs 2 or Fcab TM (See, e.g., PCT Patent Application Publication Nos. WO 2007 / 098934; WO 2006 / 072620), armadillo repeat protein (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), affibodies, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte-associated protein-4 (Weidle 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., al., Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Plückthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0141] In some embodiments, the binding domain is a single-chain T cell receptor (scTCR) comprising a V α / β and C α / β Chain (e.g., V α -C α 、V β -C β 、V α -V β ) or contain V specific for the target of interest α -C α 、Vβ -C β 、V α -V β pair (e.g., a peptide-MHC complex or a peptide-HLA complex).

[0142] 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 the amino acid sequence of TCRVα, Vβ, Cα, or Cβ, wherein each CDR comprises 0 changes or at most one, two, or three changes from a TCR or fragment or derivative thereof that specifically binds to a target of interest.

[0143] In some embodiments, when compared with the V of a known TCR α 、V β , C α or C β When compared, the binding domain V α 、V β , C α or C β The V region can be derived from or based on the V region of a known TCR (e.g., a high affinity TCR). α 、V β , C α or C β , and comprises 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 above changes. Insertions, deletions or substitutions may be in V α 、V β , C α or C β Any position in the CDR region, including at the amino or carboxyl termini or at both ends of these regions, provided that each CDR contains zero changes or at most one, two or three changes, and that a V comprising the modification is provided α 、V β , C α or C β The binding domain of the region can still specifically bind to its target with an affinity similar to 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.

[0144] In certain aspects, a fusion protein according to the invention has an extracellular component comprising a binding domain that specifically binds to a target (e.g., a ligand or receptor), wherein the extracellular component optionally comprises one or more other functional subcomponents or domains, such as a multimerization domain, a linker, connecting amino acids, or any combination thereof.

[0145] In certain embodiments, the fusion protein disclosed herein, in addition to the binding domain or the part of the molecule derived from the binding domain, also includes an additional extracellular region, such as a spacer or a multimerization domain. For example, in some aspects, the multimerization domain is included in the extracellular component of the fusion protein or as a part thereof. For example, the multimerization domain can be produced by changing (such as mutation) the extracellular component, or the multimerization domain can be produced 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 protein disclosed herein. In certain embodiments, the fusion protein expressed on the cell surface includes the multimerization domain in 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 include 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 fusion protein hydrophobic component, wherein such one or more 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.

[0146] In further embodiments, the interaction between the multimerization domains of two or more fusion proteins substantially facilitates or effectively promotes signal transduction (e.g., immune cell stimulation or activation) compared to the fusion protein monomer. In certain embodiments, the multimerization of the fusion protein promotes signal transduction in the host cell in a statistically significant manner compared to the fusion protein monomer. In further embodiments, the multimerization of the fusion protein that promotes or enhances signal transduction in the host cell is through disulfide bridges.

[0147] Exemplary polymers are "dimers", which refer to biological entities comprising two molecules (e.g., two fusion proteins) associated with each other. When two related fusion proteins have substantially similar or identical amino acid sequences, this dimer is considered to be a "homodimer". Similarly, a polymer of three substantially or identical fusion proteins is referred to as a "homodimer". In certain embodiments, a 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, a homodimer, a trimer or a homotrimer can be polymerized via a zinc finger motif or a leucine zipper motif. In other embodiments, a fusion protein comprises a plurality of multimerization domains, which can be located outside the cell, inside the cell, or both.

[0148] In some embodiments, the multimerization domain contained in the extracellular component of the fusion protein comprises an extracellular portion extending from the hydrophobic component. For example, in some embodiments, the multimerization domain contained in the extracellular component of the fusion protein comprises an 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 an amino acid sequence encoded by a nucleic acid molecule shown in SEQ ID NO.: 9. In some embodiments, the extracellular portion of CD28 comprises an amino acid sequence shown in SEQ ID NO.: 32. In other embodiments, the multimerization domain included in the extracellular component of the fusion protein includes an extracellular portion of CD137 (4-1BB) extending from the CD137 (4-1BB) transmembrane domain (e.g., a range of 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 included in the extracellular component of the fusion protein includes an extracellular portion of CD28 extending from the CD137 transmembrane domain or includes an extracellular portion of CD137 extending from the CD28 transmembrane domain. In any of the above embodiments, the multimerization domain may further include a glycosylation site.

[0149] In some embodiments, the fusion protein may include a linker or connecting amino acid, which, for example, connects the extracellular component to the multimerization domain, or connects the extracellular component to the hydrophobic component, or connects the hydrophobic component to the intracellular component. In some embodiments, the linker is Gly x Ser y , wherein x and y are independently integers from 1 to 5.

[0150] In some embodiments, the extracellular component is or comprises a molecule or portion thereof that forms a multimer, and the intracellular component is or comprises a molecule or portion thereof that forms the same number of multimers. For example, in some embodiments, the extracellular component is or comprises a molecule or portion thereof that forms a dimer, and the intracellular component is or comprises a molecule or portion thereof that also forms a dimer. In some embodiments, the extracellular component is or comprises a molecule or portion thereof that forms a trimer, and the intracellular component is or comprises a molecule or portion thereof that also forms a trimer.

[0151] Target molecules can be found on or associated with cells of interest ("target cells"), which are specifically bound by the binding domains contained in the fusion proteins of the present disclosure. Exemplary target cells include immune cells, cancer cells, cells associated with autoimmune diseases or disorders or inflammatory diseases or disorders, and infectious organisms or cells (e.g., bacteria, viruses, virus-infected cells), or any cells presenting antigens complexed with MHC or human leukocyte antigens (HLA). Cells of infectious organisms, such as mammalian parasites, 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.

[0152] Extracellular components

[0153] The intracellular components included in the fusion protein of the present disclosure will have intracellular signaling domains, such as activation domains or costimulatory domains, that can transmit functional signals to cells. In certain embodiments, the intracellular signaling domain will indirectly promote cell responses by associating with one or more other proteins, and the other proteins directly promote cell responses. The intracellular signaling domain may include one, two, three or more receptor signaling domains, costimulatory domains or a combination thereof. Any intracellular component comprising an activation domain, a costimulatory 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.

[0154] As used herein, an "intracellular signaling domain" from a cell surface receptor or ligand includes a complete intracellular domain, a portion comprising an intracellular signaling domain, or a functional (signaling) fragment thereof. In certain embodiments, the intracellular signaling domain comprises a mutated intracellular domain or a functional (signaling) fragment thereof that has higher signaling activity than a wild-type or reference intracellular signaling domain.

[0155] As used herein, "co-stimulatory molecules" refer to receptors or cell surface molecules that can conduct signals to T cells to positively regulate T cell activation (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). As background, the activation and proliferation of T cells require two signals mediated by the binding of T cell antigen-specific receptors (TCR) and co-stimulatory signals, the most typical of which are CD80 and CD86 binding CD28 (Ledbetter et al., Blood 75: 1531, 1990).

[0156] The intracellular signaling domain or its functional fragment available in the fusion protein of the present invention 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 its functional fragment does not comprise a primary signal. In some embodiments, the intracellular signaling domain does not comprise CD3ζ.

[0157] 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 by TCR (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). Due to the 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 a full-length intracellular portion of a CD28 protein, a full-length mature intracellular portion of a CD28 protein, a signaling fragment of an intracellular portion of a CD28 protein, or a signaling fragment of an intracellular portion of a CD28 protein with a CD28 transmembrane domain or a fragment thereof, or any combination thereof.

[0158] 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, wherein 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 a full-length intracellular portion of a CD137 protein, a full-length mature intracellular portion of a CD137 protein, a signaling fragment of an intracellular portion of a CD137 protein, or a signaling fragment of an intracellular portion of a CD137 protein with a CD137 transmembrane domain or a fragment thereof, or any combination thereof.

[0159] 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 further 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.

[0160] 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 connecting the extracellular and intracellular components, specifying that the fusion protein::target complex spans a distance similar to the distance between membranes in an immune synapse.

[0161] 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 an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 5. In some other embodiments, the intracellular component comprises an amino acid sequence encoded by a nucleic acid molecule as 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 an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 5 and an amino acid sequence encoded by a nucleotide sequence as shown in SEQ ID NO.: 13.

[0162] Hydrophobic component

[0163] The hydrophobic portion included 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, so that a portion of the fusion protein will be located outside the cell and a portion will be located inside the cell (e.g., the intracellular signaling domain). The hydrophobic component will generally be placed within the cell membrane phospholipid bilayer. In certain embodiments, one or more linking amino acids can be placed between the intracellular signaling domain and the hydrophobic portion and connect the intracellular signaling domain and the hydrophobic portion.

[0164] 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 replacing one or more amino acids with at least one different amino acid, or any combination thereof, such as a charged or hydrophilic residue that promotes intermolecular interactions. Thus, the term "hydrophobic domain" includes transmembrane domains with modifications that, for example, reduce hydrophobicity.

[0165] In some embodiments, the transmembrane domain includes 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 (P In certain embodiments, the hydrophobic portion is a transmembrane domain from CD28, CD4, CD8, CD27 or CD137 (4-1BB).

[0166] In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a CD28 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide of SEQ ID NO.: 4. In further embodiments, the transmembrane domain is a CD28 transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 4. In additional embodiments, the transmembrane domain is a CD28 transmembrane domain having at least 90%, 95% or higher sequence identity with the amino acid sequence of SEQ ID NO.: 27. In additional embodiments, the transmembrane domain is a CD28 transmembrane domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO.: 27.

[0167] In certain embodiments, the transmembrane domain comprises a CD137 (4-1BB) transmembrane domain. In further embodiments, the transmembrane domain comprises or consists of a CD137 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity with a polynucleotide of SEQ ID NO.: 197. In a specific embodiment, the transmembrane domain is a CD137 transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 197.

[0168] In certain other embodiments, the transmembrane domain comprises a CD200R transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a CD200R transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide of SEQ ID NO.: 3. In further embodiments, the transmembrane domain is a CD200R transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 3.

[0169] In certain embodiments, the transmembrane domain comprises a SIRPα transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a SIRPα transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide of SEQ ID NO.: 18. In further embodiments, the transmembrane domain is a SIRPα transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 18.

[0170] In certain embodiments, the transmembrane domain comprises a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a CD2 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or more sequence identity to the polynucleotide of SEQ ID NO.: 63. In further embodiments, the transmembrane domain is a CD2 transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 63.

[0171] In certain embodiments, the transmembrane domain comprises a Fas transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a Fas transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or more sequence identity to the polynucleotide of SEQ ID NO.: 77. In further embodiments, the transmembrane domain is a Fas transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 77.

[0172] In certain embodiments, the transmembrane domain comprises a TIM3 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a TIM3 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or higher sequence identity with the polynucleotide of SEQ ID NO.: 169. In a further embodiment, the transmembrane domain is a TIM3 transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 169.

[0173] In certain embodiments, the transmembrane domain comprises a LAG3 transmembrane domain. In some embodiments, the transmembrane domain comprises or consists of a LAG3 transmembrane domain having an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 95% or more sequence identity to the polynucleotide of SEQ ID NO.: 155. In further embodiments, the transmembrane domain is a LAG3 transmembrane domain comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO.: 155.

[0174] Nucleic acids and host cells

[0175] In some aspects, the invention provides nucleic acid molecules encoding one or more fusion proteins described herein, which may be immunomodulatory fusion proteins (IFPs). Such nucleic acid molecules may be inserted into suitable vectors (e.g., viral vectors or non-viral plasmid vectors) to be introduced into host cells of interest (e.g., hematopoietic progenitor cells, T cells).

