PD-1 and 4-1BB fusion protein

Abstract

To provide fusion proteins that enhance T-cell activation.SOLUTION: The invention pertains to fusion proteins comprising: (a) an extracellular domain containing a polypeptide derived from PD-1 or CD40L at its N-terminal; (b) a transmembrane domain; and (c) an intracellular domain containing a polypeptide derived from 4-1BB or CD28 at its C-terminal. Also, fusion proteins containing CD28 at the N-terminal and CD40L at the C-terminal are envisaged. The present invention also pertains to nucleic acid molecules encoding the fusion proteins, vectors, and host cells containing the vectors. The invention further pertains to a method for generating such host cells. Finally, the present invention pertains to pharmaceutical compositions that contains the fusion proteins, nucleic acid molecules, vectors, and / or the host cells, particularly for treating diseases or disorders associated with PD-1 / PD-L2 or CD40 binding and / or PD-1 / PD-L2 or CD40 expression such as cancer and chronic viral infection.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to fusion proteins comprising (a) an extracellular domain comprising at its N-terminus a polypeptide derived from PD-1 or CD40L; (b) a transmembrane domain; and (c) an intracellular domain comprising at its C-terminus a polypeptide derived from 4-1BB or CD28. Fusion proteins comprising CD28 at its N-terminus and CD40L at its C-terminus are also contemplated. The present invention also relates to nucleic acid molecules encoding such fusion proteins, vectors containing such nucleic acid molecules, and host cells containing such vectors. The present invention further relates to methods for producing such host cells. Finally, the present invention relates to pharmaceutical compositions comprising such fusion proteins, nucleic acid molecules, vectors, and / or host cells for treating diseases or disorders associated with PD-1 / PD-L2 or CD40 binding and / or PD-1 / PD-L2 or CD40 expression, particularly cancer and chronic viral infections.

[0002] Adoptive transfer of tumor-infiltrating lymphocytes (TILs) is a promising treatment option for tumors (Rosenberg et al., Science (2015), 348: 62-68). Unfortunately, isolation and expansion of TILs is not possible in all tumor entities. Therefore, the concept of genetic manipulation of T cells with transgenic T cell receptors (TCRs) was developed (Hurwitz et al., Cancer Microenviron (2014), 7: 1-9). To date, such T cells exhibit short survival times and are functionally lost in patients (Janicki et al., Cancer Res (2008), 68: 2993-3000; Bai et al., PNAS USA (2008), 105: 13003-13008; Bendle et al., Cancer Res (2004), 64: 8052-8056; Anderson et al., J Immunol (2007), 178: 1268-1276). Costimulation of the TCR signaling cascade by costimulatory receptors such as CD28 can enhance T cell proliferation, survival, and cytotoxicity (Chen et al., Nat Rev Immunol (2013), 13: 227-242). However, epithelial tumors do not express the ligands (CD80 / 86) of the costimulatory receptor CD28, and human effector T cells themselves are negative for such receptors, so costimulation cannot occur using conservative methods. To provide costimulation to T cells that does not rely on this classical method, chimeric costimulatory receptors have been created. These chimeric costimulatory receptors consist of the extracellular domain of the co-inhibitory receptor PD-1 (also known as CD279) and the signaling domain of CD28 (Ankri et al., J Immunol (2013), 4121-4129; Prosser et al., Mol Immunol (2012), 263-272; WO 2013 / 019615).

[0003] Normally, tumor-specific T cells express PD-1 on their surface, which then binds to its ligand, PD-L1, expressed on tumor cells. This binding results in blocking TCR signaling and T cell activation, thereby inhibiting tumor-specific T cells. Stimulation of CD28 is required to costimulate TCR signaling (and thus enhance T cell activation). See above.

[0004] A chimeric costimulatory receptor containing domains from PD-1 and CD28 would exhibit both related functions: the extracellular receptor function of PD-1 on the one hand, and the intracellular signaling function of CD28 on the other. In such chimeric constructs previously proposed (Ankri, inc. cit.; Prosser, inc. cit.; WO 2013 / 019615), the transmembrane domains were taken from the respective signaling molecules.

[0005] However, although such constructs have been described to reduce PD-L1-mediated T cell inhibition and enhance T cell activation, there is still room for further improvement of such constructs.

[0006] This problem has been addressed by the present invention as described and claimed herein.

[0007] The present invention provides (a) an extracellular domain (ECD) comprising a polypeptide derived from PD-1 or CD40L at its N-terminus; (b) transmembrane domain (TMD) and; (c) an intracellular domain (ICD) containing a polypeptide derived from 4-1BB or CD28 at the C-terminus; The present invention relates to a fusion protein comprising:

[0008] Preferably, according to the present invention, when the extracellular domain (ECD) comprises a polypeptide derived from PD-1 at its N-terminus, the intracellular domain (ICD) comprises a polypeptide derived from 4-1BB at its C-terminus, and vice versa. Similarly, when the extracellular domain comprises a polypeptide derived from CD40L at its N-terminus, the intracellular domain comprises a polypeptide derived from CD28 at its C-terminus, and vice versa. In a fusion protein having an ECD derived from CD40L and an ICD derived from CD28, the ICD may be located N-terminal to the TMD, while the TMD may be located at the very C-terminus of the fusion protein. See, by way of example (and not limitation), the fusion protein shown in Figure 8.

[0009] In one embodiment of the invention, the extracellular domain comprises (a) a polypeptide derived from PD-1 at its N-terminus and a polypeptide derived from 4-1BB at its C-terminus.

[0010] In contrast to CD28, 4-1BB (CD137) is a costimulatory receptor present on a subset of T cells that can promote TCR signaling. 4-1BB is a member of the tumor necrosis factor receptor (TNFR) subfamily and is not present on naive T cells, but is induced after T cell stimulation and differentiation into effector cells (Cheuk et al., Cancer Gene Ther (2004), 11: 215-226). The intracellular domain of 4-1BB contains a QEE motif, which recruits TNFR-associated factor 2 (TRAF2) upon ligation with 4-1BBL expressed on APCs (Arch et al., Mol Cell Biol (1998), 18: 558-565; Nam et al., J Immunol (2005), 174: 1898-1905). TRAF2 activates the MAPK pathway, including ERK, and activates the nuclear translocation of NFκB (Watts, Annu Rev Immunol (2005), 23: 23-68), thereby enhancing cytokine production and T cell survival. Thus, the present invention provides a fusion (also referred to herein as "chimeric") protein comprising or consisting of the extracellular domain (ECD) of PD-1 and the intracellular signaling domain (ICD) of 4-1BB.

[0011] In a further aspect of the invention, a fusion protein is contemplated that comprises at its N-terminus an ICD derived from CD28, a fragment of the ICD derived from CD40L, a TMD of CD40L, and at its C-terminus an ECD derived from CD40L (Figure 8). In one embodiment of the invention, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:29.

[0012] Furthermore, the present invention provides a fusion (chimeric) protein comprising the extracellular domain (ECD) of CD40L and the intracellular signaling domain (ICD) of CD28. It has been surprisingly discovered in the context of the present invention that, when expressed in T cells, this chimeric protein exerts dual functions upon interaction with cells expressing its receptor, CD40. In T cells, the chimeric protein should initiate costimulatory pathways (cis effect) that confer survival and enhanced effector activity. Upon interaction with CD40 cells, i.e., tumor cells, tumor endothelium (trans effect), it should cause cell death and, if the interacting cells are antigen-presenting cells, be able to induce the secretion of cytokines (e.g., IL-12) that further support T cell activity (Figure 7).

[0013] Generally, in the context of the present invention, unless otherwise specified herein, a fusion protein comprising or consisting of the extracellular domain (ECD), transmembrane domain (TMD) from PD-1, and the intracellular domain (ICD) from 4-1BB is also referred to herein as "PD-1:4-1BB" or "PD-1:BB." Similarly, a fusion protein comprising or consisting of the extracellular domain (ECD), transmembrane domain (TMD) from PD-1, and the intracellular domain (ICD) from CD28 is also referred to herein as "PD-1:CD28." Similarly, a fusion protein comprising or consisting of the extracellular domain (ECD), transmembrane domain (TMD) from CD40L, and the intracellular domain (ICD) from CD28 is also referred to herein as "CD40L:CD28" or "CD28:CD40L" (also referred to herein as "CD40L:CD28i" or "CD28i:CD40L" due to the reversed ICD of CD28; see also exemplary fusion protein embodiments shown as variant 3 in Figure 8 and evaluated in Figures 9-11), in which the ICD of CD28 forms the N-terminus of the fusion protein and the ECD of CD40L forms the C-terminus. TM " or " tm" indicates which transmembrane domain is used in each construct in relation to the fusion protein. For example, "PD-1 TM :BB" or "PD-1 tm :BB" means that the fusion protein contains the transmembrane domain of PD-1, while "PD-1:BB" means that the fusion protein contains the transmembrane domain of PD-1. TM " or "PD-1:BB tm " means that the fusion protein contains the transmembrane domain of 4-1BB. Similarly, "CD28:CD40L tm " or "CD40L tm :CD28" means that the fusion protein contains the transmembrane domain of CD40L, while "CD40L:CD28" means that the fusion protein contains the transmembrane domain of CD40L. TM " or "CD28 TM :CD40L" means that the fusion protein contains the transmembrane domain of CD28.

[0014] Provided herein are fusion proteins comprising the extracellular domain (ECD) and transmembrane domain (TMD) of PD-1 or CD40L (e.g., PD-1) and the intracellular domain (ICD) of 4-1BB. As surprisingly discovered in the context of the present invention, T cells expressing such PD-1:BB fusion proteins are expanded and exhibit a faster proliferation rate compared to T cells expressing PD-1:CD28 fusion proteins, as exemplified in human melanoma xenografts. Also surprisingly discovered in the context of the present invention is that CD40L:CD28 fusion proteins expressed on T cells can activate B cells (trans effect; Figure 10) and support T cell functions (cis effect), such as increased IFN-γ secretion (see Figure 11A) and cytotoxicity (Figure 11B).