[0176] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule or vector that includes at least one genetic change or is modified by the introduction of an exogenous nucleic acid molecule, wherein such change or modification is introduced by genetic engineering. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or the addition, deletion, substitution or other functional destruction of other nucleic acid molecules of the genetic material of the cell. Additional modifications include, for example, non-coding regulatory regions, wherein the modification changes the expression of genes or operons. In certain embodiments, cells obtained from a subject, such as T cells, can be converted into non-natural or recombinant cells (e.g., non-natural or recombinant T cells) by introducing nucleic acids encoding fusion proteins described herein, and the cells express the fusion proteins.

[0177] In certain embodiments, the nucleic acid molecule encoding the fusion protein can be codon optimized to enhance or maximize expression in certain cell types, such as T cells (Scholten et al., Clin. Immunol. 119: 135-145, 2006).

[0178] In an exemplary implementation, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28 construct (huCD200Rtm-CD28), wherein the extracellular component comprises an extracellular domain of CD200R, the hydrophobic component comprises a transmembrane domain of CD200R, and the intracellular component comprises an intracellular signaling domain of CD28. For example, in one embodiment, a nucleic acid molecule shown by SEQ ID NO.: 1 is provided. In certain embodiments, the present disclosure provides huCD200Rtm-CD28, which 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, which comprises or consists of the polynucleotide sequence of SEQ ID NO.: 1.

[0179] 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 represented by SEQ ID NO.: 6 is provided. In certain embodiments, the present disclosure provides huCD200R-CD28tm, which comprises a polynucleotide that is at least 80% or at least 90% identical 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 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.: 29.

[0180] In another exemplary implementation, the present disclosure provides a nucleic acid molecule encoding a CD200R-CD28 construct, wherein the extracellular portion of the CD200R and the CD28 are contained in the extracellular domain. For example, the CD200R extracellular domain is truncated by about 9 to about 15 amino acids. Exemplary CD200R-CD28 constructs disclosed herein 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 an amino acid sequence encoded by a nucleic acid molecule shown in SEQ ID NO.: 9. In certain embodiments, the extracellular portion of CD28tm 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 set forth in SEQ ID NO.: 32. In further embodiments, the transmembrane portion of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 32. In certain other embodiments, the huCD200R-9aas-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 set forth in SEQ ID NO.: 30. An exemplary huCD200R-9aas-CD28Cys protein comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 30. In certain other embodiments, the huCD200R-12aas-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 set forth in SEQ ID NO.: 33. An exemplary huCD200R-12aas-CD28Cys protein comprises or consists of the amino acid sequence set forth 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 set forth in SEQ ID NO.: 184. Exemplary huCD200R-15aas-CD28Cys proteins comprise or consist of the amino acid sequence set forth in SEQ ID NO.: 184.

[0181] In an 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 a nucleotide sequence shown in SEQ ID NO.: 12 or SEQ ID NO.: 14.

[0182] 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 domain of CD28 and CD137 (4-1BB). For example, in one embodiment, the nucleic acid of the present disclosure has a nucleotide sequence shown in SEQ ID NO.: 9 or SEQ ID NO.: 15.

[0183] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a SIRPα-CD28 construct. For example, the present disclosure includes a nucleic acid molecule shown in SEQ ID NO.: 16 (huSIRPαtm-CD28) or SEQ ID NO.: 19 (huSIRPα-CD28tm).

[0184] 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 an 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).

[0185] In an 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 a nucleotide sequence shown in SEQ ID NO.: 22.

[0186] 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 an intracellular signaling domain of CD28 and CD137 (4-1BB). For example, in one embodiment, the nucleic acid of the present disclosure has a nucleotide sequence shown in SEQ ID NO.: 23.

[0187] 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).

[0188] 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 an extracellular portion of CD28. For example, the PD-1 extracellular domain may be truncated by about 10 to about 25 amino acids. An exemplary PD-1-CD28 construct has a truncated 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) of the PD-1 extracellular domain.

[0189] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a CD2-CD28 construct. For example, the present disclosure includes a nucleic acid molecule shown in SEQ ID NO.: 69 (huCD2-CD28Cys).

[0190] In other exemplary embodiments, the present disclosure provides nucleic acid molecules encoding Fas-CD28 constructs. For example, the present disclosure includes a nucleic acid molecule shown in SEQ ID NO.: 83 (huFas-CD28Cys).

[0191] In other exemplary embodiments, the present disclosure provides nucleic acid molecules encoding Fas-CD28 constructs, wherein the extracellular component comprises a truncated extracellular domain of Fas and an 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).

[0192] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a Fas-4-1BB construct, wherein the extracellular component comprises a complete extracellular domain or a truncated extracellular domain of CD95 (Fas), and the intracellular component comprises a 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 a 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 a 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 more sequence identity to 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 a huFastm-41BB protein, which 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 set forth in SEQ ID NO.: 186. Exemplary huFastm-41BB proteins comprise or consist of the amino acid sequence set forth in SEQ ID NO.: 186. In certain other embodiments, the Fas-4-1BB construct is a huFas-41BBtm protein comprising 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 set forth in SEQ ID NO.: 188. Exemplary huFas-41BBtm proteins comprise or consist of the amino acid sequence set forth in SEQ ID NO.: 188.

[0193] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a Fas-CD28-41BB construct, wherein the extracellular component comprises a CD95 (Fas) extracellular domain, the hydrophobic component comprises a transmembrane domain of CD28, and the intracellular component comprises a signaling domain of CD137 (4-1BB). In some embodiments, the extracellular component comprises a truncated extracellular domain of CD95 (Fas) and an extracellular portion of CD28. For example, the CD95 (Fas) extracellular domain is truncated by about 7 to about 15 amino acids. An exemplary Fas-CD28-41BB construct has a CD95 (Fas) extracellular domain truncated by 7, 9, 12 or 15 amino acids. For example, in some embodiments, the CD95 (Fas) extracellular domain may comprise an amino acid sequence encoded by a 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 an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO.:9.

[0194] In another exemplary embodiment, the present disclosure provides a nucleic acid molecule encoding a TIM3-CD28 construct. For example, the present disclosure includes a nucleic acid molecule shown in SEQ ID NO.: 173 (huTIM3-CD28Cys). Also included within the scope of the present disclosure is a TIM3-CD28 fusion protein, wherein the extracellular component comprises a truncated extracellular domain of TIM3 and an extracellular portion of CD28. For example, the TIM3 extracellular domain 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).

[0195] In another exemplary embodiment, the present disclosure provides nucleic acid molecules encoding LAG3-CD28 constructs. For example, the present disclosure includes nucleic acid molecules set forth in SEQ ID NO.: 163 (huLAG3-CD28Cys). Also included within the scope of the present disclosure are LAG3-CD28 fusion proteins, wherein the extracellular component comprises a truncated extracellular domain of LAG3 and an extracellular portion of CD28. For example, the LAG3 extracellular domain 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).

[0196] The vector encoding the core virus is referred to herein as a "viral vector". There are a large number of available viral vectors suitable for 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 belong to this class. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Medi-Visna (sheep lentivirus). Methods for transducing mammalian host cells with viral particles containing chimeric antigen receptor transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been previously described, for example, in U.S. Pat. No. 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.

[0197] 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 to a target. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector may also include a nucleic acid sequence encoding a transduction marker. Transduction markers for viral vectors are known in the art, including screening 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 a specific embodiment, the viral vector also includes a gene 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 includes multiple nucleic acid sequences to be expressed in a host cell as separate transcripts, the viral vector may also include additional sequences between two (or more) transcripts, thereby allowing bicistronic or polycistronic expression. Examples of such sequences for viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0198] 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-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0199] Other vectors recently developed for gene therapy purposes can also be used with the compositions and methods of the present invention. These vectors include vectors derived from baculovirus and alpha virus (Jolly, DJ. 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 virus or plasmid vector also contains a gene marker for transduction (e.g., green fluorescent protein, huEGFRt).

[0200] In certain embodiments, the vector encoding the fusion protein disclosed herein can encode more than one fusion protein.For example, the vector can encode two different fusion proteins (for example, a first fusion protein comprising a PD-1 extracellular domain and a second fusion protein comprising a TIM3 extracellular domain).

[0201] In some embodiments, the vector encoding the fusion protein disclosed herein may further include an antigen-specific TCR. In some embodiments, the antigen-specific TCR is exogenous. In some embodiments, the antigen-specific TCR is specific for HLA (MHC) class I restricted antigens. 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, the vector encoding the fusion protein disclosed herein also encodes a ligand, which may be CD200, CD47, PD-L1 or CD58. In other embodiments, the vector encoding the fusion protein disclosed herein also encodes siRNA for reducing endogenous receptor expression. In some specific embodiments, the endogenous receptor is CD200R, SIRPα, CD279 (PD-1), CD95 (Fas) or CD2.

[0202] 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 TCR α and β chains are used. Such T cells may naturally lack endogenous expression of TCR α and β chains, or may have been modified to block expression (e.g., T cells from transgenic mice that do not express TCR α and β chains or cells that have been manipulated to suppress TCR α and β chain expression) or knock out TCR α chain, TCR β chain or both genes. In some embodiments, T cells can be transformed to express TCRs specific to specific antigens.

[0203] In certain embodiments, the host cells transfected to express the fusion proteins of the present invention are functional T cells, such as virus-specific T cells, tumor antigen-specific cytotoxic T cells, naive T cells, memory stem T cells, central or effector memory T cells, γδ T cells, or CD4+CD25+ regulatory T cells. In further embodiments, nucleic acid molecules encoding the fusion proteins of the present invention are introduced into bulk CD8+T cells, naive CD8+T cells, CD8+T cells, or CD4+CD25+ regulatory T cells. CM cells, CD8+T EM In a further embodiment, the nucleic acid molecules encoding the fusion proteins of the present disclosure are introduced into bulk CD4+ T cells, naive CD4+ T cells, CD4+ T cells, CM cells, CD4+T EM In other embodiments, a nucleic acid molecule encoding a fusion protein of the present disclosure is introduced into a cell line enriched for naive CD8+ T cells and CD8+ T cells. CM In some other embodiments, a nucleic acid molecule encoding a fusion protein of the present disclosure is introduced into a cell population enriched for naive CD4+ T cells and CD4+ T cells. CM T cell population of 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.

[0204] In certain embodiments, the host cell transfected to express the fusion protein of the present disclosure is a functional natural killer cell.

[0205] One or more growth factor cytokines that promote the proliferation of T cells expressing the fusion protein of the present disclosure can be added to the culture for expanding T cells. The cytokines can be human or non-human. Exemplary growth factor cytokines that can be used to promote the proliferation of T cells include IL2, IL15, etc.

[0206] In certain embodiments, the host T cells transfected to express the fusion proteins of the present disclosure are CD4 + T cells, which also express antigen-specific high-affinity TCRs specific for HLA (MHC) class I-restricted antigens (see Soto et al., Cancer Immunol Immunother. 62:359–369, 2013).

[0207] In certain embodiments, host T cells transfected to express the fusion protein of the present invention also express a recombinant TCR specific for a cancer antigen. In some embodiments, the cancer antigen is WT1. "WT1" refers to Wilm's tumor 1, a transcription factor containing four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA binding domain at the N-terminus. WT1 plays a vital role in the normal development of the urogenital system and is mutated in a small number of patients with Wilm's tumor. 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 sarcomas, and esophageal cancer. Alternative splicing of WT1 has been noted.

[0208] In certain embodiments, 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 a gene encoding a precursor protein that is cleaved into two products, megakaryocyte enhancing factor and mesothelin. Megakaryocyte enhancing 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 specific squamous cell carcinomas. Alternative splicing results in multiple transcript variants.

[0209] In certain embodiments, host T cells transfected to express a fusion protein of the present disclosure also express a recombinant TCR specific for cyclin-A1.

[0210] In certain embodiments, host T cells transfected to express a fusion protein of the present disclosure also express a CAR.

[0211] In other embodiments, the host cell expressing the fusion protein disclosed herein further comprises a ligand, which may be CD200, CD47, PD-L1 or CD58. In yet other embodiments, the host cell expressing the fusion protein disclosed herein further expresses siRNA for reducing endogenous receptor expression. In some specific embodiments, the endogenous receptor is CD200R, SIRPα, CD279 (PD-1), CD95 (Fas) or CD2.

[0212] In certain embodiments, the host cell expressing the fusion protein disclosed herein can express more than one fusion protein.For example, the host cell can express two different fusion proteins (for example, a first fusion protein comprising a PD-1 extracellular domain and a second fusion protein comprising a TIM3 extracellular domain).

[0213] use

[0214] Diseases that can be treated with cells expressing fusion proteins as described in the present disclosure include cancer, infectious diseases (viral, bacterial, protozoan infections), immune diseases (eg, autoimmunity), or diseases associated with aging (eg, senescence). Adoptive immunity and gene therapy are promising treatments 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).

[0215] 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 pulmonary bronchial lung cancer; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papilloma; APUD tumor; bud tumor; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, duct, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, sclerosis, bronchiolar, bronchial, squamous cell, and transitional cell). Other types of cancer that may be treated include histiocytic disorders; malignant histiocytic disorders; leukemias; Hodgkin's disease; immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; reticuloendothelial proliferation; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymal tumor; mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. In addition, the following cancer types are considered to be treatable: adenoma; bile duct carcinoma; cholesteatoma; cylindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; ovarian gynecoblastoma; hepatocellular carcinoma; sweat adenoma; islet cell tumor; testicular Leydig cell tumor; papilloma; testicular Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma non-pheochromocytoma. Other types of cancer that can be treated include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angiomatous sclerosis; angiomatosis; glomus tumor; hemangioendothelioma; hemangioma; hemangiopericytoma; angiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; sarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphangiosarcoma; sarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; tumor; multiple neurofibromatosis and cervical dysplasia.

[0216] Exemplary of various hyperproliferative 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's lymphoma (NHL) or central nervous system lymphomas), leukemias (such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B cell blast transformation of chronic myeloid leukemia and acute myeloid 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, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous 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 proliferation of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0217] Inflammatory and autoimmune diseases include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, 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 inflammation, leukocyte adhesion defects, 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 / angiitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemias including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, disorders involving leukocytopenia, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated disorders, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Reynaud syndrome, Sjorgen syndrome, Stevens-Jo Hnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, seronegative spondyloarthropathies, Reiter's disease, stiffness syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia, autoimmune diseases of the testicle and ovary 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 polyglandular syndrome (or polyglandular endocrine syndrome), type I diabetes mellitus also known as insulin-dependent diabetes mellitus (IDDM), and Sheehan syndrome; autoimmune hepatitis, lymphointerstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonitis (NSIP), Guillain-Barré syndrome, large vessel vasculitis (including rheumatic polyangiitis), and pulmonary embolism. Myalgia and giant cell (large-artery) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Buerger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac sprue (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan syndrome, Whiskott-Aldrich syndrome, and thromboangiitis obliterans.

[0218] In specific embodiments, methods of treating a subject with the fusion proteins disclosed herein include treating acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia (AML), and chronic myeloid leukemia.

[0219] In certain embodiments, the method of treating a subject with a fusion protein disclosed herein comprises treating pancreatic cancer.

[0220] In certain embodiments, the method of treating a subject with a fusion protein disclosed herein comprises treating ovarian cancer.

[0221] In certain embodiments, methods of treating a disease using the fusion proteins described herein are provided, wherein the disease comprises a solid tumor expressing CD200 or a solid tumor affected by CD200. + The fusion protein comprises a myeloid cell-infiltrating tumor, and the fusion protein has an extracellular component comprising the extracellular portion of CD200R.

[0222] In a specific embodiment, a method of treating a disease with a fusion protein disclosed herein is provided, wherein the disease comprises 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.

[0223] In a specific embodiment, a method of treating a disease with a fusion protein disclosed herein is provided, wherein the disease comprises a solid tumor and the fusion protein has an extracellular component comprising an extracellular portion of TIM3, CD223 (LAG3), CD95 (Fas), CD279 (PD-1) or CD2.

[0224] In specific embodiments, methods of treating diseases with the fusion proteins disclosed herein are provided, wherein the diseases include 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.

[0225] In a specific embodiment, a method for treating a disease with a fusion protein disclosed herein is provided, wherein the disease comprises ovarian cancer, pancreatic cancer or AML, and the fusion protein has an extracellular component comprising an 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 a 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 SEQ ID NO.: 186. In a further embodiment, the Fastm-4-1BB fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO.: 186.

[0226] In specific embodiments, methods of treating a disease with a fusion protein disclosed herein are provided, wherein the disease comprises ovarian cancer, pancreatic cancer or AML, and the fusion protein has an extracellular component comprising an 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 set forth 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 set forth 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 set forth in SEQ ID NO.: 188. In further embodiments, the Fas-4-1BBtm fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 188.

[0227] Infectious diseases include diseases associated with infectious agents, including any of a variety of bacteria (e.g., pathogenic Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, Vibrio cholerae, Listeria, Rickettsia, Chlamydia, etc.), mycobacteria, and parasites (including any known parasitic members of protozoa). Infectious viruses include eukaryotic viruses, such as adenoviruses, bunyaviruses, herpesviruses, papillomaviruses, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies virus), orthomyxoviruses (e.g., influenza virus), poxviruses (e.g., vaccinia virus), reoviruses, retroviruses, lentiviruses (e.g., HIV), flaviviruses (e.g., HCV, HBV), etc. In certain embodiments, fusion proteins of the present disclosure are used to treat infections with cytoplasmic pathogens that process and display their antigens using HLA (MHC) class I molecules.

[0228] The fusion proteins of the present disclosure can be used in cell-bound form (e.g., gene therapy of target cell populations (mature T cells (e.g., CD8 + or CD4 + In one embodiment, the cells of the T cell lineage expressing the fusion protein administered to the subject are syngeneic, allogeneic or autologous cells.

[0229] The pharmaceutical composition comprising the fusion protein of the present disclosure can be administered in a manner suitable for the disease or condition to be treated (or prevented) (determined by a technician in the medical field). The appropriate dosage, appropriate duration and frequency of administration of the composition will be determined by factors such as the patient's symptoms, 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 a cell 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.

[0230] In some embodiments, the disclosure relates to increasing immune cell activity, enhancing or prolonging immune response, stimulating antigen-specific T cell response, inhibiting immunosuppressive signaling pathways, treating cancer or tumors, inhibiting cancer cell immune resistance, or treating infection methods, including administering an effective amount of host cells expressing fusion proteins as described herein to subjects in need thereof. In a further embodiment, the host cells used for any of the above methods further express engineered antigen-specific TCRs, engineered antigen-specific high-affinity TCRs, CARs, costimulatory molecules, or any combination thereof. In a specific embodiment, a method for treating leukemia is provided, including co-expressing fusion proteins and recombinant antigen-specific TCRs as disclosed herein.

[0231] In some embodiments, a CD4 + A method for inducing or enhancing a class I HLA response in T cells, comprising administering to a subject in need thereof an effective amount of a CD4 T cell expressing a fusion protein as described herein. + In a further embodiment, for inducing or enhancing CD4 + The host cell of the present invention further expresses an engineered antigen-specific TCR, an engineered antigen-specific high-affinity TCR, a CAR, a co-stimulatory molecule, or any combination thereof.

[0232] In any of the above embodiments, the method is effective in the absence of administration of exogenous IL-2.

[0233] In some embodiments, methods for increasing immune cells (e.g., CD4 + T cells or CD8 + A method for regulating cytokine production in T cells) 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.

[0234] In some embodiments, methods for increasing immune cells (e.g., CD4 + T cells or CD8 + A method for increasing cytokine production in T cells) comprising contacting an immune cell with a fusion protein as disclosed herein or a vector encoding a fusion protein. In further embodiments, the immune cell further expresses 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).

[0235] In other embodiments, the subject of any of the above methods is further treated with adjuvant therapy, 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, carbaquinone, meturedopa and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, trivinylphosphoramide, triethylenethiophosphoramide and trihydroxymethylamine; nitrogen mustards such as chlorambucil, chlorpheniramine, chlorphosphamide, estramustine, ifosfamide, nitrogen mustard, oxychloramine hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorfenapyr, fomustine, lomustine, nimustine, maleyl antibiotics such as aclacinomycins, actinomycins, authramycins, azaserine, bleomycin, actinomycins, calicheamicins, carabicin, caminomycins, carzinophilins, chromomycins, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, zinstatin ostatin), 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, thiamine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, 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 RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins, capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0236] 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.

[0237] Examples

[0238] Example 1

[0239] CD200R-CD28 fusion protein construct

[0240] use Figure 1A The schematic diagram in the figure illustrates 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 , Construct IV). The hydrophobic component may include CD200R ( Figure 1A , construct I) or CD28 ( Figure 1A , constructs II-V) of the transmembrane domain or a portion thereof. In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component also includes the extracellular portion of CD28, particularly the extracellular cysteine ​​residues 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 part 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, which can be manipulated by adjusting 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. In addition, the CD200R-CD28 construct has the ability to convert the inhibitory signal that normally comes from CD200R binding to its target into a positive signal generated by the CD28 intracellular signaling domain.

[0241] An exemplary nucleic acid molecule encoding a CD200R-CD28 fusion protein comprises the following elements (5' to 3'): extracellular component (CD200R) - multimerization domain (CD28 cysteine) - hydrophobic component (CD28 transmembrane) - intracellular component (CD28 intracellular). In some embodiments, the nucleic acid molecule encoding a 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 or 15.

[0242] Nucleic acid encoding the construct can be ordered from Invitrogen or generated in the laboratory by PCR and then directionally TOPO cloned into pENTR TM / vector (Invitrogen) and used Technology (Invitrogen) into the retroviral vector pMP71-attR. In certain embodiments, the nucleic acid molecules encoding the IFPs of the present disclosure are codon optimized before being cloned into the pMP71-attR retroviral vector.

[0243] Example 2

[0244] Transgenic expression of CD200R-CD28 constructs

[0245] Based on murine C57BL / 6Friend virus-induced erythroleukemia (FBL) and TCR gag A transgenic mouse preclinical disseminated leukemia mouse model was used to determine whether the CD200R-CD28 chimeric receptor could improve T cell function.

[0246] Generation of TCR transgenic mice to produce TCR specific for the gag epitope gag )Specific CD8 + T cells. C57BL / 6 (B6) mice were purchased from Jackson Laboratory. TCR gag Transgenic mice with CD8 + T cells expressing a TCR transgene specific for the Friend virus gag epitope ( et al., J. Immunol. 166: 2863-2870, 2001). All animal studies performed were approved by the Washington University Institutional Animal Care and Use Committee protocol (protocol number 2013-01). Murine B6Friend virus-induced erythroleukemia (FBL) expressed the gag epitope encoded by F-MuLV (peptide CCLCLTVFL (SEQ ID NO.: 213)).

[0247] The murine gene-based CD200R-CD28 chimeric construct was inserted into the pMP71 retroviral vector and used to transduce primary mouse 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 TOPO cloned into pENTR TM / vector (Invitrogen) and used The retroviral vector was transferred into the retroviral vector pMP71-attR using Qiagen technology (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 effector transduction reagent (Qiagen). Viral supernatants were collected on days 2 and 3 and then used to transduce TCR gag T cells.

[0248] One day before transfection, stimulate TCR with anti-CD3 / CD28 and 100U / mL rhIL-2 gag T cells were transfected by spinfection at 1000 g for 90 min in 12-well plates in the presence of IL-2 and polybrene. gag Transduction of T cells. Similar to T cell transduction, FBL cells were transfected with CD200 using polybrene centrifugal transfection and then sorted to generate a homogenous population.