[0015] That is, as found in the context of the present invention, fusion proteins comprising the ICD of 4-1BB (CD137) exhibit superior efficacy in the form of increased proliferation rates when expressed in T cells, for example, in human melanoma xenografts, compared to constructs comprising the ICD of CD28.

[0016] Thus, the ECD of the fusion proteins described and provided in connection with the present invention having an ECD derived from PD-1 preferably functions to bind to PD-L1 / 2 on the surface of tumor cells that express PD-L1 as part of an escape mechanism, as is known in the art. Upon binding of the ECD of the fusion protein of the present invention, the ICD of the fusion protein comprising a polypeptide derived from 4-1BB preferably functions to activate signaling molecules, thereby activating the signaling molecules of the host cell (e.g., CD8 + Increases proliferation and / or cytokine secretion of T cells (e.g., T cells).

[0017] Similarly, in accordance with the present invention, the ECD of the fusion proteins described and provided in connection with the present invention having an ECD derived from CD40L preferably functions to bind to CD40 on the surface of tumor cells expressing CD40 as part of an escape mechanism, as known in the art. When bound by the ECD of the fusion protein of the present invention, the ICD of the fusion protein comprising a polypeptide derived from CD28 preferably functions to activate signaling molecules, thereby activating the host cell (e.g., CD8 + Increases proliferation and / or survival of T cells, such as T cells, and / or cytokine secretion and / or cytotoxicity.

[0018] The fusion protein provided in accordance with the present invention may further comprise a CD3ζ domain. This may be particularly applicable when the fusion protein is not expressed in T cells or generally TCR-negative cells, or when a TCR and / or CAR are not co-transduced (or generally not co-expressed) in cells expressing the fusion construct of the present invention. The ICD amino acid sequence of CD3ζ can be obtained from a database known in the art (NP_932170). Generally, CD3ζ can be introduced preferably after the ICD of the 4-1BB or CD28 protein.

[0019] The fusion proteins provided herein may, in particular, comprise an N-terminal ECD containing a polypeptide derived from PD-1, preferably an ECD of PD-1 (e.g., human or mouse, preferably human PD-1), or an ECD containing a polypeptide derived from CD40L. In this context, the term "derived from" specifically means that the polypeptide contained in the ECD comprises at least a portion of PD-1 (e.g., human or mouse, preferably human PD-1), preferably the ECD of PD-1, or at least a portion of CD40L, respectively. As used herein, the term "derived from" PD-1 or CD40L also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, deletions, and / or insertions compared to the native sequence of PD-1 (human or mouse, preferably human PD-1) or CD40L, or a portion thereof (e.g., the ECD). For example, when the ECD is derived from CD40L, it is contemplated in the present invention that the ECD derived from CD40L can comprise or consist of the ECD highlighted in SEQ ID NO: 30 in Figure 1. In this regard, in one embodiment, it can be a soluble portion of CD40L (e.g., amino acids 113-261 of SEQ ID NO: 30; Figure 8). As those skilled in the art will readily recognize, in the ECD of a fusion protein, a signal peptide sequence (recognizable to those skilled in the art and also described in the specific SEQ ID NOs referred to herein) is typically cleaved in the mature protein before, during, or after the fusion protein is integrated into the membrane. Also, according to the present invention, when the ECD is derived from CD40L, it is also possible that the signal peptide can be derived from PD-1 (Figure 8). That is, when referring to the fusion proteins described and provided herein or host cells expressing the fusion proteins, it is always encompassed in accordance with the present invention that the ECD of the fusion protein may lack the respective signal peptide.

[0020] In one embodiment of the invention, the fusion protein comprises an ECD containing a polypeptide derived from human or mouse PD-1, e.g., human PD-1 as set forth in SEQ ID NO: 2 (with or without the signal peptide shown in Figure 1 for SEQ ID NO: 2) or CD40L (highlighted in SEQ ID NO: 30 in Figure 1; see SEQ ID NO: 30 in Figure 1; see SEQ ID NO: 30 in Figure 1; for details, see SEQ ID NO: 30). The fusion protein exhibits binding affinity to PD-L1 / 2 or CD40, respectively, and the fusion protein exhibits binding affinity to native PD-1 (e.g., human PD-L1 / 2), PD-L2, PD-L3, PD-L4, PD-L5, PD-L6, PD-L7, PD-L8, PD-L9, PD-L10, PD-L11, PD-L12, PD-L13, PD-L14, PD-L15, PD-L16, PD-L17, PD-L18, PD-L19, PD-L20, PD-L21, PD-L22, PD-L23, PD-L24, PD-L25, PD-L26, PD-L27, PD-L28, PD-L29 ...9, PD-L29, PD-L29, PD-L20, PD-L21, PD-L22, PD-L23, PD-L24, PD- 1) or CD40L to its respective ligands, PD-L1 / 2 or CD40. In the context of the present invention, for purposes of determining whether a given polypeptide exhibits binding affinity to PD-L1 / 2 or CD40, a polypeptide that has a binding affinity to native human PD-L1 / 2 or CD40 that is at least 0.8-fold, preferably at least 0.9-fold, or more preferably at least 1.0-fold, compared to the binding affinity of native human PD-1 to PD-L1 / 2 or CD40L to CD40, respectively, is considered to exhibit binding affinity to PD-L1 / 2 or CD40, respectively. In this context, the binding affinity of a given polypeptide to PD-L1 / 2 or CD40 can be measured by methods known in the art, and is typically and preferably expressed as a K D This involves measuring the dissociation constant (K D Such methods for measuring protein interactions in this regard are well known in the art and include, for example, ELISA, flow cytometry, surface plasmon resonance, biacore measurements, and the like.

[0021] In one embodiment of the invention, the fusion protein comprises an ECD comprising or consisting of the amino acid sequence of the ECD of PD-1 according to SEQ ID NO: 2 (with or without the signal peptide shown in Figure 1 for SEQ ID NO: 2). In another embodiment of the invention, the fusion protein comprises an ECD comprising or consisting of the amino acid sequence of the ECD of CD40L as highlighted in SEQ ID NO: 30 in Figure 1 (either the entire polypeptide according to SEQ ID NO: 30 or only the soluble fragment according to amino acids 113 to 261 of SEQ ID NO: 30).

[0022] The ECD of the fusion proteins described and provided herein may further comprise a hinge and / or linker region at the C-terminus of the ECD (e.g., between the ECD and TMD of the fusion protein, or between the ECD and ICD of the fusion protein when the TMD is located C-terminal to the ICD) to make the ECD more flexible. Exemplary hinge and / or linker regions are known in the art and include those derived from the constant region (Fc) of an antibody (e.g., IgG1, CD8 alpha) (see, e.g., Shirasu et al., Anticancer Res (2012), 32: 2377-2383 and Cartellieri et al., J Biomed Biotechnol (2010), 956304) (e.g., an IgGFc spacer), a Gly / Ser linker, or a filamin (e.g., a Fil3 spacer). Furthermore, because such linker or hinge regions may cause side effects due to activation of NK cells, which secrete large amounts of inflammatory cytokines, it may be desirable in the context of the present invention for the fusion proteins described and provided herein to not contain either a linker or hinge region. That is, in one embodiment of the present invention, the ECD of the fusion protein does not contain a linker or hinge region. In another embodiment, when the ECD is derived from CD40L and the ICD is derived from CD28, one or more linker or hinge regions, e.g., a Gly / Ser linker plus Fc spacer (e.g., an IgGFc spacer) and / or a filamin linker (e.g., Fil3), are present at the C-terminus of the ECD, e.g., as appropriately highlighted in SEQ ID NO: 27 or 28 in Figure 1.

[0023] The fusion proteins provided herein further comprise a TMD operably linked between the ECD and ICD or linked to the C-terminus of the ICD (e.g., when the ECD is derived from CD40L and the ICD is derived from CD28). Generally, the TMD is not limited to a particular TMD. Preferably, the TMD is capable of stably anchoring the fusion protein to the membrane of a cell (e.g., a T cell) expressing the fusion protein and further capable of binding the ECD to PD-L1 / 2 or CD40, respectively, and, upon binding to PD-L1 / 2 or CD40, capable of inducing signaling of an ICD containing a polypeptide derived from 4-1BB, CD28, or CD40L as described and exemplified herein. In the context of this specification, TMDs may include those derived from CD8(alpha), CD28, ICOS, PD-1, or 4-1BB, among others. In one embodiment, when the ECD is derived from CD40L and the ICD is derived from CD28, the TMD is CD28 (CD40L:CD28 tm ) or CD40L (CD28:CD40L tm ) For example, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27, 28, or 29. The TMD generally may be of any origin, but is preferably murine or human, more preferably human.

[0024] In one embodiment of the invention, the TMD of the fusion protein is not derived from CD8(alpha) and / or ICOS. When the ECD is derived from PD-1 and the ICD is derived from 4-1BB, in one embodiment the TMD is also not derived from CD28.

[0025] In one embodiment of the invention, the TMD of the fusion protein comprises a polypeptide derived from PD-1, 4-1BB (e.g., human or mouse), particularly when the ECD is derived from PD-1 and the ICD is derived from 4-1BB, or when the TMD is derived from CD28, particularly when the ECD is derived from CD40L and the ICD is derived from CD28 (e.g., as highlighted in SEQ ID NO: 27 or 28 in Figure 1). In a specific embodiment of the invention, the TMD of the fusion protein comprises a polypeptide derived from PD-1, e.g., human or mouse PD-1, particularly human PD-1. In this context, the term "derived from" specifically means that the polypeptide contained in the TMD is the TMD of PD-1 or 4-1BB (e.g., human or mouse, preferably human PD-1 or 4-1BB), comprising at least a portion of PD-1, 4-1BB (e.g., human or mouse, preferably human PD-1 or 4-1BB) or CD28, preferably PD-1 or 4-1BB (e.g., PD-1), particularly when the ECD is derived from PD-1 and the ICD is derived from 4-1BB. As used herein, the term "derived from" PD-1, 4-1BB, or CD28 also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions, and / or insertions compared to the native sequence of PD-1, 4-1BB (human or mouse, preferably human PD-1 or 4-1BB) or CD28, or a portion thereof (e.g., the TMD).

[0026] As demonstrated in the context of the present invention, fusion proteins having an ICD derived from 4-1BB generally exhibit increased affinity to host cells (e.g., CD8) compared to similar constructs having an ICD derived from CD28 and an ECD derived from PD-1. + PD-1-derived TMDs exhibit superior proliferation rates of T cells (e.g., T cells). Furthermore, fusion proteins comprising a TMD derived from PD-1 induce faster cytokine (e.g., IFNγ or IL-2) secretion rates in transduced T cells compared to fusion proteins comprising a TMD derived from 4-1BB. Thus, in certain embodiments of the invention, the TMD of the fusion protein comprises or consists of a polypeptide derived from PD-1, e.g., human or mouse PD-1, particularly human PD-1.

[0027] In one embodiment of the invention, the fusion protein comprises a TMD containing a polypeptide derived from human or mouse 4-1BB, e.g., 4-1BB, comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the TMD of human 4-1BB set forth in SEQ ID NO: 6, and the fusion protein is capable of inhibiting CD8 + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + When stimulated with target cells, the CD8 + The proliferation rate of T cells can be increased by measuring CFSE dye dilution, i.e., a decrease in CFSE fluorescence intensity as measured by flow cytometry (as exemplified in FIG. 5 and in the method description "....TCR-D115 T cell proliferation"), or any other suitable method known in the art for determining proliferation, e.g., H 3 It can be quantified by thymidine incorporation, BrdU incorporation, etc. A given fusion protein can be used to quantify retrovirally transduced CD8 + T cell proliferation was observed in CD8 T cells not transduced with the fusion protein. + A fusion protein is considered to be capable of increasing proliferation if its mean fluorescence activity (MFI) is greater than 1.0, e.g., at least 1.2-fold, preferably at least 1.3-fold, and more preferably at least 1.5-fold higher than that of T cells. Using the CFSE dilution method, the difference in proliferation can be calculated as the ratio of mean fluorescence activity (MFI) between T cells without chimeric receptors (mock) and T cells expressing PD-1:BB or PD-1:CD28 mutants. An MFI ratio of 1.0 indicates no difference in proliferation, while an MFI ratio of greater than 1.0 indicates proliferation.

[0028] In one embodiment of the invention, the fusion protein comprises a TMD comprising or consisting of the amino acid sequence of the TMD of 4-1BB according to SEQ ID NO: 6, particularly when the ECD is derived from PD-1 and the ICD is derived from 4-1BB.

[0029] In one embodiment of the invention, particularly when the N-terminal ECD is derived from CD40L and the C-terminal ICD is derived from CD28, the fusion protein comprises a TMD (which may be located at the N-terminus or C-terminus of the ICD, preferably at the N-terminus of the ICD) containing a polypeptide derived from CD28 comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the TMD of CD28 highlighted in SEQ ID NO: 27 or 28 in Figure 1, and the fusion protein is capable of increasing the rate of B cell activation when expressed in TCR-T58 cells, as shown in Figure 10. A fusion protein is considered capable of increasing activation if the increase in B cell activation is more than 1.2-fold, preferably 1.3-fold, and more preferably at least 1.5-fold, compared to the rate of B cell activation by TCR-T58 cells not transduced with the fusion protein.

[0030] In another embodiment of the invention, particularly when the C-terminal ECD is derived from CD40L and the N-terminal ICD is derived from CD28, the fusion protein comprises a TMD (which may preferably be located between the ICD and ECD) containing a polypeptide derived from CD40L comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the TMD of CD40L highlighted in SEQ ID NO:29 in Figure 1, and the fusion protein is capable of increasing the rate of B cell activation when expressed in TCR-T58 cells as shown in Figure 10. A fusion protein is considered capable of increasing activation if the increase in B cell activation is more than 1.2-fold, preferably at least 1.3-fold, and more preferably at least 1.5-fold compared to the rate of B cell activation by TCR-T58 cells not transduced with the fusion protein.

[0031] In one embodiment of the invention, the fusion protein comprises a TMD containing a polypeptide derived from human or mouse PD-1, e.g., PD-1, comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of human PD-1 set forth in SEQ ID NO: 8, and the fusion protein is capable of inhibiting CD8 + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + Upon stimulation with target cells, it is possible to increase the secretion of IFNγ and / or IL-2. + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + The evaluation of whether a given fusion protein can increase the secretion of IFNγ and / or IL-2 upon stimulation with target cells can be performed by methods known in the art and also described and exemplified herein (see also the "Co-culture and Cytokine Assay" exemplified herein below). To evaluate whether a given fusion protein can increase the secretion of IFNγ and / or IL-2, the fusion protein is transduced into retrovirally transduced CD8 + The secretion levels of IFNγ and / or IL-2 of T cells were compared with those of comparable CD8 T cells not transduced with the fusion protein. + The levels of IFNγ and / or IL-2 secreted by CD8 T cells are compared. + T cells (one transduced with the fusion protein and the other untransduced) were transduced with PD-L1 / 2 T cells as described and exemplified herein. + The target cells are stimulated with CD8 transduced with the fusion protein, and the secretion levels of IFNγ and / or IL-2 are subsequently measured. + T cells and untransduced control CD8 +T cells are usually derived from the same donor. For example, transgenic human T cells may be retrovirally transduced to express a fusion protein containing the 4-1BB-derived polypeptide and then cultured with HEK / Tyr or HEK / Tyr / PD-L1 cells at a 1:2 ratio. Coculture supernatants may then be collected after 16 hours and analyzed by sandwich ELISA (BD) or Bio-Plex (Bio-Rad) according to the manufacturer's protocol. Transduced TCRs may express CD8 + It only functions in T cells and CD8 + / CD4 + When the T cell ratio is varied, the following formula can be applied to calculate the amount of measured cytokines relative to the TCR ratio in the cell suspension: + CD8 + Normalization can be performed to the percentage of T cells (determined by flow cytometry):

number

[0032] Methods for measuring the secretion levels of IFNγ and IL-2 are well known in the art and are also exemplified herein, and include, among others, ELISA, Bio-Plex, intracellular flow cytometry (ICS), etc. A given fusion protein can be used to measure the secretion levels of CD8 + The secretion levels of IFNγ and IL-2 of T cells were significantly higher in CD8 T cells that were not transduced with the fusion protein. + A fusion protein is considered to be capable of increasing the secretion levels of IFNγ and IL-2 if they are at least 1.2-fold, preferably at least 1.3-fold, more preferably at least 1.5-fold higher compared to T cells.

[0033] In one embodiment of the invention, the fusion protein comprises a TMD comprising or consisting of the amino acid sequence of the TMD of PD-1 according to SEQ ID NO: 8. In another embodiment, particularly when the N-terminal ECD is derived from CD40L and the C-terminal ICD is derived from CD28, the fusion protein comprises a TMD (which may be located N- or C-terminal to the ICD, preferably N-terminal to the ICD), which comprises or consists of the amino acid sequence of the TMD of CD28 highlighted in SEQ ID NO: 27 or 28 in Figure 1. In yet another embodiment of the invention, particularly when the C-terminal ECD is derived from CD40L and the N-terminal ICD is derived from CD28, the fusion protein comprises a TMD (which may preferably be located between the ICD and ECD), which comprises or consists of the amino acid sequence of the TMD of CD40L highlighted in SEQ ID NO: 29 in Figure 1.

[0034] The fusion proteins provided herein further comprise an ICD operably linked to the C-terminus of the TMD (e.g., in the case of a fusion protein in which the ECD is derived from PD-1 and the ICD is derived from 4-1BB) or operably linked to the C-terminus or N-terminus of the TMD (e.g., in the case of a fusion protein in which the ECD is derived from CD40L and the ICD is derived from CD28). The ICD of the fusion proteins of the present invention contains a polypeptide derived from 4-1BB (CD137) or CD28. In this context, the term "derived from" specifically means that the polypeptide contained in the ICD comprises at least a portion of 4-1BB (e.g., human or mouse, preferably human 4-1BB), preferably the ICD of (human) 4-1BB, or at least a portion of CD28. As used herein, the term "derived from" 4-1BB or CD28 also allows for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or fewer amino acids to be substituted, deleted, and / or inserted compared to the native sequence of 4-1BB (human or mouse, preferably human 4-1BB) or CD28, or a portion thereof (e.g., ICD). In certain embodiments of the invention, the ICD of the fusion protein comprises a polypeptide derived from the ICD of 4-1BB, e.g., human or mouse 4-1BB, specifically human 4-1BB. In another embodiment of the invention, particularly when the ECD is derived from CD40L and the ICD is derived from CD28, the fusion protein comprises a polypeptide derived from the ICD of CD28, e.g., comprising or consisting of an amino acid sequence highlighted in SEQ ID NO: 27, 28, or 29 of Figure 1 (particularly when the ECD is located at the N-terminus of the fusion protein and the ICD is located at the C-terminus of the fusion protein (the C-terminus or N-terminus of the TMD, preferably the C-terminus of the TMD); and particularly when the ICD is located at the N-terminus of the fusion protein and the ECD is located at the C-terminus of the fusion protein, according to SEQ ID NO: 29).

[0035] In one embodiment of the present invention, the fusion protein comprises an ICD containing a polypeptide derived from human or mouse 4-1BB, e.g., 4-1BB, comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the ICD of human 4-1BB set forth in SEQ ID NO: 4, and the fusion protein is capable of inhibiting CD8 + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + When stimulated with target cells, the CD8 + The rate of T cell proliferation can be increased.