[0249] Five days after transduction, CD8 + T cell construct expression ( Figure 1B ). A vector encoding green fluorescent protein (GFP) was used as a control. Transduction efficiencies ranged from 4-36%, and mean fluorescence intensity (MFI) of transduced cells was similar between constructs.

[0250] Example 3

[0251] CD200R-CD28 constructs promote proliferation, accumulation, and effector function of transduced T cells in vitro

[0252] Evaluation of the CD200R-CD28 constructs described in Examples 1 and 2 for promoting TCR gag The capacity of T cells to proliferate, accumulate and exert effector functions.

[0253] Expansion of effector cells in vitro

[0254] Effector cells were generated in vitro as described previously (Stromnes et al., J. Clin. Invest. 120:3722-34, 2010). Irradiated antigen-presenting splenocytes (5×10 ) were cultured in 10 mL of culture medium (IMDM supplemented with nonessential amino acids, 2 μM glutamine, 100 U / mL penicillin / streptomycin, 10% FBS, and 50 μM 2-mercaptoethanol). 6 ), irradiated FBL (3×10 6 ) and TCR gag Tg cells (10 6 ) were cultured with IL-2 (50 U / mL). T cells were restimulated weekly 5-7 days after the last stimulation and evaluated by flow cytometry.

[0255] In vitro T cell proliferation assay

[0256] As in Example 2, the TCR transduced gag To assess in vitro T cell proliferation, TCR was expressed using CellTrace Purple (CTV, Life Technologies) according to the manufacturer's protocol. gag T cells were stained with titrated amounts of CD200 - FBL or CD200 + FBL cells stimulated CTV-labeled Tg T cells (10 5 ) and GFP control T cells. After 3 days, TCR was evaluated by flow cytometry gag CTV dilution of T cells.

[0257] Flow cytometry results showed that titrated numbers of CD200-FBL cells (upper) or CD200 + TCR after FBL (lower) stimulation gag T cell count Figure 2A Compared with GFP control-transduced T cells (red line), four of the five CD200R-CD28 constructs tested significantly improved TCR gag T cells respond to CD200 + Proliferation of FBL (blue line).

[0258] In vitro T cell accumulation assay

[0259] To determine whether the enhanced proliferation also resulted in increased accumulation of transduced cells, the expression of CD200 + During multiple stimulation cycles performed by FBL, total TCR gag The proportion of transduced cells in the population.

[0260] Several constructs promoted the accumulation of transduced T cells, including CD200R-CD28tm, CD200R-CD28Cys, CD200R-3aas-CD28Cys, and CD200R-9aas-CD28Cys ( Figure 2B Of these constructs, CD200R-9aas-CD28Cys showed the greatest increase in transduced T cells over multiple stimulations, resulting in more than 3-fold expansion in 3 stimulations.

[0261] In vitro T cell enrichment assay

[0262] With CD200 + or CD200 - Irradiated FBL cells were expressed in transduced and non-transduced CD8 + Mixed populations of T cells were restimulated to determine whether restimulation would enrich for transduced CD200R-9aas-CD28Cys IFP + Compared with wild-type T cells, irradiated CD200 + Repeated restimulation of tumor cells enriched for cells transduced with IFP, suggesting that recognition of targets expressing the CD200R-9aas-CD28Cys IFP ligand enhances responses ( Figure 2C ).

[0263] In vitro colocalization assay

[0264] Transduced T cells were imaged by microscopy to determine whether CD200R-9aas-CD28Cys IFP colocalizes with its cognate ligand in the immune synapse (IS) during T cell activation. CTxB was used to stain lipids in the cell membrane that are enriched at the synapse ( Figure 2D , Figure I). Targeting CD200 expressed by FBL cells ( Figure 2D , Figure II) or CD200R expressed by T cells ( Figure 2D , Figure III) were used to visualize the location of the molecules relative to the IS. CD200 ligand and CD200R colocalize within the IS ( Figure 2D , Figure IV), indicating that the construct is of appropriate size to be accommodated by the immune synapse.

[0265] CFSE-based cytotoxicity assay.

[0266] 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 (CFSE) in PBS at room temperature. hi ) or 0.25 μM (CFSE lo )CFSE for 10 min. Excess dye was removed by washing tumor cells in serum-containing medium. A 1:1 mixture of EL4 and FBL tumor cells was incubated with titrated amounts of TCR transduced with CD200R-CD28 or GFP vectors. gag In vitro expanded effector T cells were incubated in 96-well round-bottom plates at 37°C and 5% CO2 for 4 hours. The % CFSE in the total CFSE-positive cells (FBL+EL4) remaining in the wells was analyzed by flow cytometry. hi (FBL), to determine specific FBL cleavage.

[0267] TCR transduced with empty vector gag Compared to T cells transduced with the CD200R-CD28 construct, gag T cells showed a 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 EL4 cells (CTV + )、CD200 + FBL(CFSE hi ) and nonspecific EL4 (CFSE lo ) 1:1:1 mixture of control target ( Figure 2F ). In addition, the control GFP-transduced TCR gag T cells cleave CD200 with equal efficiency - FBL and CD200 + FBL( Figure 2G ). In contrast, TCR transduced with CD200R-9aas-CD28Cys showed gag T cells showed expression of CD200 + Increased killing of FBL cells by more than 40% CD200 lysis at the lowest E:T ratio tested + FBL( Figure 2G ).

[0268] Taken together, these data indicate that the CD200R-CD28 construct functions to increase the accumulation and lytic activity of transduced T cells in response to tumor cell stimulation.

[0269] Example 4

[0270] CD200R-9AAS-CD28CYS-transduced T cells showed enhanced in vivo accumulation in response to recognition of FBL

[0271] As described above, B6 mice were injected intraperitoneally (ip) with 4 × 10 6 After allowing FBL to disseminate for 5 days, mice received 180 mg / kg of cyclophosphamide (Cy, which can be used as For survival studies, 10 cells that had been stimulated 1-3 times in vitro were injected intraperitoneally. 5 TCR gag T cells were transferred into tumor-bearing mice. To evaluate short-term proliferation and accumulation, 2 × 10 fusion protein-transduced T cells and GFP control-transduced T cells were each 6 Co-injected into tumor-bearing mice, mice were euthanized 8 days later for analysis. Mice were regularly monitored for tumor burden and euthanized within 24-48 hours if evidence of tumor progression indicated death.

[0272] To evaluate whether CD200R-9aas-CD28Cys fusion protein-transduced T cells exhibit greater proliferation and accumulation in vivo in response to FBL recognition, mixed populations of fusion protein-transduced and control cells were transferred into tumor-bearing mice, and the ratios of cells were compared by ex vivo analysis 8 days after transfer ( Figure 3A By using the cognate marker, transduced T cells were detected in spleen and lymph nodes at 1.2-1.4-fold higher levels than control cells, relative to the ratio injected ( Figure 3B ). Transduced CD200R-9aas-CD28Cys + TCR gag T cells showed reduced CD62L expression 3 days after transfer into tumor-bearing mice, indicating an effector T cell phenotype ( Figure 3C By day 15, transduced T cells and control T cells displayed 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 response to tumor stimulation in vivo. In addition, they exhibited a protein expression pattern consistent with an effector T cell phenotype for at least 3 days after transfer into tumor-bearing mice.

[0273] Example 5

[0274] CD200R-CD28 + Adoptive immunotherapy with T cells shows greater activity in the treatment of disseminated leukemia

[0275] Adoptive immunotherapy with CD200R-CD28-transduced T cells mediated enhanced therapeutic activity in a preclinical mouse model of disseminated leukemia.

[0276] Mice were injected with a lethal dose of CD200 + FBL leukemia, five days later, the group of mice that received Cy treatment received 10 5 Additional treatment with T cells ( Figure 4A ).like Figure 1A As shown, by comparing T cells transduced with CD200R-CD28tm, CD200R-9aas-CD28Cys and GFP control constructs, the contribution of CD28 cysteine ​​bonds to the efficacy mediated by the CD200R-CD28 construct was evaluated. IL-2 was given to a group of mice as an additional therapeutic agent for 10 days to promote the activity of T cells (Stromnes et al., J. Clin. Invest. 120: 3722-34, 2010). Before injection, various surface proteins of T cells were evaluated by flow cytometry. Transduced and control TCR gag T cells showed similar phenotypes, indicating that transduction did not alter the phenotype of cells prior to injection ( Figure 4B ).

[0277] In the small group of mice that received the IL-2 injection, the T cells improved survival, but no significant differences were detected in the survival of mice that received the different groups of T cells ( Figure 4C However, in the group of mice that did not receive IL-2 injection, the survival rate of mice that received T cells transduced with the CD200R-CD28 construct, which is sized to fit within the immune synapse ( Figure 4D Most mice that received no T cells but instead received T cells transduced with a GFP control vector or T cells transduced with the largest extracellular domain (CD200R-CD28Cys IFP) did not survive beyond 30 days ( Figure 4C and 4D, black solid line, dashed line, and orange line, respectively). In contrast, the CD200R-CD28tm + 71% of mice receiving CD200R-9aas-CD28Cys + 83% of the mice with T cells survived more than 100 days after treatment ( Figure 4C and 4D , green and red lines, respectively). These data suggest that transduction of T cells with a CD200R-CD28 construct spanning a distance similar to the distance between membranes in an immune synapse provides sufficient costimulation to overcome the dependence of T cell immunotherapy on exogenous IL-2 injection. Furthermore, despite differences in proliferation and accumulation between the CD200Rtm-CD28 and CD200R-9aas-CD28Cys constructs tested in mice that did not receive exogenous IL-2 injections, both IFPs effectively enhanced T cell immunotherapy, significantly improving clinical outcomes from otherwise progressive leukemias.

[0278] Example 6

[0279] CD200R-9AAS-CD28CYS + T cells do not cause spontaneous reactions with endogenous tissues and do not show infiltration of normal tissues in vivo

[0280] To determine the TCR gag Whether transduction of T cells can sufficiently lower the activation threshold to result in autoreactivity with endogenous tissues, autoimmune toxicity was assessed in transgenic mice engineered to express FBL gag tumor Ag in hepatocytes as a self-antigen ( Figure 5A ). TCR was generated in vitro gag effector, and 10 6 The strain was transferred to Alb with disseminated leukemia treated with cyclophosphamide: Gag Liver damage was assessed by quantifying serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) three and seven days after transfer. + TCR gag Adoptive transfer of cells did not affect serum levels of AST or ALT at 3 or 7 days after transfer, indicating that CD200R-9aas-CD28Cys does not induce detectable autoimmune liver injury in Alb:Gag mice ( Figure 5B ).

[0281] IFP-transduced T cells did not show an increase in normal tissue infiltration compared to control T cells. 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 liver tissue was observed, CD200R-9aas-CD28Cys + or control TCR gag There were no significant differences between the receptors, indicating that there was no increase in lymphocyte infiltration due to IFP expression ( Figure 5C ).

[0282] Example 7

[0283] The 4-1BB co-stimulatory signaling domain promotes the accumulation of transduced T cells in vitro

[0284] The co-stimulatory 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). In order to evaluate whether the intracellular signaling domain of 4-1BB with or without the intracellular signaling domain of CD28 can induce increased 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 using the method described in Example 2 ( Fig. 6A ). TCR was transduced as in Example 2 gag T cells, and TCRs were generated in vitro as in Example 3 gag Effector cells.

[0285] As observed with CD200R-9aas-CD28Cys, T cells transduced with the 4-1BB construct accumulated over multiple rounds of stimulation in vitro ( Figure 6B ). These data suggest that 4-1BB IFP also promotes the proliferation and survival of T cells.