[0036] In another embodiment of the present invention, the fusion protein comprises an ICD containing a polypeptide derived from CD28 comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the ICD of CD28 highlighted in SEQ ID NO: 27, 28, or 29 in FIG. 1 (particularly when the ECD is located at the N-terminus of the fusion protein and the ICD is located at the C-terminus of the fusion protein (the C-terminus or N-terminus of the TMD, preferably the C-terminus of the TMD); and particularly when the ICD is located at the N-terminus of the fusion protein and the ECD is located at the C-terminus of the fusion protein, according to SEQ ID NO: 29), and the fusion protein is capable of increasing the rate of B cell activation when expressed in TCR-T58 cells shown in FIG. 10. A fusion protein is considered to be capable of increasing activation if the increase in B cell activation is greater than 1.2-fold, preferably at least 1.3-fold, and more preferably at least 1.5-fold compared to the rate of B cell activation by TCR-T58 cells not transduced with the fusion protein.

[0037] In one embodiment of the invention, the fusion protein comprises an ICD comprising or consisting of the amino acid sequence of the ICD of 4-1BB according to SEQ ID NO:4.

[0038] In one embodiment of the invention, the fusion proteins provided and described herein comprise or consist of an ECD derived from PD-1, a TMD derived from 4-1BB, and an ICD derived from 4-1BB. In one embodiment, all domains are derived from the corresponding human domains. In a specific embodiment, the fusion proteins of the invention comprise or consist of an amino acid sequence comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 22 (with or without the signal peptide as shown in Figure 1 for Figure 22), wherein the fusion proteins exhibit PD-L1 / 2 binding affinity as described herein, and wherein the fusion proteins bind to CD8 + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + When stimulated with target cells, the CD8 + The proliferation rate of T cells can be increased. In a further specific embodiment of the invention, the fusion protein comprises or consists of amino acids according to SEQ ID NO: 22 (with or without the signal peptide shown in Figure 1 for SEQ ID NO: 22).

[0039] In another embodiment of the invention, the fusion proteins provided and described herein comprise or consist of an ECD derived from CD40L, a TMD derived from CD28 or CD40L (CD28 when the ECD is located at the N-terminus of the fusion protein and the ICD is located at the C-terminus of the fusion protein (either the C-terminus or the N-terminus of the TMD, preferably the N-terminus of the TMD); CD40L when the ICD is located at the N-terminus of the fusion protein and the ECD is located at the C-terminus of the fusion protein), and an ICD derived from CD28. In one embodiment, all domains are derived from the corresponding human domains. In particular embodiments, a fusion protein of the invention comprises or consists of an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO:27, 28, or 29 in Figure 1 (for SEQ ID NOs:27 and 28, with or without the signal peptide shown in Figure 1 for 27 or 28; for SEQ ID NO:29, compared to the complete polypeptide of SEQ ID NO:29 or a soluble fragment thereof of amino acids 113 to 261 of SEQ ID NO:29), wherein the fusion protein exhibits a CD40 binding affinity as described herein, and wherein the fusion protein is capable of increasing the rate of B cell activation when expressed in TCR-T58 cells as shown in Figure 10. In further particular embodiments of the invention, a fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:27, 28, or 29 (with or without the signal peptide shown in Figure 1 for SEQ ID NO:27, 28, or 29).

[0040] In one embodiment of the invention, the fusion proteins provided and described herein comprise or consist of an ECD derived from PD-1, a TMD derived from PD-1, and an ICD derived from 4-1BB. In one embodiment, all domains are derived from the corresponding human domains. In a specific embodiment, the fusion proteins of the invention comprise or consist of an amino acid sequence comprising an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO:24 (with or without the signal peptide as shown in Figure 1 for Figure 24), wherein the fusion proteins exhibit PD-L1 / 2 binding affinity as described herein, and wherein the fusion proteins bind to CD8 + When T cells are retrovirally transduced, the CD8 + T cells expressing PD-L1 / L2 + Upon stimulation with target cells, it is possible to increase the secretion of IFNγ and / or IL-2. In this regard, a given fusion protein may be capable of binding to CD8 + In addition to increasing the proliferation rate of T cells, the fusion protein also enhances the proliferation of retrovirally transduced CD8 + To assess whether the fusion proteins can increase the secretion levels of IFNγ and / or IL-2 in T cells, we transfected equivalent non-transduced CD8 T cells with the fusion proteins. + The proliferation rate of T cells is compared with the secretion levels of IFNγ and / or IL-2. Methods for assessing proliferation and IFNγ / IL-2 secretion rates are known in the art and are described and exemplified herein above and below.

[0041] In a further particular embodiment of the invention, the fusion protein comprises or consists of amino acids according to SEQ ID NO: 24 (with or without the signal peptide shown in Figure 1 for SEQ ID NO: 24).

[0042] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described herein.

[0043] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0044] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0045] The terms "about" or "approximately," as used herein, mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0046] Throughout the following specification and claims, unless the context otherwise requires, the term "comprises" and variations thereof will be understood to mean the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprises" may be replaced by the terms "containing" or "including," or sometimes, as used herein, the term "having."

[0047] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0048] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0049] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0050] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), cited throughout the document herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is contradictory or inconsistent with the present specification, the present specification supersedes any such material.

[0051] As used herein, a "conservative" substitution refers to a substitution listed under the heading "exemplary substitutions" in Table 1 below. A "highly conservative" substitution refers to a substitution shown under the heading "preferred substitutions" in Table I below.

[0052] [Table 1]

[0053] The term "polypeptide" is used herein equivalently to the term "protein." Proteins (including fragments thereof, preferably biologically active fragments and peptides, usually having fewer than 30 amino acids) comprise one or more amino acids linked together via covalent peptide bonds (resulting in a chain of amino acids). The term "polypeptide," as used herein, describes a group of molecules typically comprising more than 10 amino acids. Polypeptides can also form multimers, such as dimers, trimers, and higher oligomers, i.e., consisting of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. Accordingly, the corresponding higher-order structures of such multimers are referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins that are modified, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well known in the art.

[0054] The term "amino acid" or "amino acid residue," as used herein, refers to an amino acid having an art-recognized definition, such as a proteinogenic amino acid typically selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. Generally, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, VaI); negatively charged side chains (e.g., Asp, GIu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, GIy, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0055] Generally, as used herein, the term "fusion protein" refers to a protein made up of polypeptide moieties derived from different sources. It can therefore also be understood as a "chimeric protein," "chimeric construct," "fusion construct," etc. Fusion proteins generally refer to proteins made through the joining of two or more genes (or preferably cDNAs) that originally encoded separate proteins. Translation of this fusion gene (or fusion cDNA) preferably results in a single polypeptide that possesses functional properties derived from each of the original proteins. Recombinant fusion proteins are artificially produced by recombinant DNA technology for use in biological research or treatment. Further details regarding the production of the fusion proteins of the present invention are known in the art and are described and exemplified herein.

[0056] The present invention further relates to nucleic acid molecules encoding the fusion proteins provided and described herein. The present invention also relates to nucleic acid molecules encoding only a portion of the fusion proteins described and provided herein, for example, encoding only the ECD, TMD, and / or ICD of the fusion protein. For example, the present invention relates to nucleic acid molecules according to SEQ ID NOs: 1, 3, 5, 7, 21, or 23, or combinations thereof linked via nucleic acid linkages, so long as they encode the fusion proteins of the present invention or portions thereof (e.g., the ECD, TMD, and / or ICD). The present invention also relates to nucleic acid molecules in which 30, 27, 24, 21, 18, 15, 12, 9, 6, 3, or 0 nucleotides have been substituted (preferably silent mutations that do not result in a change in the translated amino acid), deleted, or inserted compared to the nucleic acid molecules according to SEQ ID NOs: 1, 3, 5, 7, 21, or 23, or combinations thereof linked via nucleic acid linkages, so long as they encode the fusion proteins of the present invention or portions thereof (e.g., the ECD, TMD, and / or ICD). In principle, the present invention preferably relates to nucleic acids encoding the fusion proteins specifically described and embodied herein. Generally, SEQ ID NOs: 13 and 14 show the nucleic acid and amino acid sequences, respectively, of human CD28, with the primary transcript of CD28 being shown. Furthermore, given that CD28 also exists in additional splice variants (e.g., in-frame donor splice variants or variants lacking in-frame coding exons), such variants or portions thereof (especially their TMDs) are also encompassed as part of the fusion proteins described and provided herein. Generally, SEQ ID NOs: 13 and 14 show the nucleic acid and amino acid sequences, respectively, of human CD28, with the primary transcript of CD28 being shown.

[0057] As used herein, unless specifically defined otherwise, the terms "nucleic acid" or "nucleic acid molecule" are used synonymously with "oligonucleotide," "nucleic acid strand," "polynucleotide," etc., and refer to a polymer containing one, two, or more nucleotides. The term "nucleic acid molecule" refers to the sequence of bases, including purine and pyrimidine bases, contained in a polynucleotide, which bases represent the primary structure of a nucleic acid molecule. As used herein, the term "nucleic acid molecule" includes all types of nucleic acids, including DNA, cDNA, genomic DNA, RNA, synthetic forms of DNA, and mixed polymers containing two or more of these molecules, and preferably relates to DNA and cDNA. As those skilled in the art will readily recognize, the nucleic acid sequences provided herein represent DNA sequences and also include the corresponding RNA sequences in which T is replaced by U. The term "nucleic acid molecule" generally includes sense and antisense strands. A "nucleic acid molecule" may further include non-natural or derivatized nucleotide bases in addition to natural or artificial nucleotide analogs, e.g., to protect the nucleic acid molecule from endonucleases and / or exonucleases, as will be readily recognized by one of skill in the art.

[0058] The present invention further relates to vectors comprising the nucleic acid molecules described and provided herein.