[0286] Using the CFSE-based cytotoxicity assay described in Example 3, TCR transduced with CD200R-4-1BB gag T cells showed enhanced ability to lyse FBL tumors in vitro ( Figure 6C CD200R-41BB-transduced T cells also promote survival ( Fig.6D ).

[0287] Example 8

[0288] Co-expression of CD200RTM-CD28 enhances the function of WT1-specific TCR primary T cells

[0289] A human CD200Rtm-CD28 construct (SEQ ID NO.: 1) was generated to determine whether IFP expression enhances T cell function in human primary T cells. The construct was conjugated to the HLA-A2 restricted WT1 by linking the gene to the P2A element. 126 The β and α chains of the specific TCR "C4" bind ( Fig. 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 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.

[0290] The Jurkat human T cell subline lacking endogenous TCR was used to test the expression of IFP and TCR. These Jurkat T cells were transfected with 2 × 10 cells / mL of retroviral supernatant by centrifugation at 1000 g at 32 °C. 6 Transduction of the triple gene construct of the Jurkat human T cell line resulted in high expression of IFP and expression of the TCR, with an MFI similar to that of T cells transduced with the TCR alone ( Fig. 7A ).

[0291] To transduce primary human T cells, + 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, T cells were transduced as described above for Jurkat T cells. As previously described, T cells were restimulated every 10-14 days using the Rapid Expansion Protocol (REP) (Ho et al., J Immunol Methods 310:40-52, 2006).

[0292] The human cell line T2 was used as APC because it lacks TAP and is therefore unable to present endogenous peptides, whereas low-level expression of MHCI allows presentation of exogenously loaded peptides. Expression of CD200 on T2 cells was assessed by flow cytometry ( Figure 7B ). T2 cells show low levels of endogenous CD200 expression ( Figure 7B ).

[0293] With WT1 126 The T2 cells stimulated the transduced T cells. Despite the low level 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 In addition, when exposed to CD200dim tumor cells, stimulated CD200Rtm-CD28-transduced T cells (i.e., IFP + T cells) produce increased levels of IFNγ and IL-2 ( Fig.7D ).

[0294] Overall, these results indicate that primary T cells transduced to express the human CD200Rtm-CD28 construct and WT1 126 The beta and alpha chains of the specific TCRs exhibited enhanced proliferation and increased cytokine production relative to T cells transduced with the TCR construct alone.

[0295] Example 9

[0296] SIRPα-CD28 fusion protein construct promotes accumulation of transduced T cells in vitro.

[0297] Exemplary fusion proteins as described herein also include IFPs that include the extracellular domain of SIRPα or a portion thereof and the intracellular signaling domain of CD28 ( Fig. 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 also includes the extracellular portion of CD28, especially the extracellular cysteine ​​residues adjacent to the hydrophobic component (e.g., SIRPα-CD28Cys, SIRPα-6aas-CD28Cys, SIRPα-9aas-CD28Cys, and SIRPα-9aas-CD28Cys). The extracellular component may include all or part of the extracellular domain of SIRPα. In some embodiments, the extracellular component includes the entire extracellular domain of SIRPα. In other examples, 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 immune synapse and co-localize with the TCR in the cSMAC to transmit a strong co-stimulatory signal. In some examples, the extracellular component comprises a truncated SIRPα, which can change the size of the extracellular component. For example, in order to take into account the additional extracellular amino acids (e.g., an additional 9 or 12 amino acids) of the extracellular domain of the fusion protein, SIRPα-6aas-CD28 has a truncated portion of SIRPα that retains a native N-linked glycosylation site. In another example, SIRPα-23aas-CD28 has a truncated portion of SIRPα that lacks the entire stem region of the SIRPα extracellular domain. Additionally, the SIRPα-CD28 construct has the ability to convert the signal initiated by the binding of SIRPα to its target into a positive (eg, co-stimulatory) signal generated by the CD28 intracellular signaling domain.

[0298] IFP using the extracellular component of SIRPα was generated using the method described in Example 2. Fig. 8A ). TCR was transduced as in Example 2 gag T cells, and TCRs were generated in vitro as in Example 3 gag Effector cells. Similar to T cell transduction, FBL cells were transduced with CD47 or mCherry by polybrene centrifugation and then sorted to generate a homogenous population.

[0299] As observed with CD200R-9aas-CD28Cys, T cells transduced with the SIRPα construct accumulated over multiple rounds of stimulation in vitro ( Figure 8B ). These data suggest that SIRPα-CD28 IFP also promotes the proliferation and survival of T cells.

[0300] 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 T cell-tumor cell synaptic distances showed enhanced proliferation compared to control T cells ( Figure 8C ). In addition, in the presence of SIRPα-CD28 + After 3 days of co-culture, T cells effectively killed CD47 + Tumor cells, but not control T cells or T cells transduced with a SIRPα construct lacking the intracellular signaling domain ( Fig.8D To further evaluate SIRPα-CD28 + The lytic capacity of T cells was used Assays to quantify CD47 killing + FBL kills. A total of 10 5 m's Cherry + CD47 + FBL were co-cultured with titers of human T cells transduced with SIRPα-CD28 construct in 24-well plates. The plates were placed in a cell culture incubator. (Essen BioScience) for 70 hours. Images were captured every hour to monitor tumor cell killing, as determined by the loss of red signal. Even at the lowest effector to target ratio tested, SIRPα-CD28 + T cells also kill CD47 + Tumor cells.

[0301] Example 10

[0302] PD-1-CD28 fusion protein constructs promote cytokine production in transduced T cells in vitro

[0303] Exemplary fusion proteins as described herein also include IFPs comprising the extracellular domain of PD-1 or a portion thereof and the intracellular signaling domain of CD28 ( Fig.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 also includes the extracellular portion of CD28, especially the extracellular cysteine ​​residues adjacent to the transmembrane component to promote interchain dimerization (e.g., PD1-CD28Cys, PD1-9aas-CD28Cys, and PD1-21aas-CD28Cys). The extracellular component may include all or part of the extracellular domain of PD-1, or may be truncated (e.g., -9aas in mouse constructs, -12aas or -15aas in human constructs; lacking the stem region of PD-1, -21aas) to maintain a shorter spatial distance between cells and promote the entry of receptors that have received ligands into immune synapses. Additionally, the PD1-CD28 construct has the ability to convert the inhibitory signal that normally results from PD1 binding to its target into a positive (eg, co-stimulatory) signal generated by the CD28 intracellular signaling domain.

[0304] IFP ( Fig.9A ). TCR was transduced as in Example 2 gag T cells, and TCRs were generated in vitro as in Example 3 gag Effector cells.

[0305] Mouse PD1-CD28 IFP ( Fig.9A PD1-CD28 was restimulated with FBL cells endogenously expressing PD-1 ligands (PD-L1 and PD-L2) in the presence of Brefeldin A (to preserve cytokine production). + T cells. After 5 h, cells were fixed and treated 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 intracellular cytokine production compared to control T cells ( Fig. 9B ).

[0306] Human PD1-CD28 IFP ( Fig.9A ). Vectors containing PD1-CD28IFP and C4 TCR were generated as described above. Jurkat T cells were transduced as described above. T cells transduced with TCR and PD1-12aas-CD28Cys or PD1-15aas-CD28Cys showed high transduction efficiency and expression of both proteins ( Fig.10 ).

[0307] Example 11

[0308] The FAS-CD28 fusion protein construct promoted accumulation and enhanced function of transduced T cells in vitro.

[0309] 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 CD28 ( Fig.11A ). The transmembrane component may include a domain or 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 ​​residues adjacent to the transmembrane component (e.g., Fas-CD28Cys and Fas-9aas-CD28Cys). The extracellular component may include all or part of the extracellular domain of Fas, or may be truncated to maintain a short spatial distance (-9aas) between cells during receptor-ligand interactions. In addition, the Fas-CD28 construct has the ability to convert a signal initiated by the binding of Fas to its target into a positive (e.g., co-stimulatory) signal generated by the intracellular signaling domain of CD28.

[0310] IFP including the Fas extracellular component was generated using the method described in Example 2. Fig.11A ). TCR was transduced as in Example 2 gag T cells, and TCRs were generated in vitro as in Example 3 gag Effector cells.

[0311] To determine whether expression of Fas-CD28 IFP resulted in increased accumulation of transduced cells, the expression of total TCR in transduced cells in mixed populations was measured over multiple cycles of stimulation with irradiated FBL as described in Example 3. gag All constructs promoted the accumulation of transduced T cells relative to control T cells ( Fig. 11B Expression of the Fas-CD28 construct but not full-length (FL)Fas promotes T cell survival or expansion after multiple stimulations in vitro ( Fig. 11C ).

[0312] Example 12

[0313] LAG3-CD28 fusion protein construct

[0314] Exemplary fusion proteins as described herein also include IFPs comprising the extracellular domain of LAG3 or a portion thereof and the intracellular signaling domain of CD28 ( Fig. 12A). The transmembrane component may include a domain or 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 also includes the extracellular portion of CD28, particularly the extracellular cysteine ​​residues adjacent to the transmembrane component (e.g., LAG3-CD28Cys and LAG3-9aas-CD28Cys). The extracellular component may include all or part of the extracellular domain of LAG3, or may be truncated to maintain a short spatial distance between cells during receptor-ligand interactions (e.g., -9aas). In addition, the LAG3-CD28 construct has the ability to convert the inhibitory signal that normally comes from LAG3 binding to its target into a positive (e.g., co-stimulatory) signal generated by the CD28 intracellular signaling domain.

[0315] IFP using the extracellular fraction of LAG3 was generated using the method described in Example 2. Fig. 12A ). T cells were transduced with LAG3-eGFP as described. Five days after transduction, CD8 + T cell construct expression ( Fig. 12B ). A vector encoding green fluorescent protein (GFP) alone was used as a control. All constructs showed expression of LAG3 ( Fig. 12B ).

[0316] Example 13

[0317] TIM3-CD28 fusion protein construct

[0318] Exemplary fusion proteins as described herein also include IFPs comprising the extracellular domain of TIM3 or a portion thereof and the intracellular signaling domain of CD28 ( Fig.13A ). The transmembrane component may include a domain or 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 also includes the extracellular portion of CD28, especially the extracellular cysteine ​​residues adjacent to the transmembrane component (e.g., TIM3-CD28Cys and TIM3-9aas-CD28Cys). The extracellular component may include all or part of the extracellular domain of TIM3, or may be truncated to maintain a short spatial distance between cells (e.g., -9aas). In addition, the TIM3-CD28 construct has the ability to convert the inhibitory signal that normally comes from TIM3 binding to its target into a positive signal generated by the CD28 intracellular signaling domain.

[0319] A novel IFP using the extracellular components of TIM3 was generated using the methods described in Example 2. Fig.13A). T cells were transduced with GFP-TIM3 constructs as described. Five days after transduction, CD8 + T cell construct expression ( Fig. 13B ). A vector encoding green fluorescent protein (GFP) alone was used as a control. Most constructs showed similar expression of TIM3 ( Fig. 13B ).

[0320] Example 14

[0321] CD200R-CD28 fusion protein construct can be expressed by primary T cells

[0322] In a further example, using Fig.14A The schematic diagram in the figure illustrates exemplary fusion proteins described herein. Exemplary fusion proteins include immunomodulatory fusion proteins (IFPs) comprising the extracellular domain of CD200R or a portion thereof and the intracellular signaling domain of CD28 or a portion thereof ( Fig.14A , Construct IV). The hydrophobic component may include CD200R ( Fig.14A , construct I) or CD28 ( Fig.14A , constructs II-V) of the transmembrane domain or a portion thereof. In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component also includes the extracellular portion of CD28, particularly the extracellular cysteine ​​residues adjacent to the hydrophobic component (e.g., Fig.14A Construct III, CD200R-CD28Cys; Construct IV, CD200R-3aas-CD28Cys; and Construct V, CD200R-9aas-CD28Cys). The extracellular component may comprise all or part of the extracellular domain of CD200R. In some examples, the extracellular component comprises the entire extracellular domain of CD200R ( Fig.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., Fig.14A , construct IV, CD200R-3aas-CD28Cys) or the first 229 amino acids (e.g., Fig.14A , construct V, CD200R-9aas-CD28Cys). The CD200R-CD28 constructs disclosed herein have the ability to convert the inhibitory signal normally resulting from CD200R binding to its target into a positive signal generated by the intracellular signaling domain of CD28.

[0323] The size of the extracellular component that can be manipulated by adjusting the fusion protein construct may affect the ability of the fusion protein to enter the immune synapse and co-localize with the TCR in cSMAC to transmit strong costimulatory signals. CD28 signaling occurs naturally in immune synapses, where CD28 is recruited to amplify TCR signals and reduce the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013; Yokosuka et al., Immunity 29: 589-601, 2008). In immune synapses, the spatial distance between T cells and APC is the shortest, and molecules with large extracellular domains are excluded. Therefore, the construct of the intercellular space closest to the immune synapse may be able to co-localize with the TCR in the immune synapse and transmit effective costimulatory signals. Constructs III and IV extend the CD28 transmembrane domain into the extracellular space to incorporate membrane-proximal cysteine ​​(CD28Cys), which promotes CD28 homodimerization and enhances native CD28 signaling (Lazar-Molnar et al., Cell Immunol. 244: 125-129, 2006). To account for the increased length of the nine amino acids of the extracellular CD28 domain, the CD200R extracellular domain portion of CD200R-9aas-CD28Cys is 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 is truncated by 3 amino acids. The truncated extracellular CD200R is truncated from the C-terminus to retain the N-linked glycosylation site. Therefore, in theory, the murine constructs CD200Rtm-CD28, CD200R-CD28tm, and CD200R-9aas-CD28Cys optimally maintain the required short spatial distance between T cells and APCs for colocalization with the TCR in the immune synapse.

[0324] An exemplary nucleic acid molecule encoding a CD200R-CD28 fusion protein comprises the following elements (5' to 3'): extracellular component (CD200R) - multimerization domain (CD28 cysteine) - hydrophobic component (CD28 transmembrane) - intracellular component (CD28 intracellular). In some embodiments, the nucleic acid molecule encoding a 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.

[0325] The nucleic acid encoding the construct was inserted into the pMP71 retroviral vector to transduce primary mouse splenocytes stimulated with anti-CD3 and anti-CD28 antibodies. C57BL / 6 (B6) mice were purchased from Jackson Laboratory. TCR gagTransgenic mice with CD8 + T cells express a TCR transgene specific for the Friend virus gag epitope (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). Order DNA constructs from Invitrogen or generate in the laboratory by PCR. Use The construct cloned 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 the effector transduction reagent (Qiagen). Viral supernatants were collected on days 2 and 3. One day before transfection, TCR was stimulated with anti-CD3 / CD28 and 100U / mL rhIL-2. gag T cells were transfected by centrifugation at 1000 g for 90 min in 12-well plates in the presence of IL-2 and polybrene for TCR gag T cell transduction. On day 7 after stimulation, irradiated splenocytes (5×10 6 ), irradiated FBL (3×10 6 ) and IL-2 (IU / mL), the transduced cells were restimulated.

[0326] Five days after transduction, CD8 + IFP expression in T cells ( Fig. 14B ). Fluorescent dye-conjugated antibodies were purchased from eBioscience or Biolegend. Transduction efficiencies ranged from 5-43%, and mean fluorescence intensity of transduced cells was similar between constructs, indicating similar IFP expression.

[0327] Example 15

[0328] CD200R-CD28 constructs promote proliferation, accumulation, and effector function of transduced T cells in vitro

[0329] Evaluation of the CD200R-CD28 construct described in Example 14 to promote TCR gag The capacity of T cells to proliferate, accumulate and exert effector functions.

[0330] In vitro T cell proliferation assay

[0331] CD28 signaling promotes proliferation and survival of T cells stimulated by 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 expanded for 2-3 stimulation cycles in the presence of IL-2 in vitro to generate effector T cells and mimic human adoptive immunotherapy protocols. Effector T cells were labeled with CellTrace purple (CTV) and FBL (CD200 - FBL)( Fig.15A , upper panel) or were transduced to express CD200 (CD200 + FBL cell line stimulates CTV-labeled Tg T cells (10 5 ) for 3 days and then assessed by flow cytometry ( Fig.15A , figure below).

[0332] At a low T cell to FBL ratio of 25:1, GFP control transduced T cells ( Fig.15A , blue line) shows the response to CD200 - or CD200 + In contrast, four of the five tested constructs ( Fig.15A , red line) significantly improved the response to CD200 + FBL proliferation, but in response to CD200 - The proliferation of FBL was not improved. T cells transduced with the largest extracellular domain CD200R-CD28Cys did not improve proliferation.

[0333] To test whether the increased proliferation conveyed by CD200R interaction with leukemia-expressed CD200 reflects enhanced adhesion and / or decoy binding rather than costimulation, constructs bearing only truncated, nonsignaling versions of the CD200R extracellular and CD28 transmembrane domains were generated (“trCD200R”; Fig. 15B ). Transduced TCR expressing construct gag T cells ( Fig. 15C ) did not show any effect on CD200 + FBL enhanced proliferation ( Fig.15D ), indicating the essential role of CD28 co-stimulatory signaling.

[0334] In vitro T cell enrichment assay

[0335] Expected to use CD200 + After FBL stimulation, the expression of IFP targeting CD200 will lead to IFP + T cells relative to IFP - T cell enrichment was evaluated using irradiated CD200 - or CD200 + After multiple stimulation cycles of FBL, total TCR gag The proportion of transduced cells in the population was measured by CD200 - or CD200 + Analysis of cell composition after 3 cycles of FBL stimulation showed that TCR expressing GFP control gag The fraction of T cells did not change ( Fig.15E ). On the contrary, when using CD200 + After stimulation, TCR expressing IFP gag T cells are enriched, while CD200 - However, it was not enriched under FBL stimulation (CD200R-9aas-CD28Cys, Fig.15F ). As predicted, although several constructs promoted the accumulation of transduced T cells, this construct, which was appropriately sized for the immune synapse and included the dimeric cysteine ​​motif CD200R-9aas-CD28Cys, produced the greatest relative increase after three stimulations in three separate experiments, yielding an average >3-fold enrichment (P<0.05) ( Figure 15G , fold enrichment is shown, stimulation 3 times / stimulation 1 time, for T cells transduced with eGFP, CD200Rtm-CD28, CD200R-CD28tm, CD200R-3aas-CD28cys, and CD200R-9aas-CD28cys).

[0336] CFSE-based cytotoxicity assay.

[0337] 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.

[0338] FBL and control EL4 tumors were incubated with 2.5 μM (hi) or 0.25 μM (lo) CFSE in PBS for 10 min at room temperature. Excess dye was removed by washing tumor cells in serum-containing medium. Nonspecific EL4 control targets and CD200+ 1:1 mixture of FBL tumor cells with titrated numbers of CD200R-9aas-CD28Cys-transduced or GFP-transduced TCR gag Effector T cells (i.e., a range of effector to target (E:T) ratios) were incubated in 96-well round-bottom plates at 37°C and 5% CO2 for 5 hours. The total CFSE-positive cells remaining in the wells were analyzed by flow cytometry (FBL + % CFSE in EL4) hi (FBL), to determine specific FBL cleavage.

[0339] TCR transduced with CD200R-9aas-CD28Cys gag T cell killing CD200 + FBL outperformed control T cells, lysing more than 40% of CD200 at a low E:T ratio (0.3:1). + FBL( Fig.15H ).

[0340] In vitro cytokine production assay

[0341] To determine whether T cells transduced with CD200R-9aas-CD28Cys IFP produced an increased amount and diversity of cytokines, which would represent additional costimulatory functions, multifunctional cytokine production was assessed by flow cytometry. + CD200R IFP of IFNγ, IL-2 and TNFα produced after FBL stimulation + Higher percentage of T cells than control T cells. Lower percentage of CD200R IFP + T cells are cells that do not produce cytokines (27% vs. 51%, Fig.15I , blue), while a higher percentage of CD200R IFP + T cells are multifunctional cells that can produce all 3 cytokines (22% vs. 7%, Fig.15I , purple). Based on the mean fluorescence intensity (MFI), CD200 + FBL stimulated CD200R IFP + T cells have increased cytokines / cells ( Fig.15J ).

[0342] In vitro colocalization assay

[0343] To more closely examine the mechanism of enhancing the IFP expression function of T cells, the location of CD200R IFP on the surface of T cells was observed by microscopy. After binding to CD80 / 86, natural CD28 is localized to the immune synapse, recruiting signal transduction molecules that amplify TCR signals (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). To evaluate the movement of IFP after stimulation, FITC-coupled cholera toxin B subunit (CTxB) was used to stain lipid rafts in the cell membrane ( Figure 15K , Figure III), which is enriched at the immune synapse (Stephan et al., Nat Med. 13:1440-1449, 2007) and is used to define the site of immune synapse components. Then, using the binding of CD200 on FBL ( Figure 15K , Figure II) or CD200R on T cells (CD200R-9aas-CD28Cys, Figure 15K , Figure 1), to visualize these molecules associated with the immune synapse. gag Cells were mixed with FBL at an E:T ratio of 15:1 in 15 mL and then incubated at 37°C for 20 minutes before loading into a μ-Slide VI.4 chamber (Ibid) 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).

[0344] CD200R and enhanced lipid raft staining are located in the T cell:target contact area ( Figures 15K to 15M , Figure IV), suggesting that the size of the IFP can be regulated by the immune synapse.

[0345] LCK phosphorylation

[0346] Tyrosine kinase LCK is essential for TCR signaling, and the recruitment of LCK to the TCR signaling complex leads to phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) sequence 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 the proline motif in the CD28 signaling tail, and the T cell expression of CD28 is essential for the sustained phosphorylation of 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 ( Fig.15A The transduced T cells were treated with PMA / ionomycin, FBL or CD200 + FBL was stimulated or not for 10 min, fixed and stained for intracellular pLCKY394, and analyzed by flow cytometry. Phospho-LCK (Tyr394) antibody was purchased from R&D Systems, and intracellular staining was detected by secondary labeling with anti-mouse PE from BioLegend.

[0347] Response to strong stimulation (PMA / ionomycin) and CD200 - Upon FBL stimulation, the three T cell populations achieved similar LCK Y394 phosphorylation ( Fig.15N ). T cells transduced with GFP control or IFP with a larger extracellular domain CD200R-CD28Cys responded to CD200 - FBL and CD200 + FBL showed similar low levels of pLCK Y394 expression. + Upon FBL stimulation, CD200R-9aas-CD28Cys-transduced T cells showed a sustained increase in the phosphorylation level of LCK Y394 at 10 min, indicating that the expression of CD200R-9aas-CD28cys provided the necessary function of CD28 co-stimulation.

[0348] Summarize

[0349] Taken together, these data suggest that CD200R-CD28 constructs function 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 showed that costimulation was most effectively achieved in IFPs containing a dimerization motif and a tumor-T cell distance that facilitates localization to the immune synapse. T cells transduced with such CD200R-CD28 IFPs responded to CD200 in vitro. + Target cells exhibited enhanced proliferation and effector functions.

[0350] Example 16

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

[0352] In adoptive T cell therapy for malignant tumors, tumors usually provide limited or no co-stimulatory signals, but 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). Therefore, the most effective TCR expressing CD200R-9aas-CD28Cys IFP in vitro was evaluated. gag T cells encounter CD200 in vivo + The ability of FBL to proliferate and accumulate in leukemia.