[0059] The term "vector," as used herein, generally includes all types of linear or circular nucleic acid molecules capable of autonomous replication and suitable host cells. Such vectors include, but are not limited to, plasmids, cosmids, phages, viruses (e.g., adenoviral, adeno-associated viral, lentiviral, or preferably retroviral vectors), and other vectors or shuttles known in the art suitable for transporting and introducing genes into host cells for stable or transient translation and constitutive or conditional expression of the fusion proteins of the present invention in the host cells. Vectors are not usually integrated into the cellular genome, but may be integrated. Vectors of the present invention comprising the nucleic acid molecules described and provided herein preferably stably express the fusion proteins of the present invention in host cells (expression vectors). Vectors of the present invention may further include marker genes, promoter and / or enhancer sequences (operably linked to the nucleic acid molecules of the present invention), origins of replication suitable for the respective host cells, restriction enzyme sites, multiple cloning sites, markers, and additional functional units known in the art. A vector may be introduced into a host cell via a shuttle, such as a virus (which may itself be considered a vector), or may directly transform or transduce the host cell. A vector is preferably adapted to suit the respective host cell it will transform or transduce. Those skilled in the art will readily understand that different host cells require different types of vectors. For example, as shown herein, the vector (plasmid) pGEM is a suitable vector for transforming bacterial cells, while the retroviral vector pMP71 is suitable for transducing eukaryotic cells (e.g., T cells).

[0060] In one embodiment of the invention, the vector of the invention is a viral vector, such as a retroviral vector or a lentiviral vector, such as a retroviral vector. Examples of suitable retroviral vectors are known in the art and include, for example, pMP71-PRE (Leisegang, K Mol Med (2008), 86(5): 573-583), SAMEN CMV / SRa, LZRS-id3-IHRES (Heemskerk et al., J. Exp. Med. 186 (1997), 1597-1602), FeLV (Neil et al., Nature 308 (1984), 814-820), SAX (Kantoff et al., Proc. Natl. Acad. Seis. USA 83 (1986), 6563-6567), pDOL (Desiderio, J. Exp. Med. 167 (1988), 372-388), N2 (Kasid et al., Proc. Natl. Acad. Sei. USA 87 (1990), 473- 477), LNL6 (Tiberghien et al., Blood 84 (1994), 1333-1341), pZipNEO (Chen et al., J. Immunol. 153 (1994), 3630-3638), LASN (Mullen et al., Hum. Gene Ther. 7 (1996), 1123- 1129), pGIXsNa (Taylor et al., J. Exp. Med. 184 (1996), 2031-2036), LCNX (Sun et al., Hum. Gene Ther. 8 (1997), 1041-1048), SFG (Gallardo et al., Blood 90 (1997), LXSN (Sun et al., Hum. Gene Ther. 8 (1997), 1041-1048), SFG (Gallardo et al., Blood 90 (1997), 952- 957), HMB-Hb-Hu (Vieillard et al., Proc. Natl. Acad. Sei.USA 94 (1997), 11595-11600), pMV7 (Cochlovius et al., Cancer Immunol. Immunother. 46 (1998), 61-66), pSTITCH (Weitjens et al., Gene Ther 5 (1998), 1195-1203), pLZR (Yang et al., Hum. Gene Ther. 10 (1999), 123-132), pBAG (Wu et al., Hum. Gene Ther. 10 (1999), 977-982), rKat.43.267bn (Gilham et al., J. Immunother. 25 (2002), 139-151), pLGSN (Engels et al., Hum. Gene Ther. 14 (2003), pMP71 (Engels et al., Hum. Gene Ther. 14 (2003), 1155-1168), pGCSAM (Morgan et al., J. Immunol. 171 (2003), 3287-3295), pMSGV (Zhao et al., J. Immunol. 174 (2005), 4415-4423), or pMX (de Witte et al., J. Immunol. 181 (2008), 5128-5136). In a specific embodiment of the invention, the vector is pMP71-PRE or pMP71.

[0061] The present invention further relates to host cells comprising the nucleic acid molecules or vectors described and provided herein. In one embodiment, a host cell of the invention is transduced or transformed with a nucleic acid molecule or vector described and provided herein.

[0062] Generally, as used herein, unless specifically defined otherwise, the terms "transduced" or "transformed" (as well as "transduction" or "transformation"), etc., may be used interchangeably and generally refer to any type of introduction of nucleic acid molecules and / or vectors into host cells, regardless of the type of host cell and regardless of the method of introduction (e.g., (chemical) transformation, (viral) transformation, electroporation, transfection, etc.).

[0063] The nucleic acid molecule and / or vector may be stably integrated into the genome of the host cell or may be extrachromosomal (i.e., transient expression). Examples of suitable methods for achieving transient expression in a host cell are known in the art and include mRNA transfection. In one embodiment, the nucleic acid molecule and / or vector is stably integrated into the genome.

[0064] Host cells described and provided herein containing the nucleic acid molecules or vectors described and provided herein are preferably capable of stably or transiently (e.g., stably) expressing (constitutively or conditionally) the fusion proteins of the invention. Host cells generally can be transduced or transformed by any method with any suitable nucleic acid molecule or vector. In one embodiment, a host cell is transduced with a retroviral or lentiviral (e.g., retroviral) vector containing a nucleic acid molecule encoding the fusion protein of the invention or a portion thereof (e.g., ECD, TMD, and / or ICD).

[0065] In one embodiment, a host cell of the invention is transduced with a retroviral vector comprising a nucleic acid molecule encoding the fusion protein of the invention or a portion thereof (e.g., ECD, TMD, and / or ICD), and the fusion protein or a portion thereof is stably (constitutively or conditionally) expressed. Preferably, the host cell then stably expresses the fusion protein in its membrane, with the ECD of the fusion protein of the invention facing the surface, the TMD (predominantly) embedded in the membrane, and the ICD facing the cytoplasm.

[0066] In the context of the present invention, a host cell comprising a nucleic acid molecule or vector described and provided herein refers to a genetically engineered cell in which the nucleic acid molecule or vector has been transduced, transformed, or otherwise introduced into the host cell. As previously described, the host cell of the present invention can be a cell that transiently or stably expresses a fusion protein of the present invention. For example, a nucleic acid molecule encoding a fusion protein of the present invention can be stably integrated into the genome of the cell by retroviral or lentiviral (e.g., retroviral) transduction. The PD-1-BB fusion protein is expressed in the membrane of the transduced cells provided herein. The ECD of the PD-1 portion of the fusion protein is located on the cell surface, while the TMD and ICD intracellularly are membrane-bound but undetectable on the cell surface. Detection of the ECD of the PD-1 polypeptide can be performed, for example, by ELISA or flow cytometry, or by microscopy, using antibodies or other binding molecules that specifically bind to the ECD of PD-1 described herein. The transduced cells of the present invention can be, for example, CD8 + T cells, CD4 + In addition to T cells, double-negative α / β T cells, NK (natural killer) cells, γδ cells, macrophages, and dendritic cells, cells suitable for storing and / or replicating the nucleic acid molecules or vectors of the invention may include bacterial cells (e.g., E. coli) and further eukaryotic cells. In one embodiment, the host cell of the invention is a T cell, e.g., a CD8 + T cells.

[0067] Host cells of the invention can be transduced with a nucleic acid molecule or vector encoding a fusion protein as described and provided herein. Preferably, the host cells provided and described herein can be co-transduced with an additional nucleic acid molecule, such as a nucleic acid molecule encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Such co-transduction (or other methods of introducing nucleic acid molecules into cells as described and exemplified herein) are known in the art and are also described and exemplified herein.

[0068] Examples of suitable host cells according to the present invention are T cells, e.g., CD8 + T cells, CD4 + Suitable cells for storing and / or replicating the nucleic acid molecules or vectors of the present invention include, but are not limited to, T cells, TCR T cells such as (but not limited to) TCR-T58 or TCR-D115, double-negative α / β T cells, NK (natural killer) cells, γδ T cells, macrophages, dendritic cells, as well as bacterial cells (e.g., E. coli) and further eukaryotic cells. The cells may be autologous or non-autologous, preferably autologous. The cells may also be allogeneic or non-allogeneic, as will be readily apparent to those skilled in the art. In one embodiment, the host cells of the present invention are CD8 + T cells.

[0069] The present invention also provides a method for preparing the host cells of the invention described and provided herein, comprising the steps of: (1) transducing or transforming the host cell with a nucleic acid molecule or vector described and provided herein; (2) culturing the transduced host cells of step (1) in a suitable medium to grow the cells and express the fusion protein encoded by the nucleic acid molecule or the vector; (3) recovering the host cells from the medium; and The present invention relates to a method comprising:

[0070] In a preferred embodiment of the present invention, the host cells are transduced or transformed ex vivo. Cells (e.g., CD8 + T cells, CD4 + Methods for obtaining, isolating, and culturing T cells, such as (but not limited to) TCR T cells, including TCR-T58 or TCR-D115, are known in the art and include, inter alia, blood collection and bone marrow removal.

[0071] According to the methods of the present invention, host cells may be transduced or transformed or otherwise provided with the nucleic acid molecules or vectors described and provided herein by any method known in the art. Such methods include, inter alia, (chemical) transformation, (viral) transduction, electroporation, transfection, etc. In one embodiment, host cells are transduced with a retroviral vector.

[0072] The host cells prepared according to the present invention can be any host cell described herein. In one embodiment, the host cells are T cells, e.g., CD8 + T cells, CD4 + T cells, TCR T cells such as (but not limited to) TCR-T58 or TCR-D115.

[0073] The present invention also relates to host cells obtainable by the preparation methods provided herein.

[0074] The present invention further relates to pharmaceutical compositions comprising the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided by the present invention. Such pharmaceutical compositions are suitable for administration to patients (preferably human patients), particularly donors of said host cells. Accordingly, the present invention also relates to methods for treating diseases or disorders by administering pharmaceutical compositions comprising the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided by the present invention.

[0075] The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier and additional ingredients, such as herbal medicines. The pharmaceutical compositions are particularly useful for treating diseases or disorders associated with the expression of PD-1 ligands (e.g., PD-L1 or PD-L2) and / or CD40. Such diseases and disorders are known to those skilled in the art and specifically include (but are not limited to) various types of cancer, such as lung cancer, gastric cancer, renal cell carcinoma, colon cancer, breast cancer, ovarian cancer, urothelial carcinoma, melanoma, pancreatic cancer, myeloma, Hodgkin's lymphoma, retinoblastoma, leukemia, cervical cancer, esophageal cancer, glioma, non-Hodgkin's lymphoma, hepatocellular carcinoma, oral cancer, and others. Additional diseases and disorders that can be treated by the pharmaceutical compositions provided herein include (chronic) viral infections and (chronic) inflammation, particularly when PD-L1 / L2 and / or CD40 are expressed.

[0076] The present invention further relates to kits or kit-in-parts comprising the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided in connection with the present invention.

[0077] The present invention further relates to the following items: (1) (a) an extracellular domain comprising at its N-terminus a polypeptide derived from PD-1; (b) transmembrane domain and; (c) an intracellular domain comprising a polypeptide derived from 4-1BB at its C-terminus; A fusion protein comprising: (2) The fusion protein according to item 1, wherein the transmembrane domain comprises a polypeptide derived from PD-1 or 4-1BB, preferably PD-1. (3) The fusion protein according to item 1 or 2, further comprising a CD3ζ domain. (4) The fusion protein according to any one of items 1 to 3, wherein the extracellular domain does not include a linker or hinge domain. (5) The fusion protein according to any one of items 1 to 4, wherein the PD-1-derived polypeptide contained in the extracellular and / or transmembrane domain is a polypeptide derived from human PD-1. (6) The fusion protein according to any one of items 1 to 5, wherein the 4-1BB-derived polypeptide contained in the transmembrane and / or intracellular domain is a polypeptide derived from human 4-1BB. (7) The fusion protein of any one of Items 1 to 6, wherein the extracellular domain containing the PD-1-derived polypeptide comprises an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 2, and the fusion protein exhibits PD-L1 / 2 binding affinity. (8) The intracellular domain containing the polypeptide derived from 4-1BB comprises an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 4, and the fusion protein is + When retrovirally transduced into T cells, the CD8 + T cells expressing PD-L1 / 2 + Upon stimulation with target cells, the CD8 + 8. The fusion protein according to any one of items 1 to 7, which is capable of increasing the proliferation rate of T cells. (9) The transmembrane domain containing the PD-1-derived polypeptide comprises an amino acid sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 8, and the fusion protein is + When retrovirally transduced into T cells, the CD8 + T cells expressing PD-L1 / 2 + 9. The fusion protein according to any one of items 2 to 8, which is capable of increasing the secretion of IFNγ and / or IL-2 upon stimulation with target cells. (10) The fusion protein according to any one of items 1 to 9, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 2. (11) The fusion protein according to any one of items 1 to 10, wherein the intracellular domain comprises the amino acid sequence of SEQ ID NO: 4. (12) The fusion protein according to any one of items 1 to 11, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 8. (13) A nucleic acid molecule encoding the fusion protein according to any one of items 1 to 12. (14) A vector comprising the nucleic acid molecule according to item 13. (15) A host cell comprising the nucleic acid molecule according to Item 13 or the vector according to Item 14. (16) The host cell according to item 15, transfected with the nucleic acid molecule according to item 13 or the vector according to item 14. (17) The host cell according to item 15 or 16, wherein the nucleic acid molecule or the vector is stably integrated into the genome of the host cell. (18) The host cell according to item 16 or 17, which is transduced via retroviral transduction. (19) The host cell according to any one of items 15 to 18, which stably expresses a fusion protein encoded by the nucleic acid molecule according to item 13. (20)CD8 + 20. The host cell of any one of items 15 to 19, which is a T cell. (twenty one) (1) transducing a host cell with the nucleic acid molecule according to item 13 or the vector according to item 14; (2) culturing the transduced host cells of step (1) in a suitable medium to grow the cells and express the fusion protein encoded by the nucleic acid molecule or the vector; (3) recovering the host cells from the medium; and 21. A method for preparing the host cell according to any one of items 15 to 20, comprising: (22) A host cell obtainable by the method according to item 21. (23) A pharmaceutical composition comprising the fusion protein according to any one of items 1 to 12, the nucleic acid molecule according to item 13, the vector according to item 14, and / or the host cell according to any one of items 15 to 20 or 22. (24) The fusion protein according to any one of items 1 to 12, the nucleic acid molecule according to item 13, the vector according to item 14, the host cell according to any one of items 15 to 20 or 22, or the pharmaceutical composition according to item 23 for use in the treatment of cancer and chronic viral infections. (25) A kit or kit-in-part comprising the fusion protein according to any one of items 1 to 12, the nucleic acid molecule according to item 13, the vector according to item 14, and / or the host cell according to any one of items 15 to 20 or 22. [Brief explanation of the drawings]