[0353] Transduced TCRs were generated as described in Example 15 gag B6 mice were injected intraperitoneally (ip) with 4×10 6 Live CD200 + After allowing FBL to disseminate for 5 days, mice were given 180 mg / kg cyclophosphamide (Cy) intraperitoneally 6 hours before transfer of effector T cells to reduce tumor burden and induce lymphopenia, similar to an artificial adoptive immunotherapy regimen. To assess short-term proliferation and accumulation, 2 × 10 6 IFP-transduced Thy1.1 + T cells were transduced with an equal number of innate different GFP control Thy1.1 + ×Thy1.2 + T cells were co-injected into tumor-bearing mice so that each mouse could serve as an internal control ( Fig.16A Both T cell populations were generated in vitro and expanded using three stimulation cycles by the same method and were phenotypically similar on the day of injection and 5 days after the third stimulation ( Fig. 16B IL-2 (2×10 4U / dose). On day 8 after T cell transfer, mice were euthanized, and spleens and inguinal lymph nodes were collected.

[0354] For some studies, negative selection was performed using EasySep TM Mouse CD8 + T cell enrichment kit (STEMCELL) to isolate CD8 + T cells. Mice were monitored regularly for increasing tumor burden and were euthanized if evidence of tumor progression indicated mortality within 24-48 hours.

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

[0356] In conclusion, in an in vivo study of adoptive therapy for disseminated leukemia, CD200R-CD28-transduced leukemia-specific T cells eradicated an otherwise lethal disease more efficiently than wild-type cells and circumvented the requirement for IL-2 administration to maintain activity in vivo.

[0357] Example 17

[0358] CD200R-CD28 + Adoptive immunotherapy with T cells shows greater activity in the treatment of disseminated leukemia

[0359] Whether costimulation provided to cells expressing CD200R-9aas-CD28Cys IFP leads to enhanced activity of therapeutic T cells was evaluated in a preclinical mouse model of disseminated leukemia, which requires T cell responses to persist for >25 days to eradicate leukemia (Cheever et al., J Immunol. 125:711-714, 1980). As described previously, B6 mice were injected intraperitoneally with a lethal dose (4×10 6 )CD200 + FBL leukemia cells (Stromnes et al., J Clin Invest. 120:3722-3734, 2010). Five days later, the mouse groups received 180 mg / kg cyclophosphamide (Cy) intraperitoneally and 10 5 TCR gag Effector T cells to metabolize drugs (Cheever et al., 1980). gag The therapeutic efficacy of T cells transduced with CD200R-9aas-CD28Cys was compared with that of T cells expressing GFP control ( Fig.17A , 17B ). This approach 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) ( Fig.17A ).

[0360] Injection of IL-2 and immunotherapy with control T cells cured 67% of the mice, while CD200R-9aas-CD28Cys + The T cells cured 100% of the mice, which was not statistically significant ( Fig.17A Subsequent studies were performed with larger cohorts and omitting the IL-2 injections. In these studies, only 40% of mice treated with T cells transduced with the GFP control vector survived beyond 30 days ( Fig. 17B , blue line). In contrast, cells that received CD200R-9aas-CD28Cys + 89% of mice expressing T cells survived 100 days after FBL transfer ( Fig. 17B , red line, P < 0.05). These results suggest that IFPs with CD200R provide costimulatory signals that can not only enhance T cell immunotherapy for progressive leukemia but also largely bypass the need for IL-2 administration.

[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 be optionally 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 normally drive the oncogenic phenotype. WT1-specific CD8 + T cells have shown 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 used in patients with leukemia, lung cancer, or mesothelioma. + T cells were tested (clinicaltrials.gov NCT01640301, NCT02408016), and the WT1 is a transcription factor overexpressed in many malignant tumors (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 at the same time as triggering antigen receptors (Chen et al., Nat Rev Immunol. 13: 227-242, 2013). Unlike CARs containing co-stimulatory domains in chimeric signal proteins, cells with introduced TCRs require independent triggering of co-stimulatory receptors. However, tumor cells often not only rarely (if at all) express ligands for co-stimulatory receptors, but also often upregulate inhibitory receptor ligands that interfere with co-stimulation and block T cell activation (Driessens et al., Immunol Rev. 229: 126-144, 2009). Therefore, strategies to overcome inhibitory signaling and increase co-stimulatory / activating 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 therapy is 26% (Society AC, Cancer Facts & Figures 2016. Atlanta: American Cancer Society, 2016). Since T cells naturally flow to the hematopoietic site where AML is located, T cell therapy has 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). CD200, a type 1 membrane protein that is 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 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 to engineer T cells not only with tumor-reactive receptors, but also with molecules that eliminate negative signals and replace them with activating signals. To overcome the inhibitory CD200R signaling associated with AML and simultaneously direct CD8 + T cells provide missing costimulatory signals, and immunomodulatory fusion proteins (IFPs) have been designed, which consist of the CD200R extracellular domain fused to the intracellular T cell costimulatory signaling domain so that IFPs can utilize leukemia cells expressing CD200 by binding to this inhibitory ligand but generating costimulatory signals. Fusion proteins containing the PD-1 extracellular domain have been shown to be able to provide costimulatory signals (Prosser et al., Mol Immunol. 51: 263-272, 2012), but the principles for designing molecules that generate or even optimize costimulatory signals have not yet been defined.

[0366] Therapy using TCR-transduced T cells was investigated in an AML clinical trial (registered as NCT01640301 at Clinicaltrials.org). All clinical studies were conducted in accordance with the principles of the Declaration of Helsinki. Protocol 2498 was approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board (IRB) and the U.S. Food and Drug Administration (FDA). Treatment of AML patients with TCR-transduced T cells. Peripheral blasts were obtained from 4 patients who progressed / relapsed after T cell therapy. The AML maintenance subset LSCs are located on the CD45 dim CD34 + CD200 expression was compared with CD34 obtained from mobilized leukocytes of three healthy donors (Bachas et al., Leukemia 26:1313-1320, 2012; Ho et al., 58th ASH Annual Meeting, San Diego, CA, 2016). +cells (used to generate T cells for infusion). + CD200 expression was not detected on AML cells, but CD200 was expressed on the majority of AML blasts in each patient tested (range 42-97%). + )( Fig.18A ), which is 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 in the immune synapse ( Fig.18B ). Human primary T cells were transduced to determine whether expression of this IFP could enhance function. The constructs were inserted into the WT1 with HLA-A2 restriction by linking each gene to the P2A element. 126 -Specific TCR C4 The β and α chains of α-antigens were expressed in a single lentiviral vector construct (Stromnes et al., Immunol Rev. 257:145-164, 2014) for transduction of T cells used in a clinical trial for the treatment of AML ( Fig.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 The medium was changed on day 1 after transfection, and the supernatant containing virus was collected on days 2 and 3, and aliquots were frozen for future use. + 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. 6T 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 (see Ho et al., J. Immunol. Methods 310:40-52, 2006).

[0369] Compared with TCR alone C4 Transduced T cells, transduced to express TCR C4 Human primary T cells expressing CD200R and CD28 fusion protein showed high levels of CD200R expression and equivalent levels of TCR C4 Express.

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

[0371] In conclusion, transduction of human primary T cells with human IFP also increased the response to CD200 + Proliferation of leukemic cells and production of cytokines. This study focused on the generation of IFPs to target the inhibitory molecule CD200, which is often upregulated in cancer cells (especially AML and LSC cells) and is known to inhibit T cell immune responses. In addition to AML, CD200 expression has been reported to be increased in other heme malignancies and solid tumors (such as breast cancer, colon cancer, ovarian cancer, and prostate cancer). In certain embodiments, CD200 IFPs can be used to treat heme malignancies and solid tumors, including breast cancer, colon cancer, ovarian cancer, and prostate cancer. These results show that genetic engineering of tumor-specific T cells with IFPs containing the CD200R extracellular domain can effectively convert inhibitory signals transmitted by leukemic cells into co-stimulatory signals in a cell-intrinsic manner, thereby avoiding the requirement for overall blockade of this inhibitory receptor and avoiding the associated risks of promoting activation of endogenous autoreactive T cells. In addition, IFPs can be used to increase sensitivity without manipulating the TCR.

[0372] Example 19

[0373] Coexpression of CD200RTM-CD28 or CD200R-9AAS-CD28CYS with WT1-specific TCR enhances the function of primary T cells

[0374] In a further example, using Fig.19A The schematic diagram in the figure illustrates an exemplary fusion protein described herein. Exemplary fusion proteins include IFP, which comprises the extracellular domain of human CD200R or a portion thereof and the intracellular signaling domain of human CD28 or a portion thereof ( Fig.19A , constructs II-VII). The hydrophobic component may include CD200R ( Fig.19A , constructs I, II and VIII) or human CD28 ( Fig.19A , constructs III-VII) of the transmembrane domain or a portion thereof. In some exemplary CD200R-CD28 fusion proteins, the hydrophobic component includes the transmembrane domain of CD28, and the extracellular component also includes the extracellular portion of human CD28, particularly the extracellular cysteine ​​residues adjacent to the hydrophobic component (e.g., Fig.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 ( Fig.19A The extracellular component may comprise all or part of the extracellular domain of human CD200R. In some embodiments, the extracellular component comprises the entire extracellular domain of CD200R ( Fig.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., Fig.19A , construct V, CD200R-9aas-CD28Cys), the first 231 amino acids (e.g., Fig.19A , construct VI, CD200R-12aas-CD28Cys), or the first 228 amino acids (e.g., Fig.19A , Construct VII, CD200R-15aas-CD28Cys). The human CD200R-CD28 constructs disclosed herein have the ability to convert the inhibitory signal normally resulting from CD200R binding to its target into a positive signal generated by the intracellular signaling domain of CD28.

[0375] The size of the extracellular component that can be manipulated by adjusting the fusion protein construct may affect the ability of the fusion protein to enter the immune synapse and co-localize with the TCR in cSMAC to transmit strong costimulatory signals. CD28 signaling occurs naturally in immune synapses, where CD28 is recruited to amplify TCR signals and reduce the activation threshold (Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013; Yokosuka et al., Immunity 29: 589-601, 2008). In immune synapses, the spatial distance between T cells and APC is the shortest, and molecules with large extracellular domains are excluded. Therefore, the construct of the intercellular space closest to the immune synapse may be able to co-localize with the TCR in the immune synapse and transmit effective costimulatory signals. Constructs IV-VII extend the CD28 transmembrane domain into the extracellular space to incorporate a membrane-proximal cysteine ​​(CD28Cys), which promotes CD28 homodimerization and enhances native CD28 signaling (Lazar-Molnar et al. al., Cell Immunol. 244: 125-129, 2006). In some embodiments, in order to illustrate the increased length of the additional amino acids of the extracellular CD28 domain, the CD200R extracellular domain portion is truncated by an equal number, for example, the CD200R extracellular domain portion of CD200R-9aas-CD28Cys is truncated by 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 by 12 amino acids, and the extracellular CD200R of CD200R-15aas-CD28Cys is truncated by 15 amino acids. In constructs V-VII, the truncated extracellular CD200R is truncated from the C-terminus to retain the N-linked glycosylation site. In order to represent Fig.19A The fusion proteins shown in , CD200Rtm-CD28, CD200R-CD28tm, and CD200R-12aas-CD28cys, theoretically optimally maintain the short spatial distance required between T cells and APCs 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 was approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board (IRB) and the U.S. Food and Drug Administration (FDA). This trial is 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 ( Fig.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 ( Fig.19B ). The constructs were inserted into the WT1 with HLA-A2 restriction by connecting each gene to 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 ( Fig.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 targeting IFP relative to IFP - T cell enrichment

[0383] For testing, use CD200 + Does cell stimulation to express CD200-targeted IFP lead to IFP + T cells relative to IFP - T cell enrichment, using CD200 + Measurement of CD200R before and after LCL cell stimulation + The relative proportion of cells.