[0078] General legend for arrays, unless otherwise specified: Bold: signal peptide (usually cleaved in the mature protein); underlined: TMD; italic and bold: ICD; capital letters in nucleotide sequence: codon-optimized sequence [Figure 1]SEQ ID NO: 1: nucleic acid sequence ECD human PD-1 SEQ ID NO: 2: amino acid sequence ECD human PD-1 SEQ ID NO: 3: nucleic acid sequence ICD human 4-1BB SEQ ID NO: 4: amino acid sequence ICD human 4-1BB SEQ ID NO: 5: nucleic acid sequence TMD human 4-1BB SEQ ID NO: 6: amino acid sequence TMD human 4-1BB SEQ ID NO: 7: nucleic acid sequence TMD human PD-1 SEQ ID NO: 8: amino acid sequence TMD human PD-1 SEQ ID NO: 9: nucleic acid sequence human PD-1 SEQ ID NO: 10: amino acid sequence human PD-1 SEQ ID NO: 11: nucleic acid sequence human 4-1BB SEQ ID NO: 12: amino acid sequence human 4-1BB SEQ ID NO: 13: nucleic acid sequence human CD28 SEQ ID NO: 14: amino acid sequence human CD28 SEQ ID NO: 15: nucleic acid sequence mouse PD-1 SEQ ID NO: 16: amino acid sequence mouse PD-1 SEQ ID NO: 17: nucleic acid sequence mouse 4-1BB SEQ ID NO: 18: amino acid sequence mouse 4-1BB SEQ ID NO: 19: nucleic acid sequence mouse CD28 SEQ ID NO: 20: amino acid sequence mouse CD28 SEQ ID NO: 21: nucleic acid sequence human PD-1:BBTM SEQ ID NO: 22: amino acid sequence human PD-1:BBTM SEQ ID NO: 23: nucleic acid sequence human PD-1TM:BB SEQ ID NO: 24: amino acid sequence human PD-1TM:BB SEQ ID NO: 25: nucleic acid sequence human PD-1:CD28TM SEQ ID NO: 26: amino acid sequence human PD-1:CD28TM SEQ ID NO: 27: amino acid sequence CD40L:CD28tm with Gly / Ser (G / S) linker and IgGFc spacer;PD1SP (PD-1-derived signal peptide, first underlined sequence portion), CD40L-derived ECD (aa 113 to 261 of SEQ ID NO: 30), GS-linker (second underlined sequence portion), IgGFc (bold spacer), CD28TM (third underlined sequence portion), CD28-derived ICD (aa 148 to 220, bold italics) SEQ ID NO: 28: Gly / Ser CD40L:CD28tm amino acid sequence with (G / S) linker and Fil3 spacer: PD1SP (signal peptide from PD-1, first underlined sequence portion), ECD from CD40L (aa 113-261 of SEQ ID NO: 30), GS-linker (second underlined sequence portion), Fil3 (bold spacer), CD28TM (third underlined sequence portion), ICD from CD28 (aa 148-220, bold italics). SEQ ID NO: 29: CD28 with reverse CD28 ICD at the N-terminus, a short CD40 ICD fragment, TMD from CD40L, and ECD from CD40L at the C-terminus: CD40Ltm, CD40L (aa 14-261 of SEQ ID NO: 30), CD28ICD reverse (bold italics; aa 180-220). SEQ ID NO: 30: native CD40L sequence·ICD (bold italics)·TM (underlined)·ECD; [Figure 2] Design of Chimeric PD-1 Costimulatory Receptors Amino acids (aa) correspond to the respective human parent proteins. Note that for the PD-1 ECD, the signal peptide sequence has been removed to show the mature protein. [Figure 3]Expression of chimeric receptors in TCR-T58+ CD8+ T cells after retroviral transduction. Human T cells stably expressing the HLA-A2-restricted tyrosinase-specific T cell receptor TCR-T58 were retrovirally transduced with vectors encoding the indicated chimeric receptors. For later use, T cells were frozen 15 days after transduction. After thawing the T cells, and before using them in coculture experiments (cultured in medium containing 50 U / mL IL-2 for 3 days), receptor expression on the T cell surface was assessed by flow cytometry. Representative FACS histograms showing the surface expression of PD-1:28tm, PD-1:BBtm, and PD-1tm:BB, as determined by anti-PD-1 staining, are shown. Numbers indicate the % of receptor-positive cells and the corresponding MFI. The black histograms and black numbers correspond to isotype staining, while the red histograms and red numbers correspond to PD-1 staining. [Figure 4] Effect of Chimeric Receptors on TCR-Induced Cytokine Secretion. TCR-T58+ T cells expressing the indicated chimeric receptors (see Figure 3) were co-cultured with HEK / tyr or HEK / tyr / PD-L1 for 16 hours. Supernatants were removed, and IFNγ and IL-2 contents were determined by ELISA. Graphs show the effect of chimeric receptor expression on IFNγ (left) and IL-2 (right) secretion. For each experiment, the x-fold change in cytokines between co-culture with HEK / tyr and co-culture with HEK / tyr / PD-L1 was calculated. The mean x-fold change was determined from all experiments performed (n=2-3) and presented in a bar graph. Error bars represent SEM. Statistical analysis was performed using two-way ANOVA (acc. to Sidak). *p<0.02 ***p<0.0002; ****p<0.0001; ns=not significant. [Figure 5]Effect of chimeric receptors on T cell proliferation in the setting of human melanoma xenografts in NSG mice. SK-Mel23 human melanoma cells expressing peptide-MHC complexes (pMHC) for TCR-D115 T cells (HLA-A2 / tyrosinase) were injected sc into the flanks of immunodeficient NSG mice. CFSE-labeled TCR-T58+ T cells lacking co-receptor expression (mock transduction) and expressing the chimeric receptors PD-1:BB or PD-1:CD28, respectively, were injected intratumorally into established xenografts (802 mm3, SEM ± 83, approximately 17 days old). At 1, 2, 4, 6, and 11 days after T cell injection, tumors were harvested, dissociated into single-cell suspensions, and used for flow cytometry. As a means of determining the proliferation history in the tumor environment, we assessed the intensity of CFSE staining in T cells (CD8+, CD4+, and CD4-CD8- double-negative (dbl-) cells). TCR-D115+ CD8+ T cells are activated by tumor cells and can acquire the dbl- phenotype upon activation. CD8+ and dbl- T cells exhibit comparable CFSE dilution and are represented as a single population. CD4+ T cells are unable to recognize melanoma cells and therefore are unable to proliferate in a pMHC-specific manner. CFSE dilution in CD4+ T cells was not observed until 6 days after T cell infusion and was most likely due to cytokines produced by activated CD8+ T cells (not shown). Representative histograms of CFSE staining of CD8+ / dbl- T cells 1 and 2 days after T cell infusion are shown. Each histogram corresponds to one tumor from one mouse. Numbers indicate the mean CFSE fluorescence intensity. The vertical axis indicates the original CFSE intensity, which is indicative of cells that had not yet initiated proliferation. On days 1 and 2, CD8+ / dbl- T cells that co-expressed the chimeric receptor PD-1tm:BB were observed to have lower CFSE intensity compared to CD8+ / dbl- T cells lacking the chimeric receptor or expressing the PD-1:CD28tm receptor, respectively. On day 4, CD8+ / dbl- T cells showed comparable CFSE dilution independent of chimeric receptor expression (not shown).The early dilution of CFSE by T cells expressing PD-1tm:BB indicates that these T cells reacted more strongly with tumor cells and initiated proliferation earlier than T cells without the chimeric receptor or T cells expressing the chimeric receptor PD-1:CD28tm. The top two lines (green) are TCR-D115 / PD-1tm:BB, the middle two lines (red) are TCR-D115 / PD-1:CD28tm, and the bottom two lines (blue) are TCR-D115 / Mock (control). [Figure 6] SK-Mel23 human melanoma cells expressing peptide-MHC complexes (pMHC) for TCR-D115 and TCR-T58 T cells (HLA-A2 / tyrosinase) were injected sc into the flanks of immunodeficient NSG mice. CFSE-labeled TCR-D115 T cells lacking co-receptor expression (mock) and those expressing the chimeric receptor PD-1tm:BB were injected intratumorally into established xenografts (802 mm3, SEM ± 83, approximately 17 days old). Tumor volume was measured using calipers before T cell injection and on days 2 and 7 after injection. Tumor volume was calculated using the modified ellipsoid formula: volume = (length × width 2) × 0.52. Mice were sacrificed, and tumors were dissociated into single-cell suspensions, and T cells per tumor were counted by flow cytometry (CD45+ cells (g tumor). N = 3 mice per time point and T cell. A). As shown, TCR-D115 / PD-1tm:BB T cells reached significantly higher intratumoral cell numbers than T cells without chimeric receptors on day 2, and this number was still higher on day 7. Concomitant with the higher cell numbers, TCR-D115 / PD-1tm:BB T cells achieved better tumor control, with tumor volume reduced compared to the starting volume on day 2 and still well controlled on day 7 (mean fold change in tumor volume on day 7 of 1.7 compared to 2) (B). [Figure 7]Expected Effects of CD40L:28 Chimeric Proteins Expressed in Human T Cells: 1) Cis-activating and supportive effects on transgene-expressing T cells: Ligation of the fusion protein in T cells induces CD28 signaling, which in turn supports TCR signaling, CTL effector function, and survival, leading to better tumor killing. 2) Trans-effects on antigen-presenting cells (APCs): Activation of CD40 on APCs stimulates them to secrete cytokines (IL-12) and chemokines that enhance CTL effector function. 3) Trans-effects on endothelium: Ligation of CD40 expressed on tumor endothelium triggers endothelial apoptosis, thereby disrupting tumor vascular support. 4) Trans-effects on tumor cells: Ligation of CD40 expressed on tumor cells triggers tumor cell apoptosis. [Figure 8] Three different constructs with different linker and domain orientations are generated: 1) CD40LFc:28; 2) CD40LFil:28; 3) CD40L:28i. [Figure 9] Expression Profile of CD40L:28 Constructs in Human T Cells After Stable Retroviral Transduction Human T cells were retrovirally transduced to express the chimeric proteins, and surface protein expression was analyzed at days 7 and 17. Surface expression was detected at day 7 but disappeared by day 17, as T cells reached a quiescent state (Figure 9A). Surface expression was regained after TCR-specific activation (Figure 9B). Thus, these chimeric proteins exhibit an inducible surface presence depending on when and where T cell support is needed. [Figure 10]Chimeric CD40L:28 protein expressed in T cells can activate B cells (trans-effect). T cells were cocultured with primary B cells at a 1:1 ratio for 24 hours. Cells were then harvested and analyzed for CD86 and Fas by flow cytometry. Bars indicate the mean fluorescence intensity of the analyzed markers in B cells. The chimeric protein CD40L-ECD was functionally active, as evidenced by the high expression of CD86 and Fas observed in B cells. A similar effect was observed with CD83, another marker of B cell activation (not shown). Three different constructs showed graded activity similar to or greater than that of the native CD40L protein. [Figure 11] The chimeric CD40L:28 protein expressed in TCR-T58 T cells supports T cell function (cis effect). TCR-T58 transgenic T cells without the chimeric protein (crtl), or expressing native CD40L or the chimeric protein after retroviral transduction, were cocultured with melanoma cell lines (SK-Mel23, FM86, positive for TCR ligands and CD40). A) IFN-γ was measured in the coculture supernatant for 48 hours. B) Cytotoxicity against melanoma tumor cells was measured (chromium release assay) after 4 hours of coculture. Results shown are from a 5:1 effector:target ratio. As can be seen from both assays, T cells expressing the chimeric protein secreted more IFN-γ and exhibited greater cytotoxicity against tumor cells compared to controls. Thus, the chimeric protein CD28-ICD is functionally active, ie, enhances the effector activity of T cells expressing the chimeric protein.

[0079] The present invention is further illustrated by the following examples. Additionally, the examples and specific embodiments described herein should not be construed as limiting the invention to such specific embodiments.

[0080] Example Plasmids encoding chimeric receptors Chimeric receptor sequences were ordered from Geneart, Life Technologies. The sequences were delivered as lyophilized powders and dissolved in nuclease-free water at a concentration of 0.5 μg / μL. For amplification, Geneart constructs were chemically transformed into TOP10 or MACH1 E. coli following standard plasmid preparation methods.

[0081] Electroporation of ivtRNA into human primary T cells To prepare ivtRNA, the chimeric receptor sequence was cloned into pGEM. Cloning into the pGEM vector (provided by S. Milosevic, Medigene GmbH, Martinsried, Germany) was performed using HindIII or HindII and EcoRI (New England Biolabs). ivtRNA was generated from the pGEM plasmid using the mMESSAGE mMACHINE kit (Ambion) according to the manufacturer's protocol. 20 μg of ivtRNA was electroporated into human primary T cells using a Gene Pulser Xcell (Bio-Rad) at 900 V for 2.3 ms.

[0082] Retroviral transduction of human primary T cells The chimeric receptor sequence was cloned into the pMP71-PRE vector (for retroviral transduction, see Leisegang 2008, in citation). Retroviral transduction of T cells was performed as described above (Leisegang 2008, in citation). 1 x 10 T cells were plated per well in RPMI 1640 supplemented with 10% human serum, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin (all Invitrogen) plus 100 U / mL IL-2 (Cancernova). 6Human PBMCs from healthy donors were plated in 24-well plates at a cell density of 1 μg / mL and activated with 5 μg / mL OKT3 (provided by E. Kremmer, Helmholtz Center Munich, Germany) and 1 μg / mL anti-CD28 (BD Pharmingen) for 2 days.

[0083] Retroviruses encoding amphotropic chimeric receptors were generated as described (Leisegang et al., Clin Cancer Res (2010), 16(8): 2333-2343) using TransIT®-LT1 reagent (Mirus) according to the manufacturer's protocol. Viral supernatants were collected after 48 hours and bound to RetroNectin® (10 μg / mL, Takara)-coated plates by centrifugation.

[0084] PBMCs activated for 2 days were added to virus-coated plates for 24 hours, then split to freshly virus-coated plates and cultured for an additional 3 days. Transduced PBMCs were transferred to uncoated plates and cultured for at least 12 more days, at which time the amount of IL-2 was reduced to 50 U / mL. Receptor expression was determined 12 days post-transduction using an anti-PD-1 antibody (BioLegend).

[0085] cell culture HLA-A2 +HEK / Tyr and HEK / Tyr / PD-L1 were generated by transducing HEK293 cells to express tyrosinase (HEK / Tyr) or tyrosinase and PD-L1 (HEK / Tyr / PD-L1). After transduction, HEK293 cells were single-cell cloned, and clones were selected for equivalent HLA-A2 and tyrosinase expression. SK-Mel23 (gift from M.C. Panelli, NIH, Bethesda, USA), HEK / Tyr, and HEK / Tyr / PD-L1 were grown in RPMI-1640 supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin / streptomycin (RMPI basic) + 12% FCS.

[0086] Multiparameter flow cytometry to determine chimeric receptor expression and T cell proliferation Flow cytometry analysis was performed on a BD LSRII. Cells were stained in PBS (Invitrogen) supplemented with 2% human serum, 0.1% sodium azide, and 2 mM EDTA (all Sigma-Aldrich). Expression of human chimeric receptors and transgenic TCRs was analyzed using anti-CD3-PE-Cy7, anti-mouse TCRβ-constant region-PB (all BioLegend), anti-CD4-APC-A780, anti-PD-1-APC (all eBioscience), anti-CD8-V500 (BD), and 7-AAD (Sigma-Aldrich).