[0384] like Fig. 20A As shown, the expression of IFPCD200R-CD28tm ( Fig.19A III) resulted in IFP after restimulation of LCL transduced with CD200. + T cells (CD200R + ) relative to IFP - T cells (CD200R - ) enrichment.

[0385] With trCD200R( Fig.19A VIII) and CD200R-15aas-CD28Cys ( Fig.19A T cells transduced with construct VII) were not enriched, indicating a lack of co-stimulation ( Fig. 20B and 20C ). However, T cells expressing several other constructs were expressed on CD200 + After LCL REP, especially in CD200R-CD28tm ( Fig.19A III) and CD200R-9aas-CD28Cys ( Fig.19A The ratio after construct V in Fig. 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 - Human T cells expressing IFPCD200R-9aas-CD28Cys targeting CD200 had increased cytokine production. TAP-deficient tumor cell line T2 expressed endogenous CD200 ( Fig.21ACD200R-9aas-CD28Cys expression enhanced peptide-pulsed T2 cells (treated with 1 ug / mL WT 1-126 impact) of cytokine production ( Fig. 21B ).

[0389] To test whether the enhanced effector function associated with CD200R-9aas-CD28Cys expression reflected enhanced adhesion and / or decoy binding rather than costimulation, constructs with only a truncated, non-signaling form of the CD200R extracellular and CD28 transmembrane domains were generated (“trCD200R”; Fig.19A , construct VIII; Fig. 21C ). Compared with expressing only TCR C4 cells, transduced T cells expressing this construct did not show enhanced cytokine production ( Fig.21D ), indicating that CD200R-9aas-CD28Cys provides a co-stimulatory signal.

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

[0391] Effector function was also assessed in a flow cytometry-based cytotoxicity assay similar to that described in Example 15 ( Fig.21J T cells transduced with the CD200R-CD28 construct 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 according to Example 11 and tested in vivo in a mouse model of leukemia ( Fig.22A ). 4×10 6tumor cells (day 0) and treated with cyclophosphamide, followed by (1) no other treatment, (2) adoptive transfer for 10 6 GFP-converted TCR gag Transgenic CD8 + T cells (day 5), or (3) adoptive transfer 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 tended to eradicate the disease more quickly ( Fig. 22B ) and provide protection over time ( Fig. 22B , 22C ).

[0396] Example 23

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

[0398] Exemplary fusion proteins as described herein also include IFP, which includes the extracellular domain of Fas or a portion thereof and the intracellular signaling domain of 4-1BB. The extracellular component may include all or part of the extracellular domain of Fas. In certain embodiments, the transmembrane component may include the domain or a portion thereof of Fas, 4-1BB or CD28. 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 part of CD28, especially the extracellular cysteine ​​residues adjacent to the transmembrane component (e.g., Fas-CD28Cys-4-1BBic and Fas-9aas-CD28Cys-4-1BBic). The extracellular component may include all or part of the extracellular domain of Fas, or may be truncated to maintain a short spatial distance (-9aas) between cells when receptor-ligand interacts. In some other exemplary Fas-4-1BB fusion proteins, the transmembrane component includes the transmembrane domain of 4-1BB (e.g., Fas-4-1BBtm; Fig.23A ). In addition, 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 transgenic TCR can be co-expressed in transduced murine T cells. IFP containing the extracellular components 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 transgenic TCR (TCR gag , specific for an epitope derived from FBL leukemia transformed by Friend murine leukemia virus (Stromnes et al., J Clin Invest. 120:3722-3734, 2010). gag or TCR gag Plat-E cells were transfected with DNA constructs encoding TCR and Fas-4-1BBtm to produce retroviral supernatant. Naive P14 T cells were stimulated with anti-CD3 and anti-CD28 and then transduced with retroviral supernatant for 2 days. Five days after stimulation, transduced T cells were stained with specific antibodies against TCR and against Fas and analyzed by flow cytometry. gag P14 T cells transduced with the Fas-4-1BBtm and Fas-4-1BBtm constructs expressed similar levels of TCR and also expressed high levels of the Fas-4-1BBtm IFP construct ( Fig. 23B ).

[0400] Discovery of Fas-4-1BB + T cells showed enhanced proliferation in vitro. 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. T cells were then harvested and analyzed by flow cytometry. gag Transduced T cells only showed a lack of proliferation, as did TCR gag T cells transduced with both Fas-4-1BBtm and TCR + Fas-4-1BBtm)( Fig.23C , left). When E:T was 8:1, some TCR-only T cells showed proliferation; however, all TCR+Fas-4-1BB + T cells all showed robust proliferation, supporting increased stimulation and proliferation capacity ( Fig.23C ,right).

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

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

[0403] Example 24

[0404] 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 model of ovarian cancer.

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

[0407] In addition, mice treated with anti-mesothelin TCR+Fas-4-1BBtm transduced T cells had improved survival compared to mice treated with anti-mesothelin TCR transduced T cells alone. 6 ID8 tumor cells and were spread for 6 weeks. After cyclophosphamide treatment, the patients received 10 7 T cells and 5.0×10 8 Mice were treated with mesothelin-pulsed splenocytes and then injected with IL-2 for 10 days. Mice were treated every two weeks until euthanized according to IACUC-approved endpoint criteria. T cells transduced with TCR+Fas-4-1BBtm had improved survival ( Fig. 24B ).

[0408] Example 25

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

[0410] It has been previously shown that immunotherapy with TCR-T cells targeting mesothelin 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] The autologous KPC pancreatic cancer model is used to simulate 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 pancreatic-specific Cre recombinase ("C") to generate mutations in Kras ("K") and p53 ("P") in the pancreatic epithelium. The KPC model (i) reproduces many key features of the immune microenvironment observed in human PDA, including strong inflammatory responses and rejection of effector T cells, (ii) is the most widely studied PDA genetic model for evaluating immunotherapy, and (iii) reproduces 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 efficacy.

[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. Fig.25AUltrasound images of healthy mice with normal pancreas and pancreatic tumors in "logged-in" mice (KPC genetically engineered mice) are shown. Mice were randomly assigned to treatment groups. Mice were treated with cyclophosphamide, and after cyclophosphamide, mice receiving TCR-T cells were injected with mesothelin-specific T cells (cells transduced with anti-mesothelin TCR or anti-mesothelin TCR+Fas-4-1BBtm) and mesothelin peptide-pulsed splenocytes, each with 10 7 Starting 14 days after enrollment, T cell / APC infusions (but without cyclophosphamide) were repeated every 2 weeks for a total of 3 infusions without IL-2 injection. Mice that survived 28 days after the last T cell infusion were bled, and the persistence of the transferred T cells was assessed by detecting T cells with innate markers using flow cytometry. At the end of the study, mice were assessed for survival and euthanized according to IACUC-approved endpoint criteria. A summary of the experimental design used in this example is shown in Fig.25B shown.

[0413] 28 days after the third T cell infusion, Fas-4-1BB + T cells showed greater persistence in the blood ( Fig.25C ). Accepts Fas-4-1BB + All mice expressing T cells (100%) showed T cell persistence, whereas none of the T cells expressing TCR alone persisted ( Fig.25C ). Using mesothelin-specific TCR and Fas-4-1BB + The survival rate of mice treated with T cells was significantly higher than that of mice treated with T cell immunotherapy with mesothelin-specific TCR alone (Mantel-Cox test, P < 0.05; Fig.25D ).

[0414] Example 26

[0415] FAS-4-1BB expression enhances adoptive immunotherapy in a mouse model of AML

[0416] As shown in Example 24 for solid tumors, Fas-4-1BB + T cell therapy improves survival of liquid tumors. In a mouse 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, the cells were given or not given 10 6 T cells transduced with TCR+Fas-4-1BBtm showed improved survival compared to T cells with TCR alone ( Fig.26 ).

[0417] While particular embodiments of the present invention have been shown and described, it will be readily appreciated that the various embodiments described above may be combined to provide further embodiments, and that various changes may be made therein without departing from the spirit and scope of the present 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 No. PCT / US2016 / 021064, are incorporated herein by reference in their entirety. If it is necessary to adopt the concepts of various patents, applications, and publications to provide other embodiments, aspects of the embodiments may be modified.

[0419] These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full range of equivalents of these authorized claims. Therefore, the claims are not limited by this disclosure.

Claims

1. A fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds to a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component linking 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) is of a size or spans a distance that is: (i) up to the distance between two cell membranes of an immune synapse, (ii) up to about or substantially the same distance 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 about or substantially the same distance 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; 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 part thereof.

2. A fusion protein comprising (a) an extracellular component comprising a binding domain that specifically binds to a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component linking the extracellular and intracellular components, wherein the binding domain is an inhibitory molecule binding domain or has at least 95% identity to an inhibitory molecule binding domain, and the intracellular signaling domain is a co-stimulatory or stimulatory molecule binding domain or comprises at least 95% identity to a co-stimulatory or stimulatory molecule binding domain, and 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 from CD137 (4-1BB) or a functional part thereof.

3. The fusion protein according to claim 1 or 2, wherein the hydrophobic component comprises CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, 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), C The transmembrane domain of D274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70.

4. The fusion protein of claim 3, wherein the hydrophobic component comprises the transmembrane domain of CD95 (Fas) or the transmembrane domain of CD137 (4-1BB).

5. The fusion protein according to claim 1 or 2, wherein the target is or comprises CD95L (FasL).

6. The fusion protein according to claim 1 or 2, wherein (a) the extracellular component comprises the extracellular domain of CD95 (Fas) or a functional fragment thereof, (b) the hydrophobic component comprises the transmembrane domain of CD137 (4-1BB), and (c) the intracellular component comprises the intracellular signaling domain of CD137 (4-1BB) or a functional fragment thereof.

7. The fusion protein according to claim 1 or 2, wherein the CD95(Fas) extracellular domain comprises the entire extracellular domain of CD95(Fas). The fusion protein according to claim 1 or 2, wherein CD95 (Fas) is human CD95 (Fas).

9. The fusion protein of claim 1 or 2, wherein (a) the extracellular component comprises an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO: 71, (b) the hydrophobic component comprises an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO: 197, and (c) the intracellular component comprises an amino acid sequence encoded by a nucleic acid molecule as shown in SEQ ID NO:

13.

10. The fusion protein of claim 1 or 2, wherein (a) the extracellular component comprises the amino acid sequence shown in SEQ ID NO: 72, (b) the hydrophobic component comprises the amino acid sequence shown in SEQ ID NO: 198, and (c) the intracellular component comprises the amino acid sequence shown in SEQ ID NO:

36.

11. Fusion protein, comprising: (1) (a) an extracellular component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 71, (b) a hydrophobic component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 197, and (c) an intracellular component comprising an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 13; or (2) (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.

12. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule according to claim 12.

14. A host cell comprising a gene encoding the fusion protein according to any one of claims 1 to 11.

15. A host cell comprising the nucleic acid molecule according to claim 12.

16. Use of the fusion protein according to any one of claims 1 to 11 in treating a disease in a subject.

17. Use of the host cell according to claim 14 in treating a disease in a subject.

18. Use of the host cell according to claim 15 in treating a disease in a subject. 19.Expression of fusion protein CD8 + or CD4 + Human T cells, the fusion protein comprises 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, The fusion protein binds FasL and provides 4-1BB intracellular signaling.

20. The CD8 according to claim 19 + or CD4 + Use of human T cells in the preparation of a medicament for treating cancer of a solid tumor in which the cancer cells express FasL, the cancer being selected from ovarian cancer or pancreatic cancer or acute myeloid leukemia.

Images