[0087] HEK293 cells were analyzed using anti-HLA-A2 (ATCC Hb54) + anti-mouse IgG1-A488 (Invitrogen), anti-PD-L1-FITC (BD), anti-tyrosinase (Upstate) + anti-mouse IgG2a-A647 (Invitrogen), and 7-AAD.

[0088] T cells were analyzed after injection into xenograft tumors using CFSE, anti-CD45-PE-Cy7, anti-CD8-PB (BD), CD4-APC-A780, anti-PD-1-APC, and 7-AAD. CD45+ leukocytes were selected, and after gating on live and single cells, CD8 + , CD4 + CFSE intensity was analyzed in dbl- and dbl- cells. Data were analyzed using FlowJo 8.8.7 software.

[0089] Co-culture and cytokine assays to assess the effect of fusion proteins on T cells TCR-T58 or TCR-D115 transgenic human T cells were electroporated with ivtRNA or transduced with retroviral vectors to express the chimeric receptors and then cocultured with HEK / Tyr or HEK / Tyr / PD-L1 cells at a ratio of 1:2. Coculture supernatants were collected after 16 hours and analyzed by sandwich ELISA (BD) or Bio-Plex (Bio-Rad) according to the manufacturer's protocol. The transduced TCRs, T58 and D115, are CD8 + It only functions in T cells and CD8 + / CD4 + Because the T cell ratio varied between experiments, the amount of cytokines measured was compared with the TCR + CD8 + Normalization was performed to the percentage of T cells (determined by flow cytometry), applying the following formula:

[0090] NSG mice NSG mice were obtained from Charles River. NOD / scid IL2Rgnull (NSG) mice are a genetic breeding strategy for the non-obese diabetic (NOD) mouse background, characterized by impaired innate immunity. NSG mice harbor a loss-of-function mutation in the PRKDC gene, prkdcscid, which results in defective repair of DNA strand breaks during V(D)J recombination in developing B and T cells. This severe combined immunodeficiency (scid) is characterized by a severe reduction in T and B cells. Furthermore, NSG mice harbor a null mutation in the gamma chain of the IL-2 receptor (IL2Rgnull), which blocks NK cell differentiation. The defective innate immunity and absence of adaptive immunity make NSG mice a suitable model system for adoptive T cell therapy of human tumor xenografts.

[0091] Human melanoma xenograft model Animal experiments were approved by local authorities and conducted in accordance with legal regulations. Male mice aged 7–11 weeks were inoculated with 5 × 10 6 HLA-A2 + tyrosinase + Human melanoma cells SK-Mel23 (kindly provided by Monica C. Panelli, NIH, Bethesda, USA) were injected sc. This melanoma line was chosen because it expresses PD-L1 / L2 as well as HLA-A2 and tyrosinase, which are required to form ligands for TCR-D115 and TCR-T58 (Wilde et al., Blood (2009), 114: 2131-2139). TCR-D115-expressing T cells were selected for mouse experiments because they can recognize SK-Mel23 with low avidity, insufficient to eradicate established tumors.

[0092] 802mm 3 Tumors were allowed to grow for approximately 16 days until they reached a size of (SEM = ±83). Tumor size (mm) was calculated using the formula for determining the volume of the spheroid. 3 ) was calculated as: π / 6 × length × width × height.

[0093] Expansion of TCR-D115 T cells in the tumor environment of human SK-Mel23 xenografts Cell proliferation is assessed using a cell tracking dye, i.e., CFDA-SE. The dye permeates the cell membrane and is converted to fluorescent carboxyfluorescein succinimidyl ester (i.e., CFSE). With each cell division, the fluorescence intensity of CFSE is halved to monitor T cell proliferation.

[0094] Here, TCR-D115 T cells expressing or not expressing the chimeric receptor were labeled with 0.15 μM CFDA-SE for 8 min at 37°C. The reaction was stopped with FCS, and T cells were washed twice with PBS and then cultured at 10 × 10 7 The T cells were resuspended in PBS at a concentration of 1000 cells / mL. 50 μL of the T cell suspension was transferred to established sc SK-Mel23 xenografts (802 mm 3 , SEM = ±83).

[0095] Tumors were harvested 1, 2, 4, 6, and 11 days after it injection. Single-cell suspensions were prepared by mechanical and enzymatic disaggregation (Prinz et al., J Immunol (2012), 188: 5990-6000) and used for flow cytometry analysis.

[0096] Chromium release assay 51 Cr-labeled melanoma cells were used as targets at a constant cell number of 2000 cells / well in 96-well V-bottom plates. Experiments were performed using duplicate measurements of a four-step titration of effector cells. In parallel wells, target cells without T cells were incubated to determine spontaneous release of [51Cr]. After 4 hours, supernatants were collected and transferred to a counting plate (PerkinElmer) for cpm measurement. The maximum cpm was determined by directly transferring the labeled target cells to a counting plate for cpm measurement. The percentage of specific lysis was calculated as follows: % specific lysis = (experimental cpm - spontaneous cpm) / (maximum cpm - spontaneous cpm) × 100.

[0097] statistics Statistical tests specified in the figure legends were performed using GraphPad Prism 6 software.

[0098] result The results are shown in the figures.

Claims

1. (i) (a) a soluble CD40L as its extracellular domain (ECD) at its N-terminus, wherein the ECD includes a signal peptide sequence; (b) the transmembrane domain (TMD) of CD28; and (c) the intracellular domain (ICD) of CD28 at its C-terminus; or (ii) (a) the ICD of CD28 at its N-terminus; (b) a fragment of CD40L ICD; (c) TMD of CD40L; and (d) the ECD of CD40L at its C-terminus Including, A fusion protein comprising: When the fusion proteins of (i) and (ii) are expressed on T cells, they increase the rate of B cell activation. Fusion proteins.

2. The ECD is a linker and / or hinge region at the C-terminus of the ECD; The fusion protein (i) according to claim 1.

3. The signal peptide sequence is a PD-1 signal peptide sequence. The fusion protein of claim 1 (i), comprising:

4. The fusion protein of claim 1 (i), wherein the ECD comprises a Gly / Ser linker and an Fc spacer or a filamin spacer at the C-terminus of the ECD.

5. The fusion protein of claim 4 , wherein the Fc spacer is an IgGFc spacer.

6. The fusion protein of claim 4 , wherein the filamin spacer is a Fil3 spacer.

7. i) the soluble CD40L as its ECD is human soluble CD40L; ii) the CD28 ICD is a human CD28 ICD; and / or iii) the CD28 TMD is the human CD28 TMD, or the CD40L TMD is the human CD40L TMD; The fusion protein according to any one of claims 1 to 6.

8. The fusion protein of any one of claims 1 to 7 (i), wherein the TMD of CD28 comprises the amino acid sequence KPFWVLVVVGGVLACYSLLVTVAFIIFWV contained in SEQ ID NO:

14.

9. The fusion protein of claim 1 or 7 (ii), wherein the TMD of CD40L comprises the amino acid sequence IFMYLLTVFLITQMIGSALFAVYL contained in SEQ ID NO:

30.

10. The fusion protein of any one of claims 1 to 8 (i), wherein the soluble CD40L comprises the amino acid sequence of amino acids 113 to 261 of SEQ ID NO:

30.

11. The fusion protein of any one of claims 1, 7 and 9 (ii), wherein the CD40L ECD comprises the amino acid sequence HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL contained in SEQ ID NO:

30.

12. The fusion protein of (ii) according to any one of claims 1, 7, 9 and 11, wherein the fragment of CD40L ICD comprises the amino acid sequence of amino acids 14 to 22 of SEQ ID NO:

30.

13. The fusion protein of any one of claims 1 to 12, wherein the ICD of CD28 comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS contained in SEQ ID NO:

14.

14. A fusion protein according to any one of claims 1 to 5, 7, 8, 10 and 13 (i) comprising or consisting of SEQ ID NO:

27.

15. A fusion protein according to any one of claims 1 to 4, 6 to 8, 10 and 13 (i) comprising or consisting of SEQ ID NO:

28.

16. 14. A fusion protein of (ii) according to any one of claims 1, 7, 9, 11, 12 and 13, comprising or consisting of SEQ ID NO:

29.

17. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 16.

18. A vector comprising the nucleic acid molecule of claim 17.

19. 19. A host cell comprising the nucleic acid molecule of claim 17 or the vector of claim 18.

20. 20. A host cell according to claim 19, transfected with the nucleic acid molecule of claim 17 or the vector of claim 18.

21. 21. The host cell of claim 19 or 20, wherein the nucleic acid molecule or the vector is stably integrated into the genome of the host cell.

22. 22. The host cell of claim 20 or 21, transduced via retroviral or lentiviral transduction.

23. A host cell according to any one of claims 19 to 22, which stably expresses a fusion protein encoded by the nucleic acid molecule according to claim 17.

24. CD8 ＋ The host cell of any one of claims 19 to 23, which is a T cell.

25. (1) transducing a host cell with the nucleic acid molecule of claim 17 or the vector of claim 18; (2) culturing the transduced host cells of step (1) in a suitable medium to grow the cells and express the fusion protein encoded by the nucleic acid molecule or the vector; (3) recovering the host cells from the medium; and A method for preparing the host cell of any one of claims 19 to 24, comprising:

26. A fusion protein according to any one of claims 1 to 16, a nucleic acid molecule according to claim 17, a vector according to claim 18, and / or a host cell according to any one of claims 19 to 24.

10. A pharmaceutical composition comprising:

27. A fusion protein according to any one of claims 1 to 16, a nucleic acid molecule according to claim 17, a vector according to claim 18, and / or a host cell according to any one of claims 19 to 24.

1. A pharmaceutical composition for cancer and chronic viral infections comprising:

28. A fusion protein according to any one of claims 1 to 16, a nucleic acid molecule according to claim 17, a vector according to claim 18, and / or a host cell according to any one of claims 19 to 24. Kits or kit-in-parts including:

